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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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Alterations in the kinetics and voltage dependence of the closing transitions associated with the F401I mutation. (A and B) Deactivation families from wt (A) and F401I (B) patches were obtained with brief (8–10 ms) depolarizations to +50 mV, followed by steps to voltages between −60 and −160 mV in 10-mV increments, as indicated schematically above the traces. (C) Relaxations of the tail currents were fitted with a single exponential function (see methods). Means and standard errors of deactivation time constants, τ, from the fits are plotted against tail potential for patches containing wt (n = 8) and F401I (n = 6) channels. Average τ vs. voltage curves were computed and fitted with an exponential over the voltage range below −60 mV. Fits are shown as solid (F401I) and broken (wt) lines. The apparent charge associated with reverse transitions, zr, was calculated from the steepness of the exponential voltage dependence of τ. zr for the wt is 1.30 e0 whereas zr for F401I is 0.68 e0. (Inset) Shown are superimposed wt and mutant tail currents recorded at −60 (left) and −160 (right) mV. The currents were scaled to match their peak amplitudes during the +50-mV prepulse. Note that the time scales are different so as to enable comparison of kinetic detail for the two species at both voltage extremes.
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Figure 3: Alterations in the kinetics and voltage dependence of the closing transitions associated with the F401I mutation. (A and B) Deactivation families from wt (A) and F401I (B) patches were obtained with brief (8–10 ms) depolarizations to +50 mV, followed by steps to voltages between −60 and −160 mV in 10-mV increments, as indicated schematically above the traces. (C) Relaxations of the tail currents were fitted with a single exponential function (see methods). Means and standard errors of deactivation time constants, τ, from the fits are plotted against tail potential for patches containing wt (n = 8) and F401I (n = 6) channels. Average τ vs. voltage curves were computed and fitted with an exponential over the voltage range below −60 mV. Fits are shown as solid (F401I) and broken (wt) lines. The apparent charge associated with reverse transitions, zr, was calculated from the steepness of the exponential voltage dependence of τ. zr for the wt is 1.30 e0 whereas zr for F401I is 0.68 e0. (Inset) Shown are superimposed wt and mutant tail currents recorded at −60 (left) and −160 (right) mV. The currents were scaled to match their peak amplitudes during the +50-mV prepulse. Note that the time scales are different so as to enable comparison of kinetic detail for the two species at both voltage extremes.

Mentions: Whereas the voltage dependence of the forward rates and, therefore, the amount of charge movement before the transition state, appears unaffected by the F401I mutation, the voltage dependence of the closing (deactivation) transitions, reflecting the charge movement “after” the transition state, is very sensitive to this change. The kinetics of deactivation were studied from currents recorded during channel closing (tail currents) at hyperpolarized potentials (negative to −60 mV) after maximally activating prepulses (Fig. 3A and Fig. B). Deactivation follows a nearly single-exponential time course in both channels, with the time constants from the fits displayed in Fig. 3 C against tail voltage. wt tail currents are not simply slower compared with F401I; the difference is greatest at −60 mV, but diminishes at very negative voltages and largely disappears below −160 mV (Fig. 3 C, inset). Kinetics of the tail currents in the wt are steeply potential dependent, with the apparent charge associated with the backward transitions, zr , of 1.2 e0, consistent with a previous report (Zagotta et al. 1994a). This number may be an overestimate of the actual charge associated with the rate of any one individual backward transition because of the tendency of channels to reopen in a voltage-sensitive fashion at all but the most negative tail voltages (Schoppa and Sigworth 1998a). In contrast to the wt, the apparent valence derived from voltage dependence of tail time constants in F401I is only 0.68 e0. Thus, while the F401I mutant appears to move roughly the same amount of charge during the forward transitions late in the activation process, the mutation nearly halves the apparent charge movement associated with the early backward transitions. Therefore, the dominant effect on the kinetics of the F401I channels' return to the closed state is the speeding of the tail currents over all but the most negative voltages at which the determination of the tail time constant can become limited by the clamp response time. Our ability to observe deactivation in the absence of superimposed fast N-type inactivation allows us to study reverse transitions in relative isolation from other kinetic processes in the channel. Our results lend direct support to the earlier proposal by Zagotta and Aldrich 1990b that the Sh5 mutation affects the magnitude and voltage dependence of the reverse rate.


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)

Alterations in the kinetics and voltage dependence of the closing transitions associated with the F401I mutation. (A and B) Deactivation families from wt (A) and F401I (B) patches were obtained with brief (8–10 ms) depolarizations to +50 mV, followed by steps to voltages between −60 and −160 mV in 10-mV increments, as indicated schematically above the traces. (C) Relaxations of the tail currents were fitted with a single exponential function (see methods). Means and standard errors of deactivation time constants, τ, from the fits are plotted against tail potential for patches containing wt (n = 8) and F401I (n = 6) channels. Average τ vs. voltage curves were computed and fitted with an exponential over the voltage range below −60 mV. Fits are shown as solid (F401I) and broken (wt) lines. The apparent charge associated with reverse transitions, zr, was calculated from the steepness of the exponential voltage dependence of τ. zr for the wt is 1.30 e0 whereas zr for F401I is 0.68 e0. (Inset) Shown are superimposed wt and mutant tail currents recorded at −60 (left) and −160 (right) mV. The currents were scaled to match their peak amplitudes during the +50-mV prepulse. Note that the time scales are different so as to enable comparison of kinetic detail for the two species at both voltage extremes.
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Related In: Results  -  Collection

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Figure 3: Alterations in the kinetics and voltage dependence of the closing transitions associated with the F401I mutation. (A and B) Deactivation families from wt (A) and F401I (B) patches were obtained with brief (8–10 ms) depolarizations to +50 mV, followed by steps to voltages between −60 and −160 mV in 10-mV increments, as indicated schematically above the traces. (C) Relaxations of the tail currents were fitted with a single exponential function (see methods). Means and standard errors of deactivation time constants, τ, from the fits are plotted against tail potential for patches containing wt (n = 8) and F401I (n = 6) channels. Average τ vs. voltage curves were computed and fitted with an exponential over the voltage range below −60 mV. Fits are shown as solid (F401I) and broken (wt) lines. The apparent charge associated with reverse transitions, zr, was calculated from the steepness of the exponential voltage dependence of τ. zr for the wt is 1.30 e0 whereas zr for F401I is 0.68 e0. (Inset) Shown are superimposed wt and mutant tail currents recorded at −60 (left) and −160 (right) mV. The currents were scaled to match their peak amplitudes during the +50-mV prepulse. Note that the time scales are different so as to enable comparison of kinetic detail for the two species at both voltage extremes.
Mentions: Whereas the voltage dependence of the forward rates and, therefore, the amount of charge movement before the transition state, appears unaffected by the F401I mutation, the voltage dependence of the closing (deactivation) transitions, reflecting the charge movement “after” the transition state, is very sensitive to this change. The kinetics of deactivation were studied from currents recorded during channel closing (tail currents) at hyperpolarized potentials (negative to −60 mV) after maximally activating prepulses (Fig. 3A and Fig. B). Deactivation follows a nearly single-exponential time course in both channels, with the time constants from the fits displayed in Fig. 3 C against tail voltage. wt tail currents are not simply slower compared with F401I; the difference is greatest at −60 mV, but diminishes at very negative voltages and largely disappears below −160 mV (Fig. 3 C, inset). Kinetics of the tail currents in the wt are steeply potential dependent, with the apparent charge associated with the backward transitions, zr , of 1.2 e0, consistent with a previous report (Zagotta et al. 1994a). This number may be an overestimate of the actual charge associated with the rate of any one individual backward transition because of the tendency of channels to reopen in a voltage-sensitive fashion at all but the most negative tail voltages (Schoppa and Sigworth 1998a). In contrast to the wt, the apparent valence derived from voltage dependence of tail time constants in F401I is only 0.68 e0. Thus, while the F401I mutant appears to move roughly the same amount of charge during the forward transitions late in the activation process, the mutation nearly halves the apparent charge movement associated with the early backward transitions. Therefore, the dominant effect on the kinetics of the F401I channels' return to the closed state is the speeding of the tail currents over all but the most negative voltages at which the determination of the tail time constant can become limited by the clamp response time. Our ability to observe deactivation in the absence of superimposed fast N-type inactivation allows us to study reverse transitions in relative isolation from other kinetic processes in the channel. Our results lend direct support to the earlier proposal by Zagotta and Aldrich 1990b that the Sh5 mutation affects the magnitude and voltage dependence of the reverse rate.

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