Ryanodine Receptors 2

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Ryanodine Receptor Calcium Release Channels

MICHAEL FILL AND JULIO A. COPELLO

Department of Physiology, Loyola University Chicago, Maywood, Illinois

I. Introduction 894II. History 894

III. Structure-Function Overview 895IV. Excitation-Contraction Coupling 897

A. DHPR-RyR interaction 897 V. Study of Intracellular Calcium Release 899

VI. Study of Single Ryanodine Receptor Channels 900 VII. Permeation Properties 901

VIII. Action of Ryanodine 902IX. Overview of Ryanodine Receptor Regulation 902 A. Cytosolic Ca 2 903B. Luminal Ca 2 903C. ATP and Mg2 903D. Redox status 903E. Cyclic ADP-ribose 904F. Phosphorylation/dephosphorylation 904

X. Regulation by Closely Associated Proteins 904 A. Calmodulin 904B. Calsequestrin 904C. FK-506 binding protein 905D. DHPR-loop peptide 905

XI. Calcium Regulation Paradox 906XII. Steady-State Calcium Dependence 907

XIII. Calcium Activation Kinetics 907 A. Rate of Ca 2 activation 907B. Rate of Ca 2 deactivation 908

XIV. Feedback Calcium Regulation 908XV. Potential Negative Control Mechanisms 908

A. Ca 2-dependent inactivation 909B. Stochastic attrition 909C. Adaptation 909D. Activation-dependent or “fateful” inactivation 910E. Depletion/luminal Ca 2 effects 910F. Allosteric or coupled gating 910

XVI. Adaptation Debate 911 A. DM-nitrophen Ca 2 overshoot 911B. Ca 2 overshoot concern 911C. Differing interpretations 912

XVII. Modal Gating 913XVIII. Concluding Remarks 914

Fill, Michael, and Julio A. Copello. Ryanodine Receptor Calcium Release Channels. Physiol Rev 82: 893–922,2002; 10.1152.physrev.00013.2002.—The ryanodine receptors (RyRs) are a family of Ca 2 release channels found onintracellular Ca 2 storage/release organelles. The RyR channels are ubiquitously expressed in many types of cellsand participate in a variety of important Ca 2 signaling phenomena (neurotransmission, secretion, etc.). In striatedmuscle, the RyR channels represent the primary pathway for Ca 2 release during the excitation-contraction coupling process. In general, the signals that activate the RyR channels are known (e.g., sarcolemmal Ca 2 influx or depolarization), but the specific mechanisms involved are still being debated. The signals that modulate and/or turnoff the RyR channels remain ambiguous and the mechanisms involved unclear. Over the last decade, studies of

Physiol Rev

82: 893–922, 2002; 10.1152/physrev.00013.2002.

www.prv.org 8930031-9333/02 $15.00 Copyright © 2002 the American Physiological Society



RyR-mediated Ca 2 release have taken many forms and have steadily advanced our knowledge. This robust field,

however, is not without controversial ideas and contradictory results. Controversies surrounding the complex Ca 2

regulation of single RyR channels receive particular attention here. In addition, a large body of information is

synthesized into a focused perspective of single RyR channel function. The present status of the single RyR channel

field and its likely future directions are also discussed.

I. INTRODUCTION

Calcium is a common second messenger (38, 42, 123)

that regulates many processes in cells (e.g., contraction,

secretion, synaptic transmission, fertilization, nuclear

pore regulation, transcription). Although cells are typi-

cally bathed in solutions containing a relatively high Ca 2

concentration (i.e., in the millimolar range), the cyto-

plasm of most cells contains much lower resting Ca 2

concentrations (i.e., in the 100 nM range). Thus Ca 2

entry across the surface membrane can substantially ele-

vate cytosolic Ca 2 levels providing the Ca 2 trigger sig-

nals for a large number of physiological processes. Thesurface membrane, however, is not the only source of

triggering Ca 2 signals. Most cells have developed an

additional pathway to generate localized and fast trigger

Ca 2 signals deep inside the cell. This other pathway

involves specialized intracellular Ca 2 storage/release or-

ganelles.

Cells have many different types of Ca 2 transporters

(channels, exchangers, pumps) that modulate cytosolic

Ca 2 levels. The rich array of Ca 2 transport proteins in

the surface and intracellular membranes provides numer-

ous potential Ca 2 signaling pathways that can respond to

many different types of stimuli. Specificity of any oneCa 2 signal pathway to a particular process is often made

possible by assembling the particular signaling proteins

into a local complex. Localizing the Ca 2 signaling effec-

tively allows Ca 2 to be effectively directed to a particular

physiological function. Certain vascular smooth muscles

are a good example of this. In these smooth muscles, large

global intracellular Ca 2 signals activate the contractile

machinery causing the cell to contract. However, small

very localized Ca 2 signals (i.e., Ca 2 sparks) that arise

from intracellular Ca 2 stores activate certain K chan-

nels in the surface membrane promoting relaxation of the

cell (219).

The primary intracellular Ca 2 storage/release or-ganelle in most cells is the endoplasmic reticulum (ER).

In striated muscles, it is the sarcoplasmic reticulum (SR).

The ER and SR contain specialized Ca 2 release channels.

There are two families of Ca 2 release channels: the

ryanodine receptors (RyRs) and inositol trisphosphate

receptors (IP3Rs). Each family of Ca 2 release channels

appears to contain three different isoforms (300, 309).

However, there may be a fourth type of RyR channel in

fish (i.e., RyR-slow; Refs. 90, 210). The Ca 2 release chan-

nels are large oligomeric structures formed by association

of either four RyR proteins or four IP3R proteins. The RyR

and IP3R proteins share significant homology, and this

homology is most marked in the sequences that may form

the channel’s pore (204, 271, 299, 315a, 367).

Calcium regulates all the RyR and IP3R channels.

However, each Ca 2 release channel has its own distin-

guishing functional attributes. The IP3R channels require

the presence of inositol 1,4,5-trisphosphate. The activity

of one type of RyR channel is coupled to a “ voltage

sensor ” in the plasma membrane. The Ca 2-induced Ca 2

release process governs the activity of the other types of

RyR channel. The distinguishing functional attributes of

each RyR or IP3R channels likely underlie the spatiotem- poral complexity of intracellular Ca 2 signaling in cells.

During the last 10 years, there have been numerous

reviews on several aspects of regulation of RyR-mediated

Ca 2 signaling and/or related topics (e.g., Refs. 18, 43, 60,

69, 76, 84, 87, 92, 112, 179, 195, 199, 202, 225, 245, 252, 253,

255, 260, 265, 281, 287, 300, 301, 307, 315a, 335, 346, 354).

Here, we focus on the RyR Ca 2 release channels found in

striated muscle. The controversial and complex Ca 2 reg-

ulation of single RyR channels is given particular atten-

tion. An attempt has been made to cover this large rapidly

growing and dynamic body of information. Regrettably, it

is impossible to describe all of the quality studies that

characterize this field. It is hoped that the single-channel

perspective of this review will be useful to readers seek-

ing to understand the complex dynamics of single RyR

channel regulation

II. HISTORY

The significance of SR Ca 2 release during skeletal

muscle contraction was recognized many decades ago

(for review, see Refs. 38, 42, 260). Electrophysiological

experiments and 45Ca autoradiography studies provided

evidence in intact muscle that Ca 2 is released from the

terminal cisternae of the SR in response to depolarizationof surface membrane invaginations called transverse tu-

bules (t tubules) (reviewed in Ref. 260). A number of

studies followed, and a putative mechanism was pro-

posed (43, 251–255, 265–267, 296), namely, that there is a

physical coupling between an integral t-tubule protein

and the SR Ca 2 release channel. The idea was that

voltage-dependent movements of charged particles in the

integral t-tubule protein activate the SR Ca 2 release

channel through this physical coupling. This mechanism

is generally referred to as depolarization-induced Ca 2

894 MICHAEL FILL AND JULIO A. COPELLO

Physiol Rev • VOL 82 • OCTOBER 2002 • www.prv.org



release (DICR). Parallel work in other experimental prep-

arations (i.e., SR vesicles and skinned muscle fibers) con-

firmed the existence and importance of the DICR mech-

anism in skeletal muscle (38, 94, 121, 127, 138, 150, 151,

183, 260, 296, 321).

In other early works, it was realized that a small

increase in cytosolic Ca 2

concentration could inducemassive intracellular SR Ca 2 release events in both skel-

etal and cardiac muscle (75, 89). This phenomenon is

often called Ca 2-induced Ca 2 release (CICR). Later,

meticulous studies by Fabiato (80, 81) established that the

CICR process was sensitive to both the speed and ampli-

tude of the applied Ca 2 trigger. These studies and others

helped reveal the complexity of the regulatory mecha-

nisms that control SR Ca 2 release.

A major advancement was the identification of the SR

Ca 2 release channel using an alkaloid called ryanodine.

Ryanodine is found naturally in the stem and roots of the

plant Ryania speciosa. The ryanodine alkaloid was first

isolated as a potential insecticide (73, 133), and its potent

paralytic action on skeletal and cardiac muscles was im-

mediately evident (73, 82, 93, 218, 311, 312). However, its

action was dif ficult to understand. Specifically, it was

dif ficult to understand how ryanodine could induce rigid

paralysis in skeletal muscle but flaccid paralysis in car-

diac muscle.

The ryanodine alkaloid was shown to inhibit SR Ca 2

release by binding with high af finity to a protein present in

the SR membrane (41, 80, 88, 93, 309 –312, 345, 347). The

ryanodine binding protein was purified (39, 88, 124, 147,

240, 241) and existed in a tetrameric complex (149) whose

shape was subsequently visualized using electron micros-copy (131, 147). Interestingly, the size and shape of the

ryanodine binding protein complex was similar to that of

the “feet” structures, which appear to physically link the t

tubule and SR membranes (83, 94). Incorporation of the

ryanodine binding protein complex into artificial planar

lipid bilayers revealed that this structure was an ion chan-

nel (124, 130, 147, 294). The RyR channel is a poorly

selective Ca 2 channel (selectivity Ca 2 /K 6) with very

high conductance (700 pS when K is charge carrier

and 100 pS when Ca 2 is charge carrier). The channel is

regulated by Ca 2, Mg2, ATP, and caffeine. Interestingly,

application of the ryanodine alkaloid locks the channel in

a slow-gating subconductance state (124, 147, 294, 337).Openings of the ryanodine-bound channel are long-lived

and have a smaller unit current (1/3 or 1/2 of control

amplitude). This action of ryanodine on single RyR chan-

nel function is quite distinctive and is now often used to

functionally identify the channel.

These early single-channel studies unambiguously

identified the RyR channel as the SR Ca 2 release channel

in striated muscle (84, 87). It also became evident how the

ryanodine alkaloid can induce rigid paralysis in skeletal

muscle but flaccid paralysis in cardiac muscle. In cardiac

muscle, ryanodine promotes Ca 2 leak from intracellular

stores because it locks the RyR channels in the long-lived

subconduction state. This “leaked” Ca 2 is rapidly re-

moved from the cell by the relatively strong surface mem-

brane Ca 2 extrusion mechanisms present in cardiac

muscle (17, 20). Intracellular Ca 2 stores eventually de-

plete, yielding the observed flaccid cardiac paralysis. Incontrast, skeletal muscle lacks the strong surface mem-

brane Ca 2 extrusion mechanisms present in cardiac

muscle. The ryanodine-induced Ca 2 leak from intracel-

lular stores causes Ca 2 to accumulate in the cytoplasm.

The result is abnormally high cytoplasmic Ca 2 levels that

induce sustained contraction (i.e., the rigid skeletal mus-

cle paralysis).

III. STRUCTURE-FUNCTION OVERVIEW

Molecular cloning studies have defined three differ-

ent RyR isoforms in fish, amphibian, birds, and mammals

(6, 60, 205, 215, 225, 245, 301, 307, 315a). Although the RyR

proteins share significant homology with the IP3R recep-

tors, they have little homology with the more widely

studied voltage-dependent Ca 2 channels found in the

surface membrane (204, 315a, 321). In mammals, the three

RyR isoforms (RyR1, RyR2, and RyR3) are encoded by

three different genes on different chromosomes (186, 205,

318, 315a, 369). The RyR proteins are in a variety of

tissues, but the highest densities are in striated muscles

(5, 6, 148, 205, 212, 225, 227, 231, 233, 299, 308). There

may also be structurally, and perhaps functionally, dis-

tinct splice variants of these channels (102, 221, 370).However, the functional significance and distribution

among fiber types of RyR splice variants are currently

unknown.

In mammalian striated muscles, the expression of the

different RyR protein isoforms is tissue specific. The pre-

dominant RyR isoform in skeletal muscle is the RyR1

protein, commonly referred to as the skeletal RyR isoform

(60, 195, 225, 245, 299). The RyR2 protein is the most

abundant isoform in cardiac muscle, and the RyR2 protein

is commonly referred to as the cardiac RyR isoform. The

RyR3 protein is also found in mammalian striated mus-

cles, but at relatively low levels (98, 308, 325). For exam-

ple, the RyR3 protein represents 5% of the overall RyR population in diaphragm (135, 213).

What is the physiological role of the RyR3 channel in

striated muscle or elsewhere? Mice missing the RyR1 and

RyR2 gene products (i.e., RyR1 or RyR2 knock-outs) die

early during embryonic development (317, 320). In con-

trast, mice missing the RyR3 gene product (i.e., RyR3

knock-out) lead relatively normal lives and have quite

normal striated muscles (55, 317). Interestingly, the CICR

process in neonate skeletal muscle fibers from RyR3-

knockout mice appears to be impaired (357). The impli-

RYANODINE RECEPTOR Ca 2 REGULATION 895




cations of this observation are not yet clear. In any event,

it appears that the physiological role of the RyR3 channel

in mammals, unlike that of the RyR1 and RyR2 channels,

can be readily compensated for both during development

and in the adult.

The RyR nomenclature and distribution are some-

what different in nonmammalian striated muscle (35, 148,212, 232, 307). Nonmammalian skeletal muscle (e.g., frog,

fish, and chicken) contains nearly equal amounts of two

different RyR proteins, named the -RyR and -RyR.

These isoforms are homologs of mammalian RyR1 and

RyR3 forms, respectively (5, 6, 148, 212, 223, 227, 233, 308,

307). It is known that embryonic chicken skeletal muscle

cells fail to develop normal excitation-contraction cou-

pling in the absence of -RyR (132), the RyR1 analog.

Although there have been no -RyR knock-out studies, all

indications are that the -RyR must play some fundamen-

tally important role in the operation of nonmammalian

skeletal muscle (307). One likely possibility is that the

-RyR isoform participates in the CICR process, effec-

tively amplifying the initial DICR mediated by the -RyR

isoform.

The RyR proteins are also expressed in smooth mus-

cle and many nonmuscle tissues (e.g., neurons). These

other tissues generally contain multiple RyR isoforms.

For example, expression of all three RyR isoforms has

been reported in aortic smooth muscle (162, 186), cere-

brum (101, 115, 220), and cerebellum (101, 115, 187).

These tissues also express multiple IP3R isoforms, and

thus one cell may contain many types of RyR and IP 3R

channels. The significance of this remarkable redundancy

in intracellular Ca 2

release channel expression in non-muscle systems is not clearly understood. One might

speculate that the rich morphological diversity implies

equally rich functional heterogeneity and that the multiple

functionally distinct Ca 2 release channels may govern

different Ca 2 signaling tasks. Consequently, Ca 2 release

channel diversity may represent one factor that allows

cells to simultaneously carry out the myriad of Ca 2

signaling tasks they need to do to survive. Even striated

muscles, which are highly specialized for the Ca 2 signal-

ing that governs contraction, must carry out other types

of Ca 2 signaling tasks to survive. Perhaps the RyR3

channel or the low density of IP3Rs expressed in striated

muscles (239, 276) carry out this “other ” Ca 2 signaling. At the amino acid level, the three mammalian RyR

isoforms share 70% identity (115, 215, 318, 315a). Anal-

ysis of the primary amino acid sequences suggests that

the membrane-spanning domains of the RyR are clustered

near its COOH terminus (271, 299, 315a, 367). Truncation

mutants, containing only these putative transmembrane

regions (TMRs), are suf ficient to form Ca 2-selective

channels when incorporated into planar lipid bilayers (23,

24). Mutation of certain regions in and around the puta-

tive TMRs generates channels with altered ion selectivity

(23, 103, 367). Thus there is reasonable certainty that this

region of the protein contains the determinants that form

the Ca 2-selective pore of the RyR channel.

Analysis of the RyR’s primary amino acid sequence

also has revealed several consensus ligand binding (e.g.,

ATP, Ca 2

, caffeine, and calmodulin) and phosphoryla-tion motifs. Experimental verification of these sequences

as potential regulatory sites (e.g., Refs. 23, 45, 47) is

ongoing. For example, substitution of alanine-3882 by a

glutamate (E3882A) in the RyR3 protein results in RyR3

channels with altered Ca 2 sensitivity (45). This implies

that this region of the protein may contain the “Ca 2

sensor ” of the channel. A gross deletion of amino acids

183– 4006 in the RyR1 protein results in channels that lack

caffeine sensitivity and do not inactivate at high Ca 2

concentrations (23). Exchange of amino acids 4187– 4628

of RyR1 for the corresponding cardiac RyR2 sequence

results in channels with increased caffeine sensitivity and

altered Ca 2 sensitivity (66, 67).

Two skeletal muscle diseases, malignant hyperther-

mia (MH) and central core disease (CCD), have also

provided useful insights into the RyR1 structure-function

relationship (180, 366). These diseases are marked by

pathologies like hypercarbia, rhabdomyolisis, and gener-

alized muscle contraction. A defect in the RyR1 channel

function was suspected to be involved in these patholo-

gies (85, 277, 338). It is now known that specific point

mutations in the RyR1 protein are associated with the MH

and CCD diseases (180, 243, 277, 366). Recent reports

describe more than 20 different point mutations in the

human RyR1 isoform that are associated with the MHand/or CCD syndromes (140, 193). The majority of the

mutations are clustered in two regions of RyR1 sequence,

residues 35– 614 (near the NH2 terminal) and 2163–2458 (a

central location). These data are important because these

mutations may reveal important points in the RyR1 struc-

ture that govern its function.

It is clear that the exploration of the RyR structure-

function relationship by mutagenesis is still in its infancy.

This is in stark contrast to the molecular study of other

types of ion channels. There are several reasons why we

have not progressed further or faster in this area. First,

the RyR channels are expressed in intracellular organelles

(ER or SR), and thus single RyR channel function cannotbe directly and easily accessed. For surface membrane

channels, function is often ef ficiently assessed by the

classical patch-clamp method on a few tagged expressing

cells. For the RyR case, function is generally defined in

vitro after the channels have been biochemically isolated

from millions of expressing cells. Second, the RyR is a

huge protein (1/10 the size of a ribosome). This large

size complicates its genetic manipulation as well as the

selection of sites to mutate. Third, recent studies suggest





that RyR1 channel function may involve inter-RyR domain

interactions (128, 279, 355), and thus some functional

attributes may be controlled by multiple and very com-

plex structural determinants. Despite these and other

technical challenges, future mutagenesis studies promise

to provide some very interesting insights into RyR struc-

ture-function in the not too distant future.Usually, ion channel structure-function studies are

interpreted in the absence of information concerning the

channel’s detailed three-dimensional (3-D) structure.

There are some exceptions where channels or channel

segments have been crystallized. However, the RyR chan-

nel is not one of these exceptions. Intriguingly, some

gross details of the 3-D RyR structure (i.e., general shape)

have been predicted using cryoelectron microscopy and

3-D reconstruction techniques. The RyR1 channel has

fourfold symmetry, presumably reflecting its formation by

four RyR protein monomers (249, 270, 342). The RyR

channel has two distinct domains (249, 270, 342). One is a

large cytoplasmic assembly (29 29 12 nm), consist-

ing of loosely packed protein densities. The other is a

small transmembrane assembly that protrudes (7 nm)

from the center of the cytoplasmic assembly (270, 342).

This transmembrane assembly appears to have a central

hole that can be occluded by a pluglike mass. This hole

and plug may correspond to the Ca 2-selective pore and

its gate (230). In general, the shapes of the RyR1 and RyR2

channels are quite similar (275). There are, however, spe-

cific points of structural heterogeneity. These points may

contribute to the isoform-specific functional attributes of

the RyR channels. Cryoelectron microscopy studies have

also revealed the sites where certain peptides (calmodu-lin, FK binding proteins, and imperatoxin) interact with

the RyR channels (263, 343). Recently, antibodies have

been used to map the location, on the 3-D structure, of a

specific domain (amino acid residues 4425– 4621) that

may be implicated in the Ca 2-dependent regulation of

the RyR channel (15).

Certain structural details of the RyR channel have

also been obtained using certain biophysical approaches.

For example, the width of the RyR2 pore has been esti-

mated from molecular sieving experiments (332). In these

studies, it was found that only cations with a predicted

circular radius smaller than 3.5 Å could permeate through

the channel. In another study, streaming potential exper-iments indicated that the RyR2 channel contains a narrow

region in which H2O (radius 1.5 Å ) and Cs (radius 2 Å )must pass in a single-file fashion (337). Combined, these

results suggest that the RyR2 channel pore is 3 Å wide.

Electrical distance measurements, using trimethylammo-

nium derivatives of varying length, indicated that the

physical length of the voltage drop along the RyR2 chan-

nel pore is 10.4 Å (331). This is consistent with steaming

potential measurements that suggest that three H2O mol-

ecules (9 Å ) occupy the single-file region of the RyR2

channel pore (337). Thus the RyR channels appear to have

a relatively short permeation pathway.

IV. EXCITATION-CONTRACTION COUPLING

A role in striated muscle excitation-contraction (E-C)

coupling is probably the RyR channel’s most notable

claim to fame. There is clear evidence that the RyR chan-

nels interact with voltage-dependent Ca 2 channels (i.e.,

dyhydropyridine receptors; DHPRs) in the nearby t-tubule

membrane (246, 247, 321, 322). This functional interaction

between the DHPR and RyR is commonly referred to as

E-C coupling (Figs. 1 and 2). Depolarization of the t-tubule

membrane (i.e., excitation) induces conformational

changes in DHPR that ultimately lead to activation of RyR

channel in the SR membrane. The activation of RyR chan-

nels leads to massive Ca 2 release from the SR, which in

turn initiates contraction. The bulk of information ad-

dressing RyR channel regulation has been obtained from

studies focused on the process of E-C coupling in striated

muscle.

More than a century ago, Ringer demonstrated that

the cardiac E-C coupling process required the presence of

extracellular Ca 2 (reviewed in Ref. 38). In cardiac mus-

cle, the DHPR receptor (an L-type Ca 2 channel) carries a

small Ca 2 influx that activates the RyR2 channel (16, 208,

250, 270, 272). A similar process governs the RyR chan-

nels in some invertebrate skeletal muscles (177, 260).

However, this is not the case in most vertebrate skeletal

muscle, where extracellular Ca 2

is not required for E-Ccoupling (9, 38, 91, 129, 177, 260). The primary role of the

DHPR in vertebrate skeletal muscles is acting as a voltage

sensor that directly (perhaps physically) modulates the

activation gate of nearby RyR1 channels.

A. DHPR-RyR Interaction

In skeletal muscle (Fig. 1), the DHPR and RyR1 are

thought to communicate via some sort of physical pro-

tein-protein linkage (39, 246, 247, 251, 253, 265). The

membranes of the t tubule and SR are juxtaposed and

separated by a small 10-nm gap. The cytosolic domain of the RyR1 channel spans this narrow gap (28, 29, 94 –97).

Electron microscopy (EM) studies show that the skeletal

DHPR in the t tubules are arranged in clusters of four

(tetrads). These tetrads are organized into distinct arrays.

The RyR1 channels in the SR membrane are arranged in a

corresponding fashion (28, 29, 94 –97, 247). The arrays of

DHPR and RyR align in certain fast-twitch skeletal muscle

such that every other RyR1 channel is associated with a

DHPR tetrad (28, 29, 247).





It is thought that the skeletal DHPR physically con-

tacts the large cytoplasmic domain of the juxtaposed

RyR1 channel to form a linkage through which t-tubule

voltage-sensing information is transduced (246, 251–255,

265–267). A distinctive feature of this transduction mech-

anism is its speed. Signal transmission between the skel-

etal DHPR and RyR1 channel must occur during the very

brief (2 ms) skeletal muscle action potential. It may be

FIG. 1. Environment of the ryanodinereceptor channel in skeletal muscle. Thet tubule contains tetrads of dihydropyri-dine receptors (DHPRs). Every other RyR1 channel is associated with a DHPRtetrad. A t-tubule membrane voltagechange (V

M) activates the DHPR. Cal-

cium entry thorough the DHPR is not

important to RyR1 channel activation inskeletal muscle. Instead, the t-tubule V Msignal is thought to be transmitted to theRyR1 channel by a direct physical DHPR-RyR1 linkage. A specific cytosolic loop of the DHPR is thought to be involved inthis physical communication. The t-tu-bule V

M signal triggers the RyR1 chan-

nel to open and release the calciumstored inside the sarcoplasmic reticulum(SR). Calsequestrin (CSQ), a low-af finity

high-capacity calcium buffer, is found in-side the SR lumen near the RyR1 chan-

nels. The SR Ca

2

-ATPase (SERCA) usesthe energy stored in the ATP to pumpcalcium back into the SR.

FIG. 2. Environment of the RyR2channel in cardiac muscle. The t tubulecontains DHPRs and calcium extrusionmechanisms like the Na /Ca 2 ex-changer (Na-CaX). A t-tubule membrane

voltage change activates the DHPR, andthe resulting calcium entry triggers un-

derlying RyR2 channels to open. Theopen RyR2 channel releases the calcium,

stored inside the SR, into the cytoplasm.CSQ, a low-af finity high-capacity calciumbuffer, buffers calcium near the RyR2channels inside the SR. The SERCA usesthe energy stored in the ATP to pumpcalcium back into the SR. The Na-CaXuses the energy stored in the sodium gra-dient to move calcium out of the cell.





this “need for speed” that has converted the physiological

role of the DHPR from Ca 2 channel to voltage sensor in

mammalian skeletal muscle.

In cardiac muscle (Fig. 2), there is about 1 DHPR for

every 5–10 RyR2 channels (29, 247, 313), and the DHPR

and RyR2 channels are not aligned in such a highly or-

dered fashion (29, 95, 247, 313). During the long cardiacaction potential (100 ms), the DHPR Ca 2 channel has

ample time to open and mediate a substantial Ca 2 influx.

This Ca 2 influx is ultimately the signal that activates the

underlying RyR2 channels via the CICR process (18, 40,

272). The involvement of a diffusible second messenger

(i.e., Ca 2) makes the DHPR-RyR2 signaling considerably

slower than the DHPR-RYR1 signaling. This lack of speed,

however, may provide a greater opportunity for regulating

DHPR-RyR2 signaling. In fact, pharmacological regulation

of DHPR-RyR2 signaling is fundamental to normal cardiac

function (18).

The expression of mutant DHPRs in mouse skeletalmuscle myotubes that lack the endogenous DHPR has

provided insights into which regions of the DHPR are

involved in DHPR-RyR1 interaction (1, 2, 106, 321). A

region within the cytosolic II-III loop of the DHPR’s -sub-

unit (residues 720 –765) appears to be critical. Further-

more, studies with myotubes lacking endogenous RyR1

channels showed that RyR2 or RyR3 could not substitute

for RyR1 in the DHPR-RyR interaction (216, 320, 356). The

expression of chimeric RyR channels (RyR1/RyR2) indi-

cates that regions R9 (2659 –3720) and R10 (1635–2636) of

RyR1 contain sequences involved in the DHPR-RyR1 in-

teraction (217, 246). Thus specific regions of the DHPRand RyR1 channels have been implicated in the DHPR-

RyR1 interaction. How these regions mediate the required

signal transduction remains to be determined.

The DHPR is not the only regulator of RyR1 channel

function. Like the RyR2 channel, the RyR1 channel (in the

absence of the DHPR) can be activated by cytosolic Ca 2

signals. This is important because more than half of the

RyR1 channels are not coupled to DHPRs (7, 183, 197,

200). These “uncoupled” RyR1 channels are thought to be

available to participate in the CICR process. It is generally

believed that CICR acts to amplify the DICR signal (i.e.,

that generated by the DHPR-RyR1 interaction). Interest-ingly, the presence of “coupled” and uncoupled RyR1

channels implies that RyR1 function is inherently hetero-

geneous “in vivo.” The amount of uncoupled RyR1 chan-

nels is not the same in all skeletal muscles. Slow-twitch

skeletal muscles may have three or more uncoupled RyR1

channels for each DHPR-linked RyR1 channel (7, 183, 197,

200). This may mean that these muscles have a greater

CICR component. This may be related to the substantially

slower rate of E-C coupling in slow-twitch muscle (104,

223, 227, 260, 370).

V. STUDY OF INTRACELLULAR

CALCIUM RELEASE

The first methods used to study Ca 2 transport

across cell membranes monitored radioisotope (45Ca) in-

flux/ef flux between two defined compartments (e.g., bath

and cytosol; Ref. 141). From these initial studies, it be-came clear that Ca 2 was sequestered into intracellular

organelles and that the cytosol represented an intermedi-

ary compartment with variable Ca 2 buffer properties.

The next generation of studies examined Ca 2 influx/

ef flux across the SR of mechanically or chemically

skinned muscle fibers. Here, the barrier of the plasma

membrane was eliminated and the cytosolic solution

could be controlled. The importance of ATP-mediated

Ca 2 uptake by the SR and some of the attributes of CICR

and even DICR were defined (80, 81, 150, 153, 151). Al-

most concurrently, methods for generating subcellular

fractionation of functional SR vesicles were developing.

Studies of Ca 2 loading and release from suspensions of

SR vesicles provided improved temporal resolution and

further revealed the intricacies of the SR Ca 2 release

process (121, 127, 138, 196, 198, 199, 234–236).

It was noticed by several investigators that the bind-

ing of the ryanodine alkaloid appeared to depend on the

activity level of the SR Ca 2 release channel (e.g., Refs.

53, 122). Experimental conditions that promoted SR Ca 2

release increased ryanodine binding. Experimental condi-

tions that inhibited SR Ca 2 release decreased ryanodine

binding. Subsequently, “nonequilibrium” ryanodine bind-

ing became a commonly used tool to assess RyR channel

activity (discussed in detail in Ref. 225). Thus a number of methods have been developed to study the SR Ca 2 re-

lease phenomenon in populations of RyR channels.

The development of the patch-clamp technique rev-

olutionized the study of ion channels, including the study

of the RyR channel. In this classic method, a small glass

pipette is placed on the surface of a cell to physically

isolate a small patch of membrane in which the function

of an individual ion channel can be measured. This method

has been widely applied to define the function of surface

membrane ion channels. The patch-clamp technique,

however, cannot be easily applied to study ion channels in

intracellular organelles. Instead, microsomes isolated

from cellular homogenates are fused into artificial planar lipid bilayers (206). The same instrumentation used to

record across the membrane patch is then used to record

across the artificial bilayer. Thus studies of single RyR

channel behavior became possible (detailed in sect. VI).

The recent development of laser scanning confocal

microscopy opened yet another chapter in the study of

intracellular Ca 2 release. Confocal imaging has made it

possible to visualize very localized intracellular Ca 2 re-

lease events (Ca 2 sparks; Refs. 50, 336). These local

events may arise from the opening of an individual chan-





nel or more likely the concerted opening of a small num-

ber of RyR channels.

VI. STUDY OF SINGLE RYANODINE

RECEPTOR CHANNELS

Defining the characteristics of single RyR channels is

fundamental to understand the origin of many intracellu-

lar Ca 2 signaling phenomena. The amplitude of single

RyR channel current reveals the number of Ca 2 passing

through the channel per second. The open probability

( P o) of single RyR channels allows us to predict how

certain agonists and antagonists regulate its activity.

Dwell-time analysis of single RyR channel activity reveals

the durations of the open and closed events. Kinetic stud-

ies of single RyR channel activity reveal how fast it can

respond to a ligand stimulus (i.e., time constants and first

latencies) as well as its specific patterns of opening (i.e.,

modal gating or bursting behavior). Additionally, single

RyR channel studies can reveal the extent of homogeneity

or heterogeneity of individual channel function in the

overall RyR channel population (56, 60, 178, 238). These

types of information are generally not available when

Ca 2 signaling phenomena are studied in a population of

RyR channels. Thus single RyR channel studies provide

detailed information about the origin of intracellular Ca 2

signaling phenomena that simply cannot be obtained by

other means.

The study of single RyR channel behavior requires

the incorporation of native SR vesicles (enriched in RyR

channel) or purified RyR channel proteins into an artifi-cial planar lipid bilayer. The artificial planar bilayer is

generally formed across a small aperture that separates

two solutions. A patch-clamp amplifier is used to monitor

electrical signals that arise from the bilayer. Single RyR

channel activity is evident as a sudden appearance of

single-channel activity in the bilayer. The sudden appear-

ance of channel activity marks the moment a channel

protein has been incorporated into the bilayer. This meth-

odology is now widely used and has been described in

detail elsewhere (206).

The environment in which the single RyR channel

operates in the cell is complex (163). The resting cytosolic

free Ca 2 concentration is near 100 nM, and the free Ca 2

concentration inside the SR is thought to be near 1 mM

(196, 274). The cytosol contains a complex mixture of

salts (140 mM K , 1 mM Mg2, 5–10 mM ATP, and

various other nucleotides; Refs. 234, 371). The cell also

contains several proteins that are known to interact with

the RyR channel (e.g., DHPR, calmodulin, FK binding

proteins, triadin, kinases-phosphatases, calsequestrin, an-

nexin, etc.). Reconstructing or mimicking, at least in part,

this complex situation in vitro (i.e., during a planar bilayer

experiment) still needs to be done. Nearly all the single

RyR channel studies have been done under very simple

experimental conditions that do not reflect the channel’s physiological reality (e.g., Refs. 54, 56, 116, 145, 147, 291).

This fact should always be taken into consideration when

interpreting single RyR channel data.

How well does single RyR channel function in thebilayer reproduce the single RyR channel behavior in the

cell? The correlation between single RyR channel func-

tion in bilayers and SR Ca 2 release phenomena observed

in cells is surprisingly strong. For example, the primary

regulatory features of SR Ca 2 release in cells (e.g., its

Ca 2, Mg2, and ATP sensitivity) are clearly evident at the

single RyR channel level in bilayers. The actions of many

secondary pharmacological regulatory agents like caf-

feine, ruthenium red, and ryanodine in the cell are mim-

icked at the single RyR channel level in bilayers. The fast

gating and high conductance of single RyR channels in

bilayers is consistent with the speed and large amplitude

of intracellular Ca 2 release phenomena in cells (173).

There is growing evidence that many regulatory factors

and closely bound regulatory proteins are actually re-

tained when single RyR channels are isolated in native

membranes (e.g., Refs. 30, 189, 287, 300, 346, 348, 365).

Thus defining single RyR channel behavior in bilayers is

likely to provide insights into the origins of RyR-mediated

Ca 2 signaling in cells.

The RyR channel is usually isolated by differential

centrifugation of muscle homogenates. The RyR channel

is found in heavy microsomal fractions. The RyR-contain-

ing vesicles (microsomes) can then be fused into planar

lipid bilayers (36, 54, 56, 258, 291, 298). Alternatively, theRyR-containing vesicles can be detergent-solubilized

(CHAPS plus phospholipids mixtures) and the resulting

“ purified” RyR channels can then be directly fused in a

lipid bilayer (124, 135, 238) or reconstituted into lipo-

somes (145, 147). These proteoliposomes can then fuse

into planar lipid bilayers. Single RyR channel function has

been examined using all these approaches. An advantage

of using the SR vesicle approach is that the RyR channel

is present in its native membrane, increasing the likeli-

hood that closely associated regulatory proteins/factors

may still be present (e.g., Refs. 189, 327). A disadvantage

of using the SR vesicle approach is that the vesicles may

contain other types of ion channels, specifically K and/or Cl channels. The presence of other channels compli-

cates things considerably. Potential interference from

other channels is minimal if Ca 2 (or Ba 2) is used as the

charge carrier (with no other permeant ions present).

Many single RyR channel studies, however, chose to use

a monovalent charge carrier to boost signal-to-noise char-

acteristics. In this case, carefully selected ionic substitu-

tions are usually made to eliminate potential contamina-

tion from other channel types.





In our experience, fusion of SR vesicles into planar

bilayers results in more stable/consistent single RyR

channel behavior compared with that obtained using

CHAPS-solubilized RyR channel preparations. As men-

tioned above, the use of SR vesicles requires that certain

specific steps be taken to avoid contaminating currents

from other ion channels. The first unitary Ca 2

currentrecordings associated with the RyR channel were made

after heavy SR vesicles were fused in a bilayer in the

presence of 50 mM Ca 2 and little else (291). The two

most common single RyR channel recording conditions

are listed below. One consists of a Ca-HEPES solution (50

mM Ca 2) on the luminal side with an isosmotic Tris-

HEPES solution on the cytosolic side of the channel. The

second consists of a Cs-methanesulfonate solution on

both sides of the channel. In both cases, contaminating

anion currents are avoided by the use of impermeable or

poorly permeable anions. In the first case, contaminating

cation currents are avoided by the presence of a nonper-

meable organic monovalent cation (Tris). In the second

case, contaminating K currents are avoided by using Cs

as a permeant monovalent cation. The use of Cs works

because SR K channels are either poorly permeable to

Cs or blocked by this ion, while RyR channels are highly

permeable to Cs (as well as other monovalent cations).

VII. PERMEATION PROPERTIES

The ability of the RyR channel to mobilize intracel-

lular Ca 2 depends on both its permeation and gating

properties. Its permeation properties include its unitaryconductance and ion selectivity. Its gating properties in-

clude its P o and the duration of individual open/closed

events. A single RyR channel with optimal permeation

properties would be ineffective at mobilizing Ca 2 if it

never opened (gated). Conversely, a single RyR channel

with optimal gating behavior would be worthless if it did

not allow Ca 2 to pass ef ficiently. The opening and clos-

ing of the RyR channel presumably involves the physical

motion of the protein. Ions are able to traverse its pore

only when the channel is in the open configuration. It is

generally assumed that movement of the gate does not

alter the nature of the pore (i.e., its diameter, length,

charge, etc.). It is in the pore where the channel discrim-ination between different ions occurs. The pore also con-

tains determinants that define how quickly particular ions

will pass. Specific RyR channel permeation properties are

described below. Specific features of single RyR channel

gating are discussed in later sections.

The permeation properties of the different RyR chan-

nel isoforms are quite similar. The RyR channels are

permeable to many different divalent and monovalent

cations (e.g., Refs. 167, 294, 330). In symmetrical solutions

containing a monovalent cation (e.g., 200 mM K , Na ,

or Cs) as the main permeant species, the unitary RyR

channel current-voltage relationship is linear with a slope

conductance usually 500 pS (167, 294). In asymmetric

solutions containing a divalent cation (e.g., 50 mM Ca 2,

Ba 2, or Sr 2) as the main permeant species, unitary RyR

channel current-voltage relationships have a slope con-ductance of 100 pS (330). These are very large conduc-

tance values. For comparison, high-conductance ion

channels of the plasma membrane such as the Ca 2-

activated K channels (i.e., maxi-K Ca channels) have a

maximal conductance of near 250 pS (157). The unitary

Ca 2 conductance of the L-type Ca 2 channel (i.e., DHPR)

in the surface membrane is no more than 20 pS (119).

Thus the RyR channels are very-large-conductance Ca 2

channels. This feature is probably fundamental to its

physiological role in cells, passing a large number of Ca 2

quickly.

The relative permeability of the RyR channels to

different monovalent and divalent cations has been de-

fined under bi-ionic conditions (e.g., Refs. 167, 294, 330).

The RyR channels show very little selectivity between

different monovalent cations. The RyR channels also

show very little selectivity between different divalent cat-

ions. However, the RyR channels are selective, albeit

poorly, between mono- and divalent cations ( P Ca / P K 6;

more permeable to Ca 2 than K ). This modest selectivity

for divalents is also unusual for a Ca 2 channel. The

typical surface membrane Ca 2 channel (e.g., DHPR) dis-

plays a much higher degree of discrimination between

monovalent and divalent cations ( P Ca / P K 20). The point

is that the RyR channel is not a highly selective Ca 2

channel. Intuitively, the apparent deficiency in Ca 2 se-

lectivity may be related to its high conductance. A high-

conductance channel (i.e., ions passing rapidly) would

have little time per ion to perform the steps needed to

discriminate between ions.

The relatively poor Ca 2 selectivity of the RyR chan-

nels does have important physiological ramifications. In

cells, Ca 2 is not the only ion that can permeate through

the open RyR channel pore. Other permeable cations

(Mg2, Na , and K ) are also present at substantial levels

(i.e., 1, 10, and 140 mM, respectively). Thus Ca 2 com-

petes with Mg2, Na , and K for occupancy of the RyR

channel pore. The consequence is that the unitary Ca 2

current carried by a single RyR channel in the cell (i.e., in

the presence of multiple permeant ions) will be consider-

ably less than measured under the traditional simple re-

cording conditions used in bilayer studies (e.g., Refs. 256,

259, 295, 330).

Tinker et al. (329) were the first to examine how

unitary Ca 2 current through the RyR2 channel is affected

by the presence of other permeant ions. They estimated

that unitary Ca 2 current (at 0 mV) under physiological





conditions would be 1.4 pA. This still quite large current

was explained using a Eyring-rate model (328) assuming

that some mechanism allows the association rate of Ca 2

at the mouth of the pore to exceed, by about an order of

magnitude, the normal diffusional limit. A more recent

examination of unitary Ca 2 current through the single

RyR2 channel suggests that unitary Ca 2

current (at 0mV) under physiological conditions may be closer to 0.5

pA (201). This is important because accurately defining

the amplitude of the unitary Ca 2 current through a single

RyR channel is fundamental to understanding the origin

of local intracellular Ca 2 signaling events (e.g., Ca 2

sparks) in cells. The unitary RyR Ca 2 current value is

central to the debate whether Ca 2 sparks arise from the

opening of one RyR channel or the concerted opening of

many RyR channels.

The larger unitary RyR channel Ca 2 current predic-

tion suggests that a mechanism to enhance permeation

may possibly exist (328). The smaller unitary Ca 2 cur-

rent prediction indicates that perhaps there is no need for

any permeation enhancement mechanism (201). What

might the permeation enhancement mechanism be? Sev-

eral possible permeability enhancement mechanisms

have been proposed. It has been argued that the RyR2

channel may contain a large charged vestibule that dielec-

trically focuses ions near the pore’s mouth (328). Alter-

natively, dielectric focusing by fixed surface charges

could occur near the RyR channel’s mouth in the absence

of a large vestibule structure (337). It has also been sug-

gested that a single RyR channel contains multiple paral-

lel conduction pathways (i.e., a multibarrel pore). The

later possibility was supported by the initial 3-D RyRchannel reconstructions that suggested the presence of

multiple conduction pathways (342). Furthermore, On-

drias et al. (228) suggested that the four commonly ob-

served subconductance states of the RyR channel might

correspond to uncoordinated opening of pores in the

individual RyR monomers of the tetrameric channel com-

plex. They suggested that the presence of the FK-binding

protein synchronizes the openings of these separate per-

meation pathways. However, recent and more detailed

3-D reconstruction data suggest that the RyR channel

structure contains a single central pore (i.e., the RyR is

not a multibarrel channel; Refs. 230, 270, 275). However,

the possibility that the channel contains a large vestibuleand/or surface charges that impact ion permeation still

exists and should not be discounted.

VIII. ACTION OF RYANODINE

The alkaloid ryanodine binds with high af finity to the

RyR proteins (225). Its binding induces a complex change

in single RyR channel function. This change is similar in

all three RyR channel isoforms. Ryanodine binding in-

duces very-long-duration open events and simultaneously

reduces ion conductance through the pore (e.g., Refs. 259,

294). This dual impact on single-channel function (i.e.,

slower gating, reduced conductance) suggests a compli-

cated mode of action. This is also evidenced by the rather

complicated ryanodine dose dependency. Low doses of

ryanodine (10 nM) are reported to increase the fre-quency of single RyR channel openings to the normal

conductance level (34). Intermediate ryanodine doses

(1 M) are reported to induce the classical ryanodine

action described above (34, 259, 294). High doses of ry-

anodine (100 M) are reported to lock the channel in a

closed configuration (368).

The fact that ryanodine binding alters single-channel

conductance suggests that ryanodine binds in or near the

RyR channel pore. In fact, the ryanodine-binding site has

been experimentally localized to the COOH terminus of

the protein (37), the region of the RyR protein thought to

contain the structural determinants of the pore. Altered

conductance suggests that ryanodine binding somehow

changes the nature of the permeation pathway. This idea

has also been experimentally confirmed. Two indepen-

dent studies have indicated that ryanodine binding actu-

ally changes the effective diameter of the RyR channel

pore (332, 337).

The complex action of ryanodine may be related to

how the alkaloid binds to the channel. It is generally

assumed that each RyR monomer contains one high-af fin-

ity ryanodine binding site (K D 50 nM; Refs. 147, 225). It

is possible that cooperative interaction between these

different ryanodine binding sites is what generates the

observed complexity (53, 242). Some ryanodine bindingstudies suggest the existence of additional lower (K D 1

M) af finity ryanodine binding sites (53, 147, 225, 242).

Thus the RyR channel complex may contain multiple

ryanodine binding sites. One interpretation suggests that

ryanodine binding to the high-af finity site depends on

whether or not the RyR channel is in the open con figura-

tion (53). It may be that conformational changes related

to the opening of the RyR channel exposes (or improves

access to) the high-af finity ryanodine binding site. This

would impart a use dependence to the kinetics of ryano-

dine binding, allowing “nonequilibrium” ryanodine bind-

ing to be used as a monitor of RyR channel function (as

described previously).

IX. OVERVIEW OF RYANODINE

RECEPTOR REGULATION

The RyR channels are modulated by numerous fac-

tors, including a number of physiological agents (e.g.,

Ca 2, ATP, and Mg2), various cellular processes (e.g.,

phosphorylation, oxidation, etc.), and several pharmaco-

logical agents (e.g., ryanodine, caffeine, and ruthenium





red). Some of these modulatory factors are discussed

below.

A. Cytosolic Ca2

The action of Ca

2

on the RyR channel is complex.The Ca 2 turns on, turns off, and conducts through this

channel. Steady-state single RyR channel activity is gen-

erally a bell-shaped function of cytosolic Ca 2 concentra-

tion (35, 46, 54, 56, 60, 135, 161, 224, 238, 285, 294, 333).

The RyR channels are activated by low Ca 2 concentra-

tions (1–10 M) and inhibited by high Ca 2 concentra-

tions (1–10 mM). It has been reported that individual

RyR1 channels do not all respond in the same way (i.e.,

they are functionally heterogeneous) to activating cytoso-

lic Ca 2 levels (56, 161, 178, 238). In contrast, individual

RyR2 and RyR3 channels respond much more homoge-

neously to cytosolic Ca 2 (46, 54, 56, 135, 161). Inhibition

at high cytosolic Ca 2

concentrations of the RyR1 is alsodifferent from that of RyR2 and RyR3 channels. The RyR1

channel is almost entirely inhibited by 1 mM Ca 2,

whereas the RyR2 and RyR3 channels are not (35, 46, 54,

56, 60, 135, 161, 224, 225, 238, 264). Substantially higher

Ca 2 levels are required to inhibit the RyR2 and RyR3

channels (46, 54, 56, 135, 161). The reported half-maximal

inhibiting concentrations for the RyR2 and RyR3 channels

range from 2 to 10 mM. It is not clear that such high

cytoplasmic Ca 2 concentrations are ever reached in the

cells. Thus the physiological role of this very high Ca 2

inhibition is not yet clear. Nevertheless, high Ca 2 inhibi-

tion of the RyR2 channels remains a controversial topic.

This is further discussed in section XV .

B. Luminal Ca2

Early Ca 2 release studies suggested that Ca 2 bind-

ing to the luminal surface of the RyR channel may regu-

late the channel (64, 65). Single RyR channel studies have

confirmed the existence of luminal Ca 2 regulation (51,

110, 286, 285, 333, 341). It appears that the sensitivity of

RyR channel to certain cytosolic agonists increases at

high luminal Ca 2 levels (110, 286). Trypsin digestion of

the luminal side of the RyR channel suggested the pres-ence of both activating and inactivating divalent sites

(51). It is possible that the luminal Ca 2 effects are me-

diated, at least in part, by associated proteins like calse-

questrin (14, 314) or junctin (364).

It was also reported that luminal Ca 2 moving

through the channel feeds back and interacts with cyto-

solic Ca 2 binding sites (333). Thus luminal Ca 2 levels

may affect single RyR channel function by interacting

with luminal binding sites, with associated luminal pro-

teins, or with cytosolic binding sites (i.e., feed-through

regulation). Distinguishing between these possibilities is

the challenge.

C. ATP and Mg2

Cytosolic ATP is an effective RyR channel activator (57, 60, 291, 297, 298, 353). Cytosolic Mg2 is a potent RyR

channel inhibitor (57, 60, 158, 291, 353). The cytosol of

most cells contains 5 mM total ATP and 1 mM free

Mg2. This means that most ATP is in its Mg2-bound

form. Free ATP (300 M in the cytosol) is the species

that binds to and activates the RyR channel (297, 298).

The action of ATP and Mg2 on single RyR channel func-

tion is isoform specific. Free ATP is a much more effec-

tive activator of the RyR1 channel than the RyR2 channel

(57, 60, 291, 297, 353). The RyR3 channel is also less ATP

sensitive than the RyR1 channel (46, 135, 297). The Mg2

action on single RyR channels is complicated (57, 158).First, Mg2may compete with Ca 2 at the Ca 2 activation

site, shifting the Ca 2 sensitivity of the channel (57, 60,

158, 353). Second, Mg2 competes with Ca 2 at the high

Ca 2 inhibition site (57, 158). The high Ca 2 inhibition site

does not discriminate between different divalent cations.

The RyR1 channel is substantially more sensitive to this

action of Mg2 than the RyR2 or RyR3 channels (57, 135).

In the presence of physiological levels of Mg2 and ATP,

the RyR1 channel requires less Ca 2 to activate than the

RyR2 or RyR3 channel (57, 257, 320, 353).

D. Redox Status

Various redox processes may also modulate single

RyR channels. The RyR monomers contain 80 –100 cys-

teine residues (315a, 318). Many of these are suitable for

modification by oxidants. There is evidence that oxidant

and reducing agents do indeed impact single RyR channel

function (182, 222, 349), including the channel’s Mg2

sensitivity. There is also evidence that oxidants may pre-

vent certain closely associated regulatory molecules, like

calmodulin, to bind to the RyR channel (363). Recently, it

has been suggested that nitric oxide (NO) may modulate

the redox regulation of the RyR channels (79, 118, 352). It

was proposed that in vivo initial NO release may activate

RyR channels and subsequent increases in NO levels in-

hibit RyR channel activity (118). Thus there is a growing

interest in identifying the cysteine residues that may be

involved in RyR channel oxidation and/or S -nitrosylation

(352). It is quite possible that redox modulation of single

RyR channels represents an important regulatory mecha-

nism. It is clear that additional information is needed in

this area.





E. Cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) appears to play an im-

portant role in many nonmuscle systems (164). The

cADPR molecule, a metabolite of NADP, has been found

to act as a powerful intracellular Ca 2 release agent in sea

urchin eggs, and its action is thought to be mediated byRyR or RyR-like channels (164). The direct action of

cADPR on RyR channels from muscle systems is contro-

versial. An early report indicated that cADPR dramatically

activates single mammalian RyR channels (203). How-

ever, subsequent studies reported only minor effects of

cADPR (284) or no impact of cADPR at all (58, 100, 209).

The controversial reports may suggest a requirement of

an unrecognized cofactor and/or a potential interaction

with some closely associated regulatory factor (i.e., cal-

modulin, FK-506 binding protein; Refs. 58, 164). When

studied in intact cellular systems (that contain endoge-

nous calmodulin and FK-binding proteins), cADPR had

only indirect action on RyR channel function (107, 126,175). It appeared that cADPR activated the Ca 2-ATPase

(175), which indirectly activated the RyR channel by in-

creasing luminal SR Ca 2 levels.

F. Phosphorylation/dephosphorylation

Analysis of the RyR protein sequence reveals that it

contains many consensus phosphorylation sites (315a,

318). For more than a decade, the effects of exogenously

applied kinases and phosphatases on single RyR channel

behavior have been examined (60, 114, 170, 189, 298, 315,

339, 348). The capacity of protein kinase A (PKA) toactivate the RyR1 channel has been reported (60, 298).

This is consistent with the observation that PKA signifi-

cantly increases depolarization-induced Ca 2 release

from skeletal muscle SR vesicles (125). However, studies

in skinned and intact skeletal muscle fibers have not

supported this idea (27). The capacity of calmodulin

kinase and PKA to activate the RyR2 channel has also

been studied. Contradictory results have been reported

for calmodulin kinase (70, 114, 170). Similarly, some re-

ports on PKA action find activation of RyR2 channels

(114), whereas others report a slight decrease in the

steady-state P o of RyR2 channels (339). Still others sug-

gest PKA destabilizes RyR2 channel openings (i.e., pro-mote substating) via phosphorylation-induced dissocia-

tion of FKBP12.6 (189). Yet another report shows that

PKA phosphorylation does not impact the RyR-mediated

Ca 2 spark in intact cardiac myocytes (166). The reason

for the appearance of such contradictory and confusing

results is unclear. It is possible that different experimen-

tal conditions are responsible. It is safe to conclude that

the impact of phosphorylation and/or dephosphorylation

on single RyR channel behavior will be debated for some

time.

X. REGULATION BY CLOSELY

ASSOCIATED PROTEINS

A number of different proteins may be associated

with and modulate the RyR channels (60, 63, 136, 142, 144,

169, 181, 183, 185, 196, 231, 257, 287, 334, 343). However,

only a few of these proteins have been thoroughly studiedat the single-channel level. These few “well-studied” pro-

teins include calmodulin, calsequestrin, FK-506 binding

protein, and certain fragments of the DHPR. A brief sur-

vey of how these proteins may interact with the RyR

channel is provided below.

A. Calmodulin

Calmodulin (CaM) was the first peptide found to

interact with single RyR channels in lipid bilayers (295).

CaM appears to bind the RyR in stoichiometric propor-

tions (11, 207, 334). It binds to sites in the RyR channelcytoplasmic assembly that are 10 nm from the putative

entrance to the transmembrane pore (343). Application of

CaM can either activate (at low Ca 2 levels) or inhibit (at

high Ca 2 levels) the RyR1 and RyR3 channels (46, 99,

295, 334). For the RyR2 channel, only inhibitory effects of

CaM have been reported (99, 295). It was reported that

oxidation modifies RyR1 channel behavior and perhaps

its interaction with CaM (117, 363). It has also been

suggested that CaM may somehow protect the RyR1 chan-

nel from oxidative modifications during periods of oxida-

tive stress (363). CaM also appears to bind to the DHPR

and consequently has also been implicated in the DHPR-

RyR1 interaction (117, 269). A potential role of CaM inmodulation of the RyR2 channel during E-C coupling is

not yet clear.

B. Calsequestrin

Calsequestrin is the main Ca 2 binding protein inside

the SR. Calsequestrin has a large number of acidic amino

acids that permit it to coordinately bind 40–50 Ca 2 /

molecule (181, 358). Calsequestrin is also known to ag-

gregate in the presence of divalent cations (190). Electron

microscopy studies suggest that calsequestrin aggregates

are present in the regions of the SR (i.e., terminal cister-nae) known to contain RyR channels (96). It has been

suggested that Ca 2- and pH-dependent conformational

changes in calsequestrin may modulate RyR channel ac-

tivity (61, 120). It also has been suggested that calseques-

trin action on the RyR channel may require the presence

of triadin, another RyR-associated protein (365). Thus

there is disagreement concerning the nature of the calse-

questrin-RyR interaction.

Some studies in lipid bilayers suggest that calseques-

trin activates the RyR channel (139, 226, 314). However,







described previously, the DHPR-RyR interaction is clearly

involved in the signaling that links surface membrane

excitation to SR Ca 2 release in striated muscle. The

nature of the DHPR-RyR interaction is tissue specific. In

skeletal muscle, the interaction is thought to involve a

direct physical link between the two proteins. In cardiac

muscle, the DHPR Ca 2

channel mediates a small Ca 2

influx that activates the RyR2 channel. Our attention here

will be focused on the physical DHPR-RyR1 link in skel-

etal muscle.

Tanabe et al. (321) showed that the cytoplasmic II-III

loop of the skeletal DHPR 1-subunit is required for the

functional DHPR-RyR1 interaction. Although other re-

gions of the DHPR may be involved (62, 165, 289), the

potential role of the II-III loop has been studied most to

date. Peptide fragments that correspond to the II-III loop

activate single RyR1 channels in planar lipid bilayers (171,

172). Interestingly, different regions of the peptide appear

to have different actions on RyR1 channel function. One

region called peptide A activates single RyR1 channel (71,

171, 172, 262). Another region called peptide C blocks the

activating action of peptide A (71, 171, 262). Additional

insights have been gained from examining the action of

the scorpion peptide toxin imperatoxin A, whose struc-

ture has similarities to that of peptide A (108). Like pep-

tide A, imperatoxin A activates single RyR1 channel in

planar bilayers (108). The toxin binds with high af finity to

the RyR1 channel but at a site that appears to be quite

distant from the RyR1 regions thought to interact with the

DHPR (263). If peptide A and imperatoxin A bind to the

same site, then the distant location of this site is dif ficult

to reconcile with our current understanding of the DHPR-RyR1 interaction. Recently, the interpretation of the phys-

iological role of peptide A on RyR1 channel function was

further complicated. Grabner et al. (106) showed that a

chimeric DHPR, lacking the peptide A region, supported

normal DHPR-RyR1 signaling when expressed in dysgenic

myotubes (i.e., myotubes lacking endogenous DHPR).

Thus the role of peptide A in the DHPR-RyR1 interaction

has yet to be clearly established.

XI. CALCIUM REGULATION PARADOX

Although the RyR channels are modulated by manyfactors, Ca 2 regulation plays a central role in all RyR-

mediated signaling cascades. In some cases (i.e., cardiac

muscle), the regulating Ca 2 signal initiates RyR channel

activity. In other cases (i.e., skeletal muscle), the regulat-

ing Ca 2 signal may amplify RyR channel activity. There is

also evidence that regulating Ca 2 signals may terminate

RyR channel activity. The importance of understanding

how Ca 2 regulates the RyR channels cannot be overstated.

Our discussion of RyR2 channel Ca 2 regulation

starts with the classical work of Fabiato (80, 81), which

defined the Ca 2 regulation of the SR Ca 2 release pro-

cess in a skinned cardiac muscle preparation. This work

showed that the amplitude and duration of trigger Ca 2

stimulus finely grades the CICR process (i.e., Ca 2-in-

duced Ca 2 release). Specifically, the amount of Ca 2

released from the SR was a bell-shaped function of trigger

Ca 2

stimulus amplitude, and that trigger Ca 2

stimulusspeed scaled this relationship.

Intuitively, the CICR process should be self-regener-

ating because the Ca 2 released by a RyR2 channel should

feedback and further activate the channel or its neigh-

bors. However, self-regeneration of CICR is not observed

in cells. This is the Ca 2 regulation paradox. The impli-

cation is that some sort of negative-feedback mecha-

nism(s) must exist to counter the inherent positive feed-

back of CICR. Fabiato (81) proposed that the negative-

feedback mechanism was Ca 2-dependent inactivation.

The idea was that two different Ca 2 binding sites (acti-

vating and inhibitory, respectively) regulate the Ca 2 re-

lease channel (i.e., RyR2). The Ca 2 activation process

was thought to have a fast action and a relatively low

af finity. The Ca 2 inactivation process had a slower ac-

tion but a relatively high Ca 2 af finity (81). Consequently,

a fast Ca 2 stimulus transiently activates the channel

because the channel would be “turned on” as Ca 2 occu-

pies the activation site and then “turned off ” as Ca 2more

slowly acts at the inactivation site. A slow Ca 2 stimulus

would not effectively activate the channel because inac-

tivation would “keep pace” with the activation process. At

very high Ca 2 levels (100 M), the inactivation sites

would be saturated, leaving the channel in an inactivated

state. The mechanism of Ca 2

-dependent inactivation asenvisioned by Fabiato (81) implies that the channel be-

comes refractory (unable to respond to further Ca 2 stim-

ulation). Recovery from the refractory state would require

removal of the Ca 2 stimulus and enough time for some

sort of recovery process (i.e., repriming) to occur. This

scheme is still widely discussed and is often applied to

explain the complex Ca 2 regulation of many RyR-medi-

ated Ca 2 signaling events.

Despite its popularity, the existence of high-af finity

Ca 2-dependent inactivation has been challenged. Studies

on patch-clamped cardiac myocytes have not conclu-

sively confirmed the existence of the Ca 2-dependent

inactivation phenomenon (208, 214, 360). For example,the capacity of a second Ca 2 stimulus to trigger SR Ca 2

release was shown to be independent of the interval

between the first and second stimuli (214). The argument

here is that if the SR Ca 2 release channels were inacti-

vated after the first stimulus, then the effectiveness of the

second stimulus should be affected. In another study

(360), a prolonged triggering Ca 2 signal was used to

inactivate the Ca 2 release process. A subsequent trigger

Ca 2 signal, however, reactivated the apparently inacti-

vated release process (i.e., the process was not refractory).





Still, it is possible that the second Ca 2 stimulus (in both

cases above) was activating neighboring Ca 2 release

sites that did not participate in the first Ca 2 stimulus. In

any event, studies at the whole cell level have not con-

clusively confirmed or dismissed the existence of Ca 2-

dependent inactivation. Studies of RyR2 channel Ca 2

regulation at the single-channel level promised to resolvethis question.

XII. STEADY-STATE CALCIUM DEPENDENCE

As discussed above, several laboratories have de-

fined the Ca 2 dependence of single RyR1 and RyR2

channels under steady-state conditions (e.g., Refs. 54, 56,

161, 256). In the absence of other modulating agents,

these studies show that there is little spontaneous chan-

nel activity at low cytosolic free Ca 2 concentrations

(100 nM). This is consistent with the rarity of sponta-

neous Ca 2

release events (i.e., Ca 2

sparks) at restingintracellular Ca 2 levels in cells (25, 50). There is substan-

tial spontaneous activity of single RyR channels at steady-

state Ca 2 concentrations in the micromolar range. The

degree of spontaneous activity peaks at 10 M. The

apparent K D of single RyR channel Ca 2 activation is

typically between 0.5 and 5 M. This is consistent with the

Ca 2 sensitivity of SR Ca 2 release activation in skinned

cardiac cells (81) and isolated cardiac and skeletal SR

vesicle preparations (54, 199, 234–236, 355).

The activity of single RyR1 channel decreases to near

zero if the steady-state Ca 2 concentration is elevated to

1 mM (54, 56, 161, 238). This is not true for the RyR2 or

RyR3 channels. Substantial inhibition of these channels isonly observed when the steady-state Ca 2 concentration

exceed 5–10 mM (54, 56, 135, 161, 238). It is unlikely that

the free Ca 2 levels in the cytosol of a cell would ever

exceed the 1 mM mark because the Ca 2 sources used to

elevate cytosolic Ca 2 (i.e., extracellular solution and SR

lumen) contain only 1 mM Ca 2. Thus the physiological

relevance of such low-af finity Ca 2 inhibition is question-

able. It is unclear how such low af finity of Ca 2 inhibition

(as observed at the single-channel level) is related to the

apparent high-af finity Ca 2-dependent inactivation ob-

served in cells (81). However, the RyR channels operate in

a dynamic (not stationary) Ca

2

signaling environment.Thus steady-state measurements of channel activity (over

minutes) may not reveal all the intricacies of single RyR

channel Ca 2 regulation in cells (on a millisecond time

scale).

XIII. CALCIUM ACTIVATION KINETICS

Several groups have defined the kinetics of single

RyR2 channel Ca 2 regulation in bilayers (e.g., Refs. 112,

113, 159, 264, 283, 339). There is considerably less data

addressing the kinetics of single RyR1 channels (e.g., Ref.

340) and none concerning single RyR3 channel function.

Consequently, the discussion here focuses mainly on the

kinetics of RyR2 channel Ca 2 regulation.

A. Rate of Ca

2

Activation

Fast Ca 2 stimuli were applied to single RyR2 chan-

nels using two different methodologies. One methodology

generates fast Ca 2 stimuli by liberating Ca 2 from caged-

Ca 2 compounds using laser flash photolysis (112, 339,

340). The other methodology generates fast Ca 2 stimuli

by mechanically changing the solution near the RyR2

channel (159, 264, 283). The RyR2 channel activates with

a time constant of 1 ms when the Ca 2 is very rapidly

changed via flash photolysis of caged-Ca 2 and slightly

slower ( 2–20 ms) when Ca 2 is changed by a mechan-

ical solution change technique. These numbers are fairly

consistent and are generally in agreement with whole cell

studies that clearly show that Ca 2 activation of SR Ca 2

release occurs on a millisecond time scale.

Interestingly, the reported RyR2 channel Ca 2 acti-

vation time constants vary with the speed of the applied

Ca 2 stimulus. For example, Laver and Curtis (159) ap-

plied relatively slow mechanical Ca 2 changes and re-

ported a time constant of 20 ms. Sitsapesan et al. (283)

applied slightly faster mechanical Ca 2 changes and re-

ported a Ca 2 activation time constant of 10 ms.

Schiefer et al. (264) applied even faster mechanical Ca 2

changes and reported a time constant between 2 and 5

ms. The fastest Ca 2

changes, however, were appliedusing the flash photolysis method. These studies report a

Ca 2 activation time constant of 1 ms (112, 113, 339).

The interpretation of the mechanical solution change

data described above must consider the presence of un-

stirred layers immediately in front of the bilayer. In some

of the mechanical solution change studies (159, 283), the

size of the unstirred layers clearly impacted the rate of

Ca 2 change in the microenvironment of the channel.

Unfortunately, concerns about the impact of unstirred

layers have rarely been discussed. Unstirred layers would

tend to slow the measured Ca 2 activation time constant.

Unstirred layers, however, do not complicate the inter-

pretation of the flash photolysis data, but there is another concern here. The Ca 2 waveform applied by flash pho-

tolysis is not a simple square step. Instead, there is a fast

Ca 2 overshoot at its leading edge (77, 78, 194, 372). This

very brief Ca 2 overshoot (lasting 150 s) likely accel-

erates the initial opening transition of the RyR2 channel.

The overshoot in these studies would tend to speed up the

measured Ca 2 activation time constant. Thus there are

studies (flash photolysis) that may overestimate the Ca 2

activation kinetics as well as studies (mechanical solution

change) that may underestimate it.





What is the real RyR2 channel Ca 2 activation kinet-

ics? The single RyR2 channel Ca 2 activation time con-

stant ( 2–5 ms) to a steplike Ca 2 stimulus is probably

best represented by the data of Schiefer et al. (264). This

study applied very fast mechanical Ca 2 changes on very

small-diameter bilayers alleviating unstirred layer con-

cerns. These types of experiments, however, have never been repeated. The physiological Ca 2 trigger in cells is

clearly not a simple square Ca 2 step. The RyR2 channels

in vivo are governed by the very fast transient Ca 2

changes that occur near DHPR (i.e., L-type) Ca 2 chan-

nels when they flicker open (302). It has been estimated

that the free Ca 2 concentration near the open mouth of

a DHPR Ca 2 channel can reach very high levels (100

M) when it is open and fall very rapidly (10 s) after

the channel closes. Interestingly, the fast Ca 2 overshoot

generated by flash photolysis has similar characteristics

(78). The rise time of the fast Ca 2 overshoot has a time

constant of 30 s, and the Ca 2 overshoot can theoret-

ically peak at 50–100 M before it decays with a time

constant of 150 s. Thus the fast Ca 2 overshoot may be

a reasonable reproduction of the free Ca 2 changes that

initiate RyR2 channel activity in the cell. Therefore, the

single RyR2 channel Ca 2 activation time constant to

physiological Ca 2 stimuli may be best represented by the

flash photolysis data ( 1 ms; Ref. 112).

B. Rate of Ca2 Deactivation

Fast Ca 2 elevations clearly turn on single RyR2

channels rapidly (112, 264). An equally important questionis, How quickly do single RyR2 channels turn off (deacti-

vate) in response to fast Ca 2 reductions? Intuitively, it

will take some time for Ca 2 to “fall off ” the Ca 2 activa -

tion site and for the channel to move into a closed con-

figuration. The Ca 2 deactivation kinetics of single RyR2

channels have been explored using both fast solution

exchanges (264, 283) and flash photolysis (340). Sitsape-

san et al. (283) reported that the time constant of RyR2

channel Ca 2 deactivation was 10 ms, about the mea-

sured rate of the Ca 2 change. Schiefer et al. (264) re-

ported that RyR2 channel Ca 2 deactivation occurred

with a time constant of 6 ms, 10 times slower than

measured Ca 2 change. Velez et al. (340) photolyzedDiazo-2 (a caged Ca 2 chelator) to reduce the local free

Ca 2 concentration near a single RyR2 channel in planar

bilayers. They reported a deactivation time constant of 5.3

ms, 10 times slower than measured Ca 2 change. Thus

there is reasonable consensus that single RyR2 channels

deactivate rapidly with a time constant of 5– 6 ms in

response to a fast Ca 2 reduction.

The point is that the Ca 2 activation and deactivation

kinetics of single RyR2 channels in vitro are suf ficient for

the channel to respond to the very brief (1 ms) trigger

Ca 2 concentration changes, the kind of local trigger

Ca 2 signals that are thought to govern RyR2 channel

activity in vivo (302).

XIV. FEEDBACK CALCIUM REGULATION

One suggestion is that the Ca 2 interacting with the

Ca 2 activation site is different from the Ca 2 that passes

through the open RyR channel pore. Another hypothesis

is that the Ca 2 activation site may “see” the Ca 2 flux

that passes through its own open pore. In other words, the

RyR channel may operate in a local “common Ca 2 pool”(302). There are several whole cell observations that are

consistent with this latter common Ca 2 pool idea (25,

168, 237, 344).

Is this type of self-feedback Ca 2 regulation seen in

single RyR channel studies? Most single RyR studies have

been done under conditions that generate superphysi-

ological unitary Ca 2

currents (285, 290 –292, 306). The pertinent single RyR channel studies were done with huge

Ca 2 driving forces that are more than 50 times greater

than those thought to exist in cells. This would increase

the possibility of feedback Ca 2 regulation in ways that

do not exist in the cell. This fact must be considered here.

The steady-state cytosolic Ca 2 sensitivity of the

RyR1 or RyR2 channels appears to be the same whether

the charge carrier is Ca 2 or a monovalent cation (58, 161,

290, 294, 306). This observation implies, but does not

prove, that charge carrier identity does not impact RyR

channel Ca 2 regulation. Sitsapesan and Williams (285)

have evaluated single RyR2 channel gating in the pres-

ence of different luminal Ca 2 concentrations. They con-cluded that P o and open/closed lifetimes of Ca 2-acti-

vated, Ca 2-conducting RyR1 channels were independent

of the Ca 2 flux through the channel. In contrast, Tripathy

and Meissner (333) reported that the duration of single

RyR1 channel openings was significantly longer in the

presence of even a relatively small Ca 2 flux through the

pore. They go on to predict that the RyR1 channel’s Ca 2

activation site must be 75 nm from the pore to explain

their results. Interestingly, the RyR channel complex is

only 25 nm square (230, 249, 342). This may indicate that

the RyR1 channel’s Ca 2 activation site may be in some

sort of protected pocket that shields the site from theCa 2 coming through the pore. In any event, it is clear that

additional data are required to establish whether or not

feedback Ca 2 regulation occurs.

XV. POTENTIAL NEGATIVE

CONTROL MECHANISMS

It is clear that some sort of RyR channel negative

control mechanism must exist to counter the inherent

positive feedback of the CICR process (81, 105, 214, 255,





265, 302, 303, 307). There is a growing consensus that the

negative control that counters the inherent positive feed-

back of CICR is actually a composite of factors/processes

that act in concert to terminate local RyR-mediated Ca 2

release. This latter idea is consistent with the fact that no

individual negative control mechanism appears suf ficient

by itself to explain the termination of local Ca 2

release.Several candidate RyR channel negative control mecha-

nisms are discussed below.

A. Ca2-Dependent Inactivation

The first negative-feedback mechanism considered

was Ca 2-dependent inactivation (81). It was reported

that the SR Ca 2 release process is substantially inacti-

vated at steady-state Ca 2 concentrations as low as 60 nM

in skinned cardiac muscle fibers (81). However, single

RyR2 channel studies in bilayers have forwarded no evi-

dence of Ca 2

-dependent inactivation at such low Ca 2

concentrations (54, 161, 256). Also, subsequent studies on

intact cells presented contradictory results (109, 208, 214,

280). For example, Lukyanenko and Gyo ¨ rke et al. (173)

imaged permeabilized ventricular myocytes using a con-

focal microscope and showed that elevation of resting

Ca 2 levels resulted in an increase in RyR2-mediated Ca 2

spark activity, not the decrease expected if Ca 2-depen-

dent inactivation exists. Thus the existence of high-af fin-

ity Ca 2-dependent inactivation is debated. The existence

of low-af finity Ca 2-dependent inactivation is not. Single

RyR1 and RyR2 channel data clearly demonstrate the

existence of low-af finity Ca 2 inhibition under steady-

state experimental conditions. Single RyR1 channelsclearly turn off as the cytosolic free Ca 2 concentration is

elevated toward the 1 mM mark (196, 253, 307). This

low-af finity Ca 2 inhibition could come into play if local

Ca 2 levels in the cell reach the 0.6 –1 mM range (156).

For the RyR2 channel, the situation is different. Single

RyR2 channels turn off only after free Ca 2 concentra-

tions reach much higher levels (5–10 mM; Refs. 12, 54, 56,

161, 199, 256). These levels are probably never realized in

a cell. Thus Ca 2-dependent inactivation as a potential

negative control mechanism that may account for (or

contribute to) the termination of local RyR-mediated

Ca

2

release events is still not clearly established.

B. Stochastic Attrition

Stochastic attrition refers to the inherent random

closing of an individual ion channel. Stern (302) demon-

strated using mathematical modeling that a SR Ca 2 re-

lease unit composed of one or relatively small number of

RyR2 channels will turn off spontaneously as a result of

reduced local Ca 2 levels resulting from the stochastic

closures of individual RyR2 channels in the cluster. Thus

stochastic attrition of single RyR2 channel activity could

contribute to terminating a local Ca 2 release event. This

process, however, would be very sensitive to the number

of channels involved and the positive feedback gain of

CICR in the channel cluster. Stern (302) showed that

stochastic attrition was suf ficient to terminate local Ca 2

release only if there were 10 RyR2 channels per cluster.If release units are composed of 10 –30 or more channels

(31, 95, 174), then the likelihood of termination solely by

stochastic attrition is low. Experimentally, Lukyanenko et

al. (176) showed that the rate of Ca 2 release termination

is proportional to the magnitude of the release event (i.e.,

large events terminate faster than small events). Addition-

ally, Sham et al. (273) demonstrated that local Ca 2 re-

lease events terminate rapidly despite the presence of a

sustained trigger Ca 2 signal. These results are inconsis-

tent with stochastic attrition playing a major role in con-

trol of the CICR process. Nevertheless, stochastic attri-

tion may still contribute to local Ca 2 release termination

if it acts in combination with other negative control mech-

anisms. It should be noted that the action of stochastic

attrition would be highly nonlinear because once the

number of active channels falls below some critical level,

the remaining channels would be increasingly impacted

by the phenomenon.

C. Adaptation

Most single RyR channels studies have focused on

defining RyR behavior under steady-state conditions.

Gyo ¨ rke and Fill (112), using laser flash photolysis of caged Ca 2, were the first to apply fast trigger Ca 2

signals to single RyR2 channels in bilayers. They reported

that single RyR2 channels activated rapidly reaching P o values that were well above those predicted from previ-

ous steady-state studies. After an initial burst of single-

channel activity, P o spontaneously decayed with a time

constant of 1 s. Application of second Ca 2 stimulus

reactivated the apparently “inactivated” channels. This

suggested that the spontaneous decay observed after the

first Ca 2 stimulus was not due to a conventional absorb-

ing Ca 2 inactivation mechanism (81) because the chan-

nels were not in a refractory state. They proposed that the

spontaneous decay may be mediated by a different pro-cess they termed adaptation (112, 230).

The RyR2 channel adaptation proposal spurred an

active debate. One point of debate centered on the rela-

tively slow time course of adaptation (1 s). The point

was that adaptation might be too slow to be a negative

control mechanism that stabilizes the CICR process in

heart. Somewhat lessening this concern, Valdivia et al.

(339) demonstrated that the RyR2 channel adaptation

phenomenon was 10-fold faster in the presence of phys-

iological Mg2. Another point of debate was that not all





investigators reported “adaptive behavior ” of single RyR2

channels. Although several groups reported that fast Ca 2

stimuli induced a transient burst of single RyR2 channel

activity (112, 159, 264, 283, 339), different groups inter-

preted their results in different ways. Still another point of

debate was centered on the nature of the Ca 2 stimulus

applied in the original adaptation study (112). It wassuggested that the fast Ca 2 overshoot at the leading edge

of the applied Ca 2 stimulus might generate the observed

adaptation phenomenon (154). As a result, the single

RyR2 channel data generated by the flash photolysis have

undergone an unusual level of scrutiny. It has endured,

has been frequently reviewed (86, 152, 288), and has

generated new insights into single RyR2 channel Ca 2

regulation. Details concerning the RyR2 adaptation de-

bate are discussed in section XVI.

We now know that the adaptation phenomenon is

due to a Ca 2- and time-dependent shift in the modal

gating behavior of single RyR2 channels (111). A fast-

sustained Ca 2 stimulus transiently drives the RyR2 chan-

nel into a high P o gating mode before it has time to

equilibrate (i.e., adapt) between all its different gating

modes. A specific kinetic scheme of RyR2 channel gating

has been developed describing this behavior (86). It is

now clear that Ca 2-dependent inactivation and adapta-

tion are not mutually exclusive mechanisms (as generally

thought). These two phenomena may simply be different

manifestations of a common underlying mechanism,

Ca 2- and time-dependent modal gating.

D. Activation-Dependent or “Fateful” Inactivation

Pizarro et al. (244) found that the global RyR1-medi-

ated Ca 2 release transient in frog skeletal muscle, trig-

gered by a maximally depolarizing stimulus, was inhibited

in proportion to the degree of Ca 2 release generated

during a prestimulation. This suggests that the RyR1 chan-

nels activated in the prestimulation were not available for

activation during the main stimulus. Thus preactivated

RyR1 channels appeared inactivated or refractory. These

authors proposed that RyR1 channel inactivation is

strictly and “fatefully” linked to their activation. In other

words, RyR1 channel inactivation is use dependent. Cer-

tain single-channel studies are consistent with this idea. Itis possible that a modal gating shift (i.e., high to low P omodes) may be a single-channel manifestation of fateful

inactivation (i.e., use dependence) observed in situ. Sev-

eral different groups have now reported modal gating

behavior of single RyR channels (8, 86, 111, 361).

E. Depletion/Luminal Ca2 Effects

When the SR is overloaded with Ca 2, periodic spon-

taneous Ca 2 waves can occur in heart muscle (304). It

appears that luminal Ca 2 levels modulate these RyR2-

mediated Ca 2 waves. The presence of a luminal Ca 2

regulation may also explain why a critical Ca 2 load must

be attained before Ca 2 can trigger SR Ca 2 release from

SR vesicle preparations (234–236). Several single RyR

channel studies have also addressed the possibility that a

luminal Ca 2

regulatory site exists (110, 341, 285, 333). Velez and Suarez-Isla (341) showed that RyR channel

activity changed as a function of luminal Ca 2 concentra -

tion and suggested this phenomenon was mediated by a

low-af finity Ca 2 binding site on the luminal side of the

channel. Later, two groups (110, 285) systematically in-

vestigated luminal RyR2 Ca 2 regulation and showed that

its extent depended on how the RyR2 channel was acti-

vated. For example, ATP-activated RyR2 channels were

more sensitive to luminal Ca 2 levels than Ca 2-activated

RyR2 channels.

The pertinent low-af finity luminal Ca 2 binding

site(s) may reside on the RyR channel or possibly on

another closely associated luminal protein. These sites

would need to sense changes in intra-SR Ca 2 levels in the

appropriate Ca 2 concentration range (0.2–20 mM). As SR

Ca 2 levels fall, the RyR channel would sense the reduced

SR Ca 2 level leading to a decrease in channel activity.

This would then represent an interesting form of RyR

negative control. There are caveats, however, in this lu-

minal Ca 2 story. Repetitive Ca 2 sparks should reduce

local SR Ca 2 load and thus downregulate RyR2 channel

activity. Yet, multiple Ca 2 sparks can occur frequently at

the same site in cells with little sign of progressive run-

down. This may suggest that SR Ca 2 levels either do not

change dramatically and/or that luminal Ca 2

may notimpact RyR2 channel activity dramatically. The spark

data, however, do not rule out the possibility that very

brief and highly localized luminal Ca 2 depletions govern

RyR2 channel function.

F. Allosteric or Coupled Gating

Recently, Stern et al. (305) evaluated several pub-

lished single RyR2 gating schemes and found that they all

generated unacceptable instability when applied in a

model of Ca 2 release local control. One reason for the

instability was the lack of a strong negative control mech-anism to minimize local regenerative Ca 2 release in clus-

ters of RyR2 channels. Another reason for the instability

was the relatively low cooperativity of the RyR2 channel

Ca 2 activation process. The low cooperativity does not

allow the RyR2 channels to adequately discriminate be-

tween trigger and background Ca 2 signals. Interestingly,

Stern et al. (305) suggested that RyR2-RyR2 allosteric

interactions could theoretically overcome these instabil-

ity problems. Recently, it has been reported that neigh-

boring RyR1 channels may be physically coupled by the





FK506-binding protein (FKBP12) and consequently gate

in synchrony (188). Is this coupled RyR channel gating

operative under physiological conditions? The answer to

this question is not yet clear (19, 305). Other investigators

have not reported coupled RyR channel gating. Addition-

ally, certain thermodynamic considerations may also be a

problem (i.e., multiple large macromolecules operatingwith microsecond synchrony). Furthermore, removal of

FKBP12.6 from the RyR channel does not abolish Ca 2

sparks, but actually increases their frequency and dura-

tion (351). Although coupled RyR channel gating is an

interesting and provocative hypothesis, its existence and

nature still require verification.

XVI. ADAPTATION DEBATE

The Gyo ¨ rke and Fill (112) RyR2 adaptation hypothe-

sis was controversial. One concern was that the decrease

in RyR2 channel activity, interpreted as adaptation, mightreflect slow Ca 2 deactivation (154) following a short-

lived Ca 2 transient (i.e., the fast Ca 2 overshoot) at the

leading edge of the applied Ca 2 stimuli. This possibility

was experimentally addressed, and the data suggest that

the brief Ca 2 overshoot has little impact on the much

slower (i.e., 1,000-fold slower) adaptation phenomenon.

The data indicate that the impact of the fast Ca 2 over-

shoot is most likely limited to accelerating the initial

closed to open transition of the RyR2 channel (i.e., it

essentially supercharges the applied Ca 2 stimuli). A de-

tailed discussion of the Ca 2 overshoot concern is pre-

sented below after a detailed description of the Ca 2

overshoot itself. Another concern regarding the originaladaptation hypothesis (112) was that some investigators

did not report adaptive behavior of single RyR2 channels

but instead forwarded alternate interpretations (49, 112,

159, 264, 283, 339). Some of these different interpretations

are also presented below.

A. DM-nitrophen Ca2 Overshoot

DM-nitrophen is an ultraviolet-sensitive high-af finity

Ca 2 buffer that changes its Ca 2 af finity from 4.8 nM to

3 mM upon flash photolysis (74, 78, 194). Ca 2 liberation

by flash photolysis of DM-nitrophen (the caged Ca 2 compound used the RyR2 adaptation studies) generates a fast

Ca 2 overshoot (78, 154, 194, 372). It arises because flash

photolysis liberates Ca 2 faster than free DM-nitrophen

can bind Ca 2. Its characteristics and how its kinetics

vary with experimental conditions are not generally un-

derstood and often the basis of debate (112, 154, 372).

The fast Ca 2 overshoot is very fast compared with

kinetics of commonly used fluorescent dyes (78). Conse-

quently, the kinetic characteristics of the fast Ca 2 over-

shoot are necessarily based on numerical calculations

from the much slower fluorescence data even in the case

of the fastest fluorescent indicators (78). The parameters

estimated for the fast Ca 2 overshoot may then reflect

more the kinetic characteristics of the indicator than the

Ca 2 liberation kinetics of the caged Ca 2 compound.

There is no debate that DM-nitrophen photolysis by a

brief flash (50 ns long) liberates Ca 2

in 30 s (74, 78,194). However, there is disagreement about the Ca 2

association rate of DM-nitrophen. Zucker (372) suggested

a relatively slow association rate (1.5 106 M1s1), and

use of this value predicts that Ca 2 overshoots should last

several milliseconds. More recently, Escobar et al. (78)

showed that the association rate was substantially faster

(3 107 M1s1). This means that the fast Ca 2 over-

shoot is more than an order of magnitude faster (100 s

vs. 2 ms) than previously thought (154, 372).

The magnitude of the overshoot depends on the free

DM-nitrophen levels. At pCa 9, only 18% of unphotol-

ysed high-af finity DM-nitrophen will be bound to Ca 2.

Flash photolysis will liberate Ca 2 from Ca 2-bound DM-

nitrophen, but much of this Ca 2 will rebind to Ca 2-free

DM-nitrophen. At pCa 7, nearly 96% of unphotolysed high-

af finity DM-nitrophen will be bound to Ca 2. The same

flash will liberate much more Ca 2 from Ca 2-bound DM-

nitrophen, but there will be less Ca 2-free DM-nitrophen

to participate in rebinding. As a result, the Ca 2 overshoot

will be smaller and slower. At pCa 6, nearly all unphoto-

lysed high-af finity DM-nitrophen will be bound to Ca 2.

There will thus be no Ca 2 rebinding and consequently no

Ca 2 overshoot.

We estimate, based on the calculations presented in

previously works (78, 86), that the Ca 2

overshoot in theoriginal Gyo ¨ rke and Fill (112) study peaked at 30 – 60 M

and lasted 100–200 s. Gyo ¨ rke and Fill (112) used a

Ca 2 electrode to measure free Ca 2 concentrations be-

fore and after photolysis. Thus the properties of the fast

Ca 2 overshoot could not be measured. These kinetic

estimates predict that the fast Ca 2 overshoot was at least

three orders of magnitude faster than the observed adap-

tation phenomenon (112, 372).

B. Ca2 Overshoot Concern

The concern was that RyR2 adaptation might simplybe deactivation following the fast Ca 2 overshoot (154).

In other words, the Ca 2 activation sites will be rapidly

occupied during the overshoot. After the overshoot, oc-

cupancy of the site will fall and deactivate the channel.

The idea was that the RyR2 deactivation after the over-

shoot (lasting 200 s) generates the adaptation ( 1

s) phenomenon. If this were the case, then the deactiva-

tion of the RyR2 would need to be very slow. Is RyR2

Ca 2 deactivation so slow? The answer is no. The rate of

RyR2 channel deactivation in response to a fast Ca 2





reduction has been experimentally defined (264, 340, 362),

and it is quite fast ( values 5 ms). Does the fast Ca 2

overshoot impact single RyR2 channel function? The an-

swer is yes. Zahradnı kova ´ et al. (362) using improved

recording conditions showed that the fast Ca 2 over-

shoot, albeit slightly smaller than those applied by Gyo ¨ rke

and Fill (112), can occasionally induce one or two brief opening events. Application of Ca 2 stimuli like those

applied by Gyo ¨ rke and Fill (112) (i.e., overshoot followed

by a sustained elevation), induce an entirely different

pattern of single RyR2 channel activity (including the

classical adaptation phenomenon). These data and more

recent single-channel modeling (86) suggest the impact of

the fast Ca 2 overshoot on single RyR2 channel function

may simply be to accelerate the initial opening transition.

Does the fast Ca 2 overshoot induce the adaptation

phenomenon? Few questions concerning single RyR

channel function have been debated more (86, 152, 288).

In principle, adaptation represents a slow transition of the

RyR channel from one state to another. There is circum-

stantial experimental evidence that suggests that this

slow transition is not simple Ca 2 deactivation following

the Ca 2 overshoot (154, 264, 340, 362). There are also

theoretical arguments that suggest that the slow transi-

tion is not induced by the fast Ca 2 overshoot. A specific

Markovian gating scheme, albeit still imperfect, has been

forwarded to describe RyR2 adaptation (see Fig. 3; Ref.

86). In this scheme, a fast Ca 2 overshoot is not required

to generate adaptation-like phenomenon. This scheme

suggests that RyR2 adaptation is due to a transient Ca 2-

dependent modal RyR2 gating shift (see sect. XVII and Fig.

3). There is, however, no experimental evidence directlyaddressing the salient point (i.e., demonstration that the

same channel response is elicited by a Ca 2 step with or

without the initial overshoot). Thus the question above

has not been definitively answered yet.

C. Differing Interpretations

Several studies have now defined the kinetics of sin-

gle RyR2 channel Ca 2 regulation (112, 113, 159, 264, 282,

283, 339, 340, 362). Although the data are generally con-

sistent, how the different data sets have been interpreted

varied a great deal. Some investigators choose to interprettheir data in terms of adaptation (112, 339, 340), whereas

others choose to interpret their data in terms of classical

Ca 2-dependent inactivation (159, 264, 283). Several fac-

tors contributed to the different interpretations. The var-

ious studies used very different techniques and conse-

quently applied very different types of Ca 2 stimuli. This

is important because even relatively small differences in

the characteristics of the Ca 2 stimulus can have a large

impact on single RyR2 channel function (81). For years,

the absence of adaptation following the mechanically in-

duced Ca 2 stimuli was construed as evidence against the

adaptation phenomenon. A great deal of attention was

focused on understanding how the Ca 2 overshoot could

generate “adaptation.” Attention is now more appropri-

ately focused on understanding how the different types of

Ca 2 stimuli may impact channel function. It is now clear

that the photolytic Ca 2 stimuli have a fast component

not present in the mechanical methods. Thus it is not

surprising that RyR channel behavior is different in the

different studies. The point is that the results of the flash

photolysis studies (112, 339, 340, 362) are not inherentlyat odds with those reported in the mechanical solution

change studies (159, 160, 264, 282, 283), and there is no

reason a priori to favor one type of study over another.

Each study should be evaluated on its own merits, for

each represents a heroic effort and makes a substantive

contribution. In our opinion, the differences in the Ca 2

stimuli make the data more (not less) interesting. The fast

photolytic Ca 2 stimuli are particularly interesting be-

cause they may mimic the Ca 2 changes that activate RyR

channels in cells.

FIG. 3. Markovian scheme of modal RyR2 channel gating. The chan-

nel recording is thought to reflect the fluctuation of the molecule be-tween four highlighted sets of states, including high and low open

probability ( P o) modes, to explain the bursting nature of the channel’sgating. Below are four simulated single channel records generated usingthis scheme. Each illustrates the predicted response to a fast steplikefree calcium change. The different colored regions of the recordings

correspond to the different colored sets of the gating scheme. [Adaptedfrom Zahradnı kova ´ and Zahradnı k (361).]





XVII. MODAL GATING

Under steady-state conditions, single RyR2 channel

activity occurs in bursts. This means that channel open-

ings tend to be clustered in time instead of being ran-

domly distributed in time. The P o during a burst can be

either high or low (Fig. 4). The high P o and low P o burstsdo not occur randomly. Instead, the bursts tend to be

clustered as well. Trains of high P o bursts characterize a

high P o gating mode. Trains of low P o bursts characterize

a low P o gating mode. Extended periods of no openings

characterize a zero P o gating mode. Several different

groups have now reported the existence of modal RyR2

channel gating (8, 86, 261, 361).

How does modal RyR2 channel gating work? This is

still an open question. The identities, transitions, and

characteristics of the different RyR2 gating modes are

currently being defined. Preliminary interpretations sug-

gest that there is a dynamic equilibrium between at least

three different gating modes under steady-state condi-

tions (i.e., at a constant Ca 2 level). High, low, and zero P omodes are clearly evident in the steady-state activity of

single RyR2 channels (8, 86, 261, 361). There also appears

to be clear spontaneous shifts between the different gat-

ing modes. Simulated single-channel data are presented in

Figure 4 to illustrate some of these points. It is thought

that the dynamic Ca 2-dependent equilibrium between

gating modes is what generates the classical bell-shaped

steady-state Ca 2 dependence of the RyR2 channel (54,

161, 353).

An interesting thing happens when the Ca 2 level

changes suddenly. Small fast Ca 2 elevations (from a low

Ca 2 level) upset the existing dynamic equilibrium mo-

mentarily. The result is a transient change in channel

activity until a new equilibrium is reached. Specifically, it

has been argued that single RyR2 channel activity momen-

tarily rises to a P o level above that predicted by steady-

state measurements before it spontaneously decays (86).Channel activity decays as the channel reequilibrates be-

tween the gating modes at the new higher Ca 2 level (Fig.

4). Subsequent small fast Ca 2 elevations would induce

further transient modal gating shifts, inducing additional

transient episodes of high channel activity. This behavior

is reminiscent of the adaptation phenomenon. The mag-

nitude of the transient modal gating shift should depend

on the speed and magnitude of the Ca 2 stimulus. A slow

Ca 2 stimulus would not induce a dramatic modal gating

shift because mode reequilibration is time dependent (i.e.,

modes will reequilibrate during the slow stimulus). Large

Ca 2 stimuli should be less effective because at high Ca 2

levels the channel will begin to frequent the inactivated

state. Consequently, a slow large Ca 2 stimulus would not

induce a substantial transient modal gating shift but may

instead induce Ca 2-dependent inactivation.

Is this consistent with the experimental record? Ap-

plications of fast Ca 2 elevations do indeed induce super

steady-state P o levels that are followed by a spontaneous

decay in channel activity (112, 159, 339). Also, repeated

episodes of transient channel activity in response to mul-

tiple incremental Ca 2 elevations have been reported

(112). Experiments have also defined the dependence of

channel activation rate on stimulus speed (159, 264, 283)

FIG. 4. Modal gating of the RyR2 channel. The Mark-ovian scheme shown in Figure 3 was used here to simulate

the single-channel recordings shown. At the top, a seriesof high P o bursts or low P o bursts are marked. Trains of high or low P o bursts correspond to the high or low P ogating modes of the RyR2 channel. At any steady-statecalcium concentration, the RyR2 channel will spontane-ously shift between its different gating modes. A samplespontaneous modal gating shift (at pCa 5) is shown in the

middle panel. In this case, the channel shifted from thelow P

o mode to the high P

o mode and back. Shifts in model

RyR2 gating can also be evoked by fast calcium concen-tration changes. A sample evoked modal gating shift is

shown on the bottom panel. A fast calcium concentrationchange (pCa 7 to 6) was introduced at the arrow. Thechannel immediately shifts into the high P o mode for a

period of time before shifting again into the low P o mode.





and clear refractory behavior at very high Ca 2 levels

(264). Thus the modal gating theory qualitatively pre-

sented above has some experimental support. It is clear,

however, that additional experimental results are needed.

For example, current modal gating theory does not yet

consider the potential impact of certain physiologically

important ligands (e.g., luminal Ca 2, cytosolic Mg2, and ATP).

Is modal RyR channel gating physiological? Modal

gating may reproduce and reconcile certain single RyR

channel phenomena in bilayers, but its physiological rel-

evance is questionable. In cells, the RyR channels gener-

ate intracellular Ca 2 signals that last less than 25 ms

(e.g., sparks). Studies of “steady-state” RyR behavior in

bilayers report that intermodal gating transitions occur

over seconds. From these data, it appears unlikely that

modal RyR gating is a physiologically relevant phenome-

non. Modal gating may simply be a biophysical ramifica-

tion of RyR channel Ca 2

regulatory complexity. Thisdoes not make it less important because understanding

the origin of modal gating may provide new insights into

RyR channel Ca 2 regulation.

XVIII. CONCLUDING REMARKS

The advent of in vitro single RyR channel recording

has provided an abundance of information. It has revealed

key properties of the RyR channel’s permeation pathway

and how (and if) particular ligands alter channel function.

Despite some amazing advances, many fundamental ques-tions about how the RyR channels are turned on and off

in vivo remain unanswered. For example, in vitro single

RyR2 channel recording has not definitively established

the identity of the mechanism(s) that terminate the SR

Ca 2 release process or revealed how movement of the

DHPR voltage sensor governs RyR1 channel activity.

Our understanding of single RyR channel regulation

is clearly incomplete. This impairs our understanding of

many intracellular calcium signaling phenomena. Long-

standing RyR channel mysteries remain and are certain to

motivate research long into the future.

We thank the reviewers for their meticulous revision of the

manuscript and for providing us with very constructive com-

ments. We also thank Dr. J. Ramos-Franco and M. Porta for

comments on the manuscript and A. Nani for technical help.

Research in the laboratory of M. Fill is supported by Na-

tional Heart, Lung, and Blood Institute Grant HL-57832. J. A.

Copello is supported by American Heart Association Grant

0130142N.

Address for reprint requests and other correspondence: M.

Fill, Dept. of Physiology, Loyola University Chicago, 2160 South

First Ave., Maywood, IL 60153.

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doi:10.1152/physrev.00013.200282:893-922, 2002.Physiol RevMichael Fill and Julio A. CopelloRyanodine Receptor Calcium Release Channels

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