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