Peripheral GABAARs: Overview the Lung, Pancreatic Islets, and

Lymphocytes

王双连 Nov 12, 2012

More Attention Has Been Paid to Peripheral GABA Since 1954

GABAergic Sigals in the CNS

GAD: glutamic acid decarboxylase

GABA-T: GABA-transaminase

GAT: GABA transporter

GABAAR is a pentameric chloride channel composed of a combination of various subunits;(α1-6, β1-3, γ1-3, δ, ε, π, θ, and ρ1-3)

GABABR is a type of G-protein coupled receptors (GPCR)

Cl-

Glutamate

GABABRGABAAR

GABA

GAD65/67

GAT

GABA-T

Succinicsemialdehyde

GABA

Cl-

phasictonic

Characteristics of GABAARs

Critical Reviews in Biochemistry and Molecular Biology, 42:3–14, 2007

extracellular

intracellular

Synaptic and Extrasynaptic GABAAR Currents

extrasynaptic

synaptic M. Wallner etc. PNAS, 2003

Cl-

Glutamate

GABA

GABA

Cl-

phasictonic

Valerie B. etc. PNAS, 2004

Modulation of GABAARs

• Agonists: GABA and muscimol• Antagonist: bicuculline (competitive inhibition) picrotoxin (non – competitive inhibition)• Benzodiazepines, pentobarbital, and ethanol• Neurosteroids and endocannabinoid 2-

arachidonoylglycerol (2-AG)• Ions: Zn2+, Mn2+, Co2+

• Posttranslational modification: protein kinases• Receptor associated proteins:

Overview of the Major GABAAR Subunits

• α1: sedative, amnesic and anticonvulsant action of BZ

• α 2: Anxiolytic and myorelaxant action of BZ

• α 3: anxiolytic and myorelaxant action of BZ

• α 4: ethanol sensitivity

• α 5: amnesic and myorelaxant action of BZ, memory enhancement

• α 6: ethanol sensitivity

Overview of the Major GABAAR Subunits

• β1: Salicylidene salicylhydrazide as selective inhibitor

• β 2: anesthetic action of etomidate

• β 3: anesthetic action of propofol and etomidate

• γ1-3: no specific properties

• δ: ethanol and neurosteroid sensitivity

Example 1: Benzodiazepines Regulation of GABAARs

Example 2: Ethanol Regulation of GABAARs

M. Wallner etc. PNAS, 2003

What Hampers us in Further Understanding GABAAR Functions?

• Easy to do: To identify sequence, expression level and localization of

indivial subunits in a neuron

• Less easier to do: To know subunits collaboration

• Hard to do: To identify the subunit arrangement of the pentamer

• Experiments have therefore been limited to the role of defined receptor subunit isoforms

GABA and the Lung Story

GABAARs in airway epithelium

Expression of GABAARs in airway epithelium

Electrophysiological characteristics

Physiological and pathophysiological significance

Expression of GAGAARs in lung epithelial cells

Electrophysiological characteristics of GABAARs in lung epithelial cells

Voltage - clamp recording Current - clamp recordingVholding = -60 mV

sweep

Perforated Whole- cell

Cl-

Electrophysiological characteristics of GABAARs in lung epithelial cells

Voltage - clamp recordingVholding = -60 mV

Electrophysiological characteristics of GABAARs in lung epithelial cells

Voltage - clamp recording (Vstep)

Vholding = from -60 to 40 mv, △ v = 20 mv, totally 6 sweeps

Electrophysiological Characteristics of GABAARs in Lung Epithelial Cells

Voltage - clamp recording Current - clamp recording

Perforated

Cl-

X

Physiological or Pathophysiological Significance of GABAARs in Lung Epithelium

GAD and β2/3 Subunit Upregulation in Asthmatic Lung in Mice and Human

Mic

eH

uman

IL-13 Mediates the Upregulation of GABA Signals in Asthmatic Mice Lung

GAD65/67 β2 subunit

In vitro

IL-13 Mediates the Up-regulation of GABA Signals in Asthmatic Mice LungIn vivo

Picrotoxin (PTXN) Protects the Lung against OVA-induced Asthma in Mice

Blocking GABAARs does not Affect OVA-induced Inflammation and Hyperreactivity in the Airway

Summary to the GABA-Lung Story

Up-regulation of the epithelial GABAergic system occurs downstream of activation of the IL-13 receptor, and that this GABAergic system plays a selective part in goblet cell metaplasia and mucus overproduction.

GABA and the Pancreas Story

Expression of GABAARs in INS-1 cells

GABA-evoked Currents in INS-1 cells

Perforated Whole- cell

16.7 mmol/l glucose0 mmol/l glucose

GABA-evoked Currents in INS-1 cells

Clinical Pharmacology & Therapeutics, 2008

Effect of GABAAR activation on the Excitability of INS-1 β cells

Reversal potential of IGABA: -43 mV

Effect of GABAAR Activation on the Excitability of INS-1 β cells

outside

inside

Cl-

membrane

0 G: Vm= -50 mV < -43 mV

Driving force

outside

inside

28 G: Vm= -20 mV > -43 mV

Driving forceCl-

GABAAR GABAAR

Effect of GABAAR Activation on the Excitability of INS-1 β cells

Modulation of Ca2+ infux by GABA in INS-1 cells

Regulation of Insulin Secretion by GABA is dependent on Glucose Concentration

Lower glucose Higher glucose

Summary to the GABA-INS-1 Story

• Activation of GABAARs in beta cells regulates insulin secretion in concert with changes in glucose levels. A mechanism involving Ca2+ movement may underplay.

• The GABA system may function as a negative feedback regulating mechanism in the islets.

glucose

GLUT2ATP/ADP

KATP

K+

Ca2+

Ca2+

Insulin release

hyperglycaemia Insulin release

GABA-GABAARs

(-)

hypoglycaemia Insulin release

GABA-GABAARs

(+)

Insulin Negatively Regulates GABAAR Function and Inhibits GABA- induced beta Cell Secretion

GABA-evoked Currents (IGABA) is Inhibited by Insulin in INS-1 cells

GABA-evoked Currents (IGABA) is Inhibited by Insulin but not Zn2+ in INS-1 cells

Insulin-induced Inhibition on IGABA is PI3-K/Akt Independent

DN: dominant negative

membrane

Wortmannin/Ly294002

insulin

Insulin receptor

inactive active

IRS

P P

PI3-K

P

P

inactive active

Akt/PKB

Insulin does not Alter the Localization of GABAARs at the INS-1 Plasma Membrane

Red: GABAAR beta2/3 SubunitBlue: DIPI

Insulin Sigaling

PD98059

Insulin Suppresses IGABA via MEK/ERK Siganling Pathway

Insulin Suppresses GABA-induced Insulin Secretion in INS-1 Cells

Summary

Insulin Insulin release

GABA - GABAARs

( － ) ( ＋ )

Physiological significance: a feedback mechanism for fine-tuning b-cell secretion

Insulin may utilize GABA-GABAARs system to inhibit further release at the peak of the exocytotic event, particularly, at very high local insulin concentration.

( － )

The Akt-GABAARs Pathway in Mediating Glucose- Induced Suppression of Glucagon Release in α Cells

GABA Exerts Protective and Regenerative Effects onIslet Beta Cells and Reverses Diabetes

GABAARs in T Lymphocytes

Different Subtypes of GABA-A Receptors Are Expressed in Human, Mouse and Rat T Lymphocytes

Different Subtypes of GABA-A Receptors Are Expressed in Human, Mouse and Rat T Lymphocytes

Vh= -80 mV

Vh= 60 mV

“Tonic” currents

An Intrinsic GABAergic System in Human Lymphocytes

(10 mM)

(10 mM)

Whole cell currents,Vh = -50 mV

GABA Channels are Activated with 1M GABA+ 100 nM THDOC on CD8+ DR+/+ T Cells

Single-channel currents

GABA Channels are Activated with 1M GABA/100 nM THDOC on CD4+/CD8+ DR+/+ T Cells

GABA

GABA

GABA +THDOC

Single-channel currents

CD8+ DRlyp/lyp T cells but not CD4+ DRlyp/lyp T cells Express Higher Level of GABAA Channel Subunits

DRlyp/lyp: diabetic prone ratsDR+/+: diabets resistant rats

DRlyp/lyp: diabetic prone ratsDR+/+: diabets resistant rats

CD8+ DRlyp/lyp T Cells Express More GABAAR Subunitsas Compared with CD4+ DRlyp/lyp T cells

CD4+ and CD8+ T cell Proliferation is Decreased by Sub-M GABA Concentration

CD8+ DRlyp/lyp T cells

?

Conclusions

• Animals that do develop type-1 diabetes do express higher levels of GABAA channel subunits in CD8+ T cells than their wild-type counterparts.

• GABA in the islets of Langerhans may diminish inflammation by inhibition of activated T lymphocytes

• Our results raise the question of whether an enhanced GABAA channel activity might delay or possibly prevent the development of type-1 diabetes.