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Residue 224 controls conductance and inward rectification in Kir2.1 channels
謝如姬生醫所
中央研究院
0.1 M KCl 0.01 KCl+ 0.09 NaCl
intra- extracellular side
0.1 M KCl 0.01 KCl+ 0.09 NaCl
intra- extracellular side
G = iGni
i = oi (T,P) + RT ln Ci + ziFV
iX
oX
][
][ln
FxZ
RT EX
ENa =+58 mV
ECl = -41 mV
ER= -75 mV
EK = -86 mV
0 mV
Ion Channel 101
Permeability
I Q/t Ohm’s law: I R = V I/V = 1/R = G
• Selectivity
• Gating
Iinward influx of cationsIoutward efflux of cationsErev: Iinward = Ioutward DepolarizationHyperpolarization
Significance of Inward Rectifier K+ Channel
1. Maintain resting membrane potential2. Control excitability3. Regulating heart rate, vascular tone, renal
salt flow, insulin release, etc.
VmEK Vres
I
Mechanisms for Inward Rectification
1. Voltage-dependent blockade of outward IKir2.1 by intracellular Mg2+ (Matsuda et al., 1987; Vandenberg 1987) and Polyamines (Fickler et al., 1994; Lopatin et al., 1994) 2. Intrinsic gating (Shieh et al., 1996; Xie et al., 2004)
M1 M2
Out
In
N C
D172
E224E299
Kuo et. al., 2003 Science, Vol 300, p:1922-1926.
Substituted-Cysteine Accessibility Method(SCAM)
Reaction of methane thiosulfonates (MTS)with a cysteinyl residue(Karlin and Akabas, 1992 Methods in Enzymology, Vol. 293)
0 2 0 4 0 6 0 8 0 1 0 0- 4
- 3
- 2
- 1
0
0 2 0 4 0 6 0 8 0 1 0 0- 1 0
- 8
- 6
- 4
- 2
0
4 s
2 0 s
2 0 s
1 0 s
4 s
= 1 3 . 7 s
I (nA
)
T im e ( s )
M T S E T ( 2 m M )M T S E T ( 2 m M )
= 7 . 8 sI (
nA)
T im e ( s )
1 0 s
B
2 n A
1 0 m s
1 n A
S p mC t r l
S p mC t r l
D 1 7 2 C
A
2 5 m s
E 2 2 4 K
M
C
M
C
M
2 5 m s
E 2 2 4 Q
E 2 2 4 CE 2 2 4 D
2 p A
C
2 p A
DC
BA
M T S E T
C t r l M T S E SC
M
1 p A
5 0 m s5 0 m s
1 p A
E 2 2 4 CIR K 1 J
C t r l
E 2 2 4 C
M
CM T S E S - M T S E T
IR K 1 J
M T S E T - M T S E SC t r lC t r l
M
CM T S E T
M T S E S
1 0 0 m M [ K + ]O
2 p A
2 p A
2 p A
M
C
M2 p A
5 0 m s
1 0 0 m M [ K + ]i
1 0 0 m M [ R b + ]i
1 0 0 m M [ N H4
+ ]O
5 0 m s
5 0 m s
5 0 m s
1 0 0 m M [ N H4
+ ]i
1 0 0 m M [ N M G + ]i
C
1 0 0 m M [ N H4
+ ]i
1 0 0 m M [ T l+ ]i
1 0 0 m M [ N a + ]i
1 0 0 m M [ K + ]i
C
0 . 0 0
0 . 0 5
0 . 1 0
0 . 1 5
0 . 2 0
* *
* # # #
* * *
1 0 0 m M [ N H4
+ ]o
1 0 0 m M [ K + ]o
* * *(O
2 -
C
2 )/i
(pA
)
Summaries
1. The single-channel inward current flickers with reduced conductance in an E224G mutant.
2. Introducing a negatively charged MTS reagent to the E224C mutant
irreversibly increases channel conductance and eliminates open-channel fluctuations.
3. The negatively charged residue 224 is located at the internal vestibule
and it is important for smooth inward K+ conduction. 4. Open-channel fluctuations are mainly attributed to the rapid transitions
between the substate and the main state 5. The open-channel fluctuations decreased in internal NH4
+ or
Tl+ as compared to internal K+.
Acknowledgement
張雪愷 葉士豪 何宜芳
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