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Selectivity changes during activation of mutant Shaker potassium channels.

Zheng J, Sigworth FJ - J. Gen. Physiol. (1997)

Bottom Line: Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics.While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel.From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal-truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179-182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in approximately 40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of approximately 1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.

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Voltage dependence of mean dwell time in the open  state and the two substates. Data points at each voltage represents  the mean value, with the number of measurements marked in parentheses (except the data at −30 mV, which represents a single  measurement). The curves are the values predicted from the kinetic scheme In Fig. 9.
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Figure 8: Voltage dependence of mean dwell time in the open state and the two substates. Data points at each voltage represents the mean value, with the number of measurements marked in parentheses (except the data at −30 mV, which represents a single measurement). The curves are the values predicted from the kinetic scheme In Fig. 9.

Mentions: The activation of a Shaker potassium channel involves many intermediate transitions, each of which is voltage dependent and therefore involves charge movement (Bezanilla et al., 1994; Zagotta et al., 1994a). We were therefore interested in testing whether transitions between the conductance states in the SNS channel were voltage dependent. From single-channel recordings at various membrane potentials, we again used a set of three thresholds to measure the dwell times in each current level and to determine the exit state following each dwell; this is illustrated for tail currents in Fig. 7. We found that the dwell times at each conductance level are voltage-dependent, as shown in Fig. 8. The open time becomes shorter at hyperpolarized voltages, with a mean value of 50.5 ms at −120 mV. The dwell times at sub1 and sub2 have their maximum values of 5.3 and 9.4 ms at −100 and −120 mV, respectively. At more hyperpolarized and more depolarized voltages, the mean dwell time at each sublevel is smaller. There is also a class of brief closures that completely interrupt the current in the open state (see for example Fig. 7 A). These are also voltage dependent, having a smaller mean duration at more hyperpolarized voltages.


Selectivity changes during activation of mutant Shaker potassium channels.

Zheng J, Sigworth FJ - J. Gen. Physiol. (1997)

Voltage dependence of mean dwell time in the open  state and the two substates. Data points at each voltage represents  the mean value, with the number of measurements marked in parentheses (except the data at −30 mV, which represents a single  measurement). The curves are the values predicted from the kinetic scheme In Fig. 9.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233792&req=5

Figure 8: Voltage dependence of mean dwell time in the open state and the two substates. Data points at each voltage represents the mean value, with the number of measurements marked in parentheses (except the data at −30 mV, which represents a single measurement). The curves are the values predicted from the kinetic scheme In Fig. 9.
Mentions: The activation of a Shaker potassium channel involves many intermediate transitions, each of which is voltage dependent and therefore involves charge movement (Bezanilla et al., 1994; Zagotta et al., 1994a). We were therefore interested in testing whether transitions between the conductance states in the SNS channel were voltage dependent. From single-channel recordings at various membrane potentials, we again used a set of three thresholds to measure the dwell times in each current level and to determine the exit state following each dwell; this is illustrated for tail currents in Fig. 7. We found that the dwell times at each conductance level are voltage-dependent, as shown in Fig. 8. The open time becomes shorter at hyperpolarized voltages, with a mean value of 50.5 ms at −120 mV. The dwell times at sub1 and sub2 have their maximum values of 5.3 and 9.4 ms at −100 and −120 mV, respectively. At more hyperpolarized and more depolarized voltages, the mean dwell time at each sublevel is smaller. There is also a class of brief closures that completely interrupt the current in the open state (see for example Fig. 7 A). These are also voltage dependent, having a smaller mean duration at more hyperpolarized voltages.

Bottom Line: Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics.While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel.From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal-truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179-182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in approximately 40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of approximately 1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.

Show MeSH
Related in: MedlinePlus