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Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

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Kinetics and voltage dependence of forward transitions in the wild-type Shaker channel and the F401I mutant. (A) The time course of activation was compared in current traces obtained as in Fig. 1, with pulse voltages indicated on the right. Currents from wild-type and mutant channels were scaled to match maximal amplitudes at each of three voltage levels and fit with a single exponential function beginning at the time point corresponding to half-maximal current amplitude (see methods). Fits are superimposed on the traces as dotted curves. (B) Voltage dependence of the activation time constant. Values of the time constant, τ, derived from single-exponential fits from a number of patches (wt: n = 7; F401I: n = 5) were averaged and plotted against pulse voltage on semilogarithmic axes. Error bars represent the standard error of the mean. The time constant versus voltage relation was fit with a decaying exponential function above −10 mV, shown as solid (F401I) and dashed (wt) lines. The apparent charge associated with forward transitions late in activation, zf, was calculated from the slope of the fitted line, and found to be 0.36 e0 for the wt and 0.31 e0 for F401I. (C) Difference in activation delay between wt and mutant currents. Sets of representative traces recorded from two patches different from those in A are shown, with pulse voltage to the right of the traces. Current amplitudes for the +20-mV traces in each family were set to unity. Arrows indicate the time points at which current amplitudes are half-maximal. (D) As a way to quantify the absolute activation delay, the mean time to reach half-maximum is displayed against pulse voltage for wt (n = 8) and F401I (n = 5) currents. The standard error of the mean is shown as error bars when it exceeds the size of a symbol.
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Figure 2: Kinetics and voltage dependence of forward transitions in the wild-type Shaker channel and the F401I mutant. (A) The time course of activation was compared in current traces obtained as in Fig. 1, with pulse voltages indicated on the right. Currents from wild-type and mutant channels were scaled to match maximal amplitudes at each of three voltage levels and fit with a single exponential function beginning at the time point corresponding to half-maximal current amplitude (see methods). Fits are superimposed on the traces as dotted curves. (B) Voltage dependence of the activation time constant. Values of the time constant, τ, derived from single-exponential fits from a number of patches (wt: n = 7; F401I: n = 5) were averaged and plotted against pulse voltage on semilogarithmic axes. Error bars represent the standard error of the mean. The time constant versus voltage relation was fit with a decaying exponential function above −10 mV, shown as solid (F401I) and dashed (wt) lines. The apparent charge associated with forward transitions late in activation, zf, was calculated from the slope of the fitted line, and found to be 0.36 e0 for the wt and 0.31 e0 for F401I. (C) Difference in activation delay between wt and mutant currents. Sets of representative traces recorded from two patches different from those in A are shown, with pulse voltage to the right of the traces. Current amplitudes for the +20-mV traces in each family were set to unity. Arrows indicate the time points at which current amplitudes are half-maximal. (D) As a way to quantify the absolute activation delay, the mean time to reach half-maximum is displayed against pulse voltage for wt (n = 8) and F401I (n = 5) currents. The standard error of the mean is shown as error bars when it exceeds the size of a symbol.

Mentions: If we consider a voltage-sensitive transition with associated charge displacement z in terms of transition-state theory, the voltage dependence of the forward and backward rates is determined by the charge movement before and after the transition state, respectively, and need not be equal. We asked if the diminished voltage dependence of the F401I mutant is associated primarily with forward or reverse transitions. A method to assess the forward rates in relative isolation from the backward transitions is illustrated in Fig. 2. The currents from wt and F401I channels activate with a sigmoidal delay, reflecting a multistep opening process. As the test potential is stepped to more positive values, channel opening kinetics accelerate for both the wt and F401I families. With sufficiently depolarizing voltage steps (i.e., more positive than −10 mV where the probability of opening for both channels nears saturation), the reverse rates can be considered negligible and the kinetics of activation are almost entirely determined by the forward rates. In this voltage range, the time course of activation has a complex multiexponential behavior but, for a class of models commonly used to describe Shaker gating, the slowest exponential component has a time constant that is the inverse of the slowest forward rate (Zagotta et al. 1994a; Schoppa and Sigworth 1998c). We find that a good single-exponential fit can be obtained to the latter phase of the trace beginning with the time at which currents reach their half-maximal amplitude (Fig. 2 A). The F401I mutant activates more rapidly and with less sigmoid delay than the wild type. Time constants are voltage dependent, but the deduced amount of charge moved for these forward transitions is small and essentially unchanged between the wt and F401I channels (Fig. 2 B): 0.36 (see also Zagotta et al. 1994a) and 0.31 e0, respectively. F401I also produces a consistent decrease in the time-to-half-maximum current over the depolarized voltage ranges (Fig. 2C and Fig. D).


Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Kinetics and voltage dependence of forward transitions in the wild-type Shaker channel and the F401I mutant. (A) The time course of activation was compared in current traces obtained as in Fig. 1, with pulse voltages indicated on the right. Currents from wild-type and mutant channels were scaled to match maximal amplitudes at each of three voltage levels and fit with a single exponential function beginning at the time point corresponding to half-maximal current amplitude (see methods). Fits are superimposed on the traces as dotted curves. (B) Voltage dependence of the activation time constant. Values of the time constant, τ, derived from single-exponential fits from a number of patches (wt: n = 7; F401I: n = 5) were averaged and plotted against pulse voltage on semilogarithmic axes. Error bars represent the standard error of the mean. The time constant versus voltage relation was fit with a decaying exponential function above −10 mV, shown as solid (F401I) and dashed (wt) lines. The apparent charge associated with forward transitions late in activation, zf, was calculated from the slope of the fitted line, and found to be 0.36 e0 for the wt and 0.31 e0 for F401I. (C) Difference in activation delay between wt and mutant currents. Sets of representative traces recorded from two patches different from those in A are shown, with pulse voltage to the right of the traces. Current amplitudes for the +20-mV traces in each family were set to unity. Arrows indicate the time points at which current amplitudes are half-maximal. (D) As a way to quantify the absolute activation delay, the mean time to reach half-maximum is displayed against pulse voltage for wt (n = 8) and F401I (n = 5) currents. The standard error of the mean is shown as error bars when it exceeds the size of a symbol.
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Related In: Results  -  Collection

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Figure 2: Kinetics and voltage dependence of forward transitions in the wild-type Shaker channel and the F401I mutant. (A) The time course of activation was compared in current traces obtained as in Fig. 1, with pulse voltages indicated on the right. Currents from wild-type and mutant channels were scaled to match maximal amplitudes at each of three voltage levels and fit with a single exponential function beginning at the time point corresponding to half-maximal current amplitude (see methods). Fits are superimposed on the traces as dotted curves. (B) Voltage dependence of the activation time constant. Values of the time constant, τ, derived from single-exponential fits from a number of patches (wt: n = 7; F401I: n = 5) were averaged and plotted against pulse voltage on semilogarithmic axes. Error bars represent the standard error of the mean. The time constant versus voltage relation was fit with a decaying exponential function above −10 mV, shown as solid (F401I) and dashed (wt) lines. The apparent charge associated with forward transitions late in activation, zf, was calculated from the slope of the fitted line, and found to be 0.36 e0 for the wt and 0.31 e0 for F401I. (C) Difference in activation delay between wt and mutant currents. Sets of representative traces recorded from two patches different from those in A are shown, with pulse voltage to the right of the traces. Current amplitudes for the +20-mV traces in each family were set to unity. Arrows indicate the time points at which current amplitudes are half-maximal. (D) As a way to quantify the absolute activation delay, the mean time to reach half-maximum is displayed against pulse voltage for wt (n = 8) and F401I (n = 5) currents. The standard error of the mean is shown as error bars when it exceeds the size of a symbol.
Mentions: If we consider a voltage-sensitive transition with associated charge displacement z in terms of transition-state theory, the voltage dependence of the forward and backward rates is determined by the charge movement before and after the transition state, respectively, and need not be equal. We asked if the diminished voltage dependence of the F401I mutant is associated primarily with forward or reverse transitions. A method to assess the forward rates in relative isolation from the backward transitions is illustrated in Fig. 2. The currents from wt and F401I channels activate with a sigmoidal delay, reflecting a multistep opening process. As the test potential is stepped to more positive values, channel opening kinetics accelerate for both the wt and F401I families. With sufficiently depolarizing voltage steps (i.e., more positive than −10 mV where the probability of opening for both channels nears saturation), the reverse rates can be considered negligible and the kinetics of activation are almost entirely determined by the forward rates. In this voltage range, the time course of activation has a complex multiexponential behavior but, for a class of models commonly used to describe Shaker gating, the slowest exponential component has a time constant that is the inverse of the slowest forward rate (Zagotta et al. 1994a; Schoppa and Sigworth 1998c). We find that a good single-exponential fit can be obtained to the latter phase of the trace beginning with the time at which currents reach their half-maximal amplitude (Fig. 2 A). The F401I mutant activates more rapidly and with less sigmoid delay than the wild type. Time constants are voltage dependent, but the deduced amount of charge moved for these forward transitions is small and essentially unchanged between the wt and F401I channels (Fig. 2 B): 0.36 (see also Zagotta et al. 1994a) and 0.31 e0, respectively. F401I also produces a consistent decrease in the time-to-half-maximum current over the depolarized voltage ranges (Fig. 2C and Fig. D).

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Show MeSH
Related in: MedlinePlus