a single charged voltage sensor is capable of gating the shaker k + channel dominique g. gagnon and...
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A single charged voltage sensor is
capable of gating the Shaker K+ channel
Dominique G. Gagnon and Francisco Bezanilla Department of Biochemistry and Molecular Biology,
The University of Chicago, Chicago, IL 60637
J. Gen. Physiol. Vol. 133 No. 5 467–483
13 de julio 2009
http://einstein.ciencias.uchile.cl
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Extracelular
Intracelular
Bicapa
Estructura de los canales de K dependientes de voltaje.
S1
S3a
S6
Doyle et al 1998 Science 280:69-77. Jiang et al 2003 Nature 423:33-41.
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S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
S1 S2 S3 S4 S5 S6
S1 S2 S3 S4 S5 S6
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
Shaker zH(6–46) AKA Shaker zH4 IR = wt
Shaker zH(6–46) R362Q, R365Q, R368N, R371Q = mut
4wt
2wt/2mut
1wt/3mut
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Simulación de canales de potasio del modelo del axón de jibia de Hodgkin y Huxley 1952.HP -140 mV pulso 20 mV, post-pulso -100 mV.
/)()0()()( tennntn )()()( 4
KKKK VVtngNtI
4/44 1)()( tentn
/444 )0()( tentn
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/)()0()()( tennntn ))(()( KKKK VVtngNtI
/1)()( tentn
/)0()( tentn
Simulación de canales de potasio del modelo del axón de jibia de Hodgkin y Huxley 1952.sin elevar n a la 4ª porencia. HP -140 mV pulso 20 mV, post-pulso -100 mV.
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Canales de potasio del modelo del axón de jibia de Hodgkin y Huxley 1952.HP -140 mV pulso 20 mV, post-pulso -100 mV.
n4
n4
n
n
4/44 1)()( tentn
/444 )0()( tentn /)0()( tentn
/1)()( tentn
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Homotetrameric Shaker R362Q/R365Q/R368N/R371Q is poorly expressed at the plasma membrane of Xenopus oocytes
Fig 1
Canales expresados en oocitos de Xenopus. Cut Open Voltage Clamp. Kin = 120 mM Kout 12 mMImágenes de la fluorescencia de los oocitos con los canales marcados con VENUS. The VENUS tag is a Green Fluorescent Protein isoform. (Nagai et al., 2002 Nat.Biotechnol. 20:87–90.),
Oocyte diameter 1.0-1.2 mm.
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VENUS Shaker IROocyte diameter 1.0-1.2 mm.
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VENUS Shaker R362Q/R365Q/R368N/R371Q
Oocyte diameter 1.0-1.2 mm.
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Comparación wt hometrámero (negro) con 4wt concatámetro (rojo)
Canales expresados en oocitos de Xenopus. Cut Open Oocyte Voltage Camp
Shaker zH4 IR and 4wt are functionally undistinguishable
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Shaker heterotetramers with three neutralized voltage sensors still gate
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Shaker heterotetramers with three neutralized voltage sensors still gate
The lines represent the global fit of a simple Boltzmann distribution for 1wt/3mut and Boltzmann distribution taken to the “nth” power for 2wt/2mut and 4wt, all sharing the parameters V1/2 and z.
n
RTVVzFeGG
/)(
max2/11
1
n = 1.00 para 1wt/3mutn = 1.93 para 2wt/2mut n = 4.97 para 4wt
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Simulación de anales de potasio del modelo del axón de jibia de Hodgkin y Huxley 1952.HP -140 mV o -60 mV, pulso 20 mV, post-pulso -100 mV.
HP -140 mV
HP -60 mV
Voltage-dependent K+ channels exhibit a delay for activation which increases when hyperpolarizing prepulses are applied (Cole and Moore, 1960. Biophys. J. 1:1–14 ).
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Reduced delay for activation in the 1wt/3mut
Voltage-dependent K+ channels exhibit a delay for activation (Hodgkin and Huxley, 1952. J. Physiol. 116:449–472) which increases when hyperpolarizing prepulses are applied (Cole and Moore, 1960. Biophys. J. 1:1–14 ).
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nteAti /1)(
Las líneas rojas de los primeros 10 ms se pueden describir bien con una función de esta clase
El exponente n de esta ecuación es una medida del número de pasos previos a la apertura del canal.
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nteAti /1)(
Las líneas rojas de los primeros 10 ms se pueden describir bien con una función de esta clase
El exponente n de esta ecuación es una medida del número de pasos previos a la apertura del canal.
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nteAti /1)(
Las líneas rojas de los primeros 10 ms se pueden describir bien con una función de esta clase
El exponente n de esta ecuación es una medida del número de pasos previos a la apertura del canal.
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Voltage-dependent K+ channels exhibit a delay for activation (Hodgkin and Huxley, 1952. J. Physiol. 116:449–472) which increases when hyperpolarizing prepulses are applied (Cole and Moore, 1960. Biophys. J. 1:1–14 ).
Reduced delay for activation in the 1wt/3mut
nteAti /1)(
Las líneas rojas de los primeros 10 ms se pueden describir bien con una función de esta clase
El exponente n de esta ecuación es una medida del número de pasos previos a la apertura del canal.
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Scheme 1
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Canales de potasio del modelo del axón de jibia de Hodgkin y Huxley 1952.HP -140 mV pulso 20 mV, post-pulso -100 mV.
n4
n4
n
n
4/44 1)()( tentn
/444 )0()( tentn /)0()( tentn
/1)()( tentn
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Fig 4
Reposo a Activación Activación a Reposo
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Fig 6Po,max = 0.73 Po,max = 0.67 Po,max = 0.43
Análisis de ruido.
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Fig 7
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Scheme 1
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Table I
-
-
-
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Fig 9
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Inactivación
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-60 a 10 en 10 mV -80 a 30 en 10 mV
-40 a 20 en 10 mV
COOVC.Cut open oocyte voltage clamp. Solución externa Na-MES isotónica. Olcese et al 1997 J. Gen. Physiol. 110:579–589.
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COOVC.Cut open oocyte voltage clamp. Solución externa Na-MES isotónica. Olcese et al 1997 J. Gen. Physiol. 110:579–589.
La inmovilización de las cargas de compuerta y la inactivación lenta tienen el mismo curso temporal.
Curso temporal de la inactivación lenta
Curso temporal de la inactivación lenta (línea delgada) y la inmovilización de cargas ( puntos y línea gruesa)
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Pulsos -70 a 17 mV en pasos de 3 mV Corriente de cola instantánea a -50 mV después de un puso de 15 ms a -1mV, habiendo estado al menos 1 min al holding potential anotado
Inactivación lenta en función del holding potential, estado estacionario
COOVC.Cut open oocyte voltage clamp. Solución externa Na-MES isotónica. Olcese et al 1997 J. Gen. Physiol. 110:579–589.
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Crystal structure of open-inactivated KcsA and the molecular basis of the K+ channel gating cycle
Luis G. Cuello, Vishwanath Jogini, D. Marien Cortes, and Eduardo Perozo.
Sneak preprint
Alteración del filtro de selectividad relacionado con la apertura de la compuerta de activación. Base estructural de la inactivación lenta.
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Fig 3
La inactivación lenta tiene le mismo curso temporal para todos los concatámeros pero se corre en el eje del potencial al neutralizar las cargas.
V1/2 mV valencia
4wt -34 5.32wt/2mut -53 2.41wt/3mut -65 1.9
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Fig 3
La inactivación lenta tiene le mismo curso temporal para todos los concatámeros.
Este resultado indica que la inactivación lenta resulta del la acción cooperativa de las compuertas de activación. Si una se mueve arrastra las otras.
Si no hubiese cooperatividad, la inactivación de 2wt/2mut y 1wt/3mut debería ser más rápida porque las compuertas mut estaría ya listas para inactivar
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Fig 8
The double mutation T449V-I470C in one wt subunit impairs slow inactivation in 1wt/3mut, but not in 4wt.
These results suggest that subunits bearing a neutral S4 segment were unable to go into the inactivated state by themselves.
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We sought to determine the contribution of an individual voltage sensor to Shaker’s function.
Concatenated heterotetramers of Shaker zH(6–46) wild type (wt) in combination with a neutral S4 segment Shaker mutant (mut)with stoichiometries 2wt/2mut and 1wt/3mut were studied and compared with the 4wt concatenated homotetramer.
A single charged voltage sensor is sufficient to open Shaker conductance withreduced delay (<1 ms) and at more hyperpolarized voltages compared with 4wt
In addition, the wt-like slow inactivation of 1wt/3mut was almost completely eliminated by mutations T449V-I470C in its single wt subunit, indicating that the subunits bearing a neutral S4 were unable to trigger slow inactivation.
Our results strongly suggest that a neutral S4 segment of Shaker’s subunit is voltage insensitive and its voltage sensor is in the activated position (i.e., ready for pore opening), and provide experimental support to the proposed model of independent voltage sensors with a final, almost voltage-independent concerted step.
Abstract
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