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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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Alanine replacements in the S5 region and the steady state activation in Shaker. On the left are representative families recorded from inside-out patches containing the mutant channels indicated. Test steps were given in the voltage ranges shown under the traces in 10-mV increments. Tail voltage was −65 mV for all families. On the right, corresponding G(V) relations are shown for each mutant; curves fitted to the wt G(V) are included for comparison (shown as broken lines). For the F416A mutant, the mean of 12 experiments with its standard error is displayed. For the other mutants, combined data from three (F404A), two (L403A), two (S411A), and three (S412A) patches are shown. Thin lines through the data represent fits of the Boltzmann function raised to the fourth power (see methods), yielding the following estimates of apparent gating valence and midpoint of voltage-dependent transition: z = 3.81 e0 and V1/2 = −55.5 mV (F404A); z = 3.97 e0 and V1/2 = −51.1 mV (F416A); z = 3.86 e0 and V1/2 = −49.1 mV (L403A); z = 4.16 e0 and V1/2 = −50.4 mV (S411A); z = 4.10 e0 and V1/2 = −49.6 mV (S412A).
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Figure 4: Alanine replacements in the S5 region and the steady state activation in Shaker. On the left are representative families recorded from inside-out patches containing the mutant channels indicated. Test steps were given in the voltage ranges shown under the traces in 10-mV increments. Tail voltage was −65 mV for all families. On the right, corresponding G(V) relations are shown for each mutant; curves fitted to the wt G(V) are included for comparison (shown as broken lines). For the F416A mutant, the mean of 12 experiments with its standard error is displayed. For the other mutants, combined data from three (F404A), two (L403A), two (S411A), and three (S412A) patches are shown. Thin lines through the data represent fits of the Boltzmann function raised to the fourth power (see methods), yielding the following estimates of apparent gating valence and midpoint of voltage-dependent transition: z = 3.81 e0 and V1/2 = −55.5 mV (F404A); z = 3.97 e0 and V1/2 = −51.1 mV (F416A); z = 3.86 e0 and V1/2 = −49.1 mV (L403A); z = 4.16 e0 and V1/2 = −50.4 mV (S411A); z = 4.10 e0 and V1/2 = −49.6 mV (S412A).

Mentions: F401 is one of five phenylalanines in the Shaker S5 sequence (at positions 401, 402, 404, 410, and 416; see Fig. 1 C). To investigate whether other amino acid substitutions in S5 have similar effects on activation gating, we conducted alanine mutagenesis of the four phenylalanines downstream (towards the carboxyl terminus) of F401 as well as other S5 hydrophobic residues (leucines at positions 396, 398, 399, 403, and 409, and serines at positions 411 and 412), noting that it was an alanine substitution at F401 that resulted in the greatest effects (see below). Only F404A, F416A, L403A, S411A, and S412A gave rise to reliable ionic current expression. The results are shown in Fig. 4. The mutants' steady state activation voltage dependence shows few differences from the wt other than a small 1–10-mV depolarizing shift in most of the G(V) curves. The apparent valence of activation was not altered in any of the mutants. For L403A, these findings confirm earlier observations on channels with intact inactivation (Lopez et al. 1991; McCormack et al. 1991) that this residue, the fifth leucine in a putative heptad motif spanning the S4–S5 regions, plays at most a minor role in voltage-dependent gating. Results from the two serine substitutions imply that removal of the hydroxyl groups from the respective side chains does not alter the activation process.


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)

Alanine replacements in the S5 region and the steady state activation in Shaker. On the left are representative families recorded from inside-out patches containing the mutant channels indicated. Test steps were given in the voltage ranges shown under the traces in 10-mV increments. Tail voltage was −65 mV for all families. On the right, corresponding G(V) relations are shown for each mutant; curves fitted to the wt G(V) are included for comparison (shown as broken lines). For the F416A mutant, the mean of 12 experiments with its standard error is displayed. For the other mutants, combined data from three (F404A), two (L403A), two (S411A), and three (S412A) patches are shown. Thin lines through the data represent fits of the Boltzmann function raised to the fourth power (see methods), yielding the following estimates of apparent gating valence and midpoint of voltage-dependent transition: z = 3.81 e0 and V1/2 = −55.5 mV (F404A); z = 3.97 e0 and V1/2 = −51.1 mV (F416A); z = 3.86 e0 and V1/2 = −49.1 mV (L403A); z = 4.16 e0 and V1/2 = −50.4 mV (S411A); z = 4.10 e0 and V1/2 = −49.6 mV (S412A).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230647&req=5

Figure 4: Alanine replacements in the S5 region and the steady state activation in Shaker. On the left are representative families recorded from inside-out patches containing the mutant channels indicated. Test steps were given in the voltage ranges shown under the traces in 10-mV increments. Tail voltage was −65 mV for all families. On the right, corresponding G(V) relations are shown for each mutant; curves fitted to the wt G(V) are included for comparison (shown as broken lines). For the F416A mutant, the mean of 12 experiments with its standard error is displayed. For the other mutants, combined data from three (F404A), two (L403A), two (S411A), and three (S412A) patches are shown. Thin lines through the data represent fits of the Boltzmann function raised to the fourth power (see methods), yielding the following estimates of apparent gating valence and midpoint of voltage-dependent transition: z = 3.81 e0 and V1/2 = −55.5 mV (F404A); z = 3.97 e0 and V1/2 = −51.1 mV (F416A); z = 3.86 e0 and V1/2 = −49.1 mV (L403A); z = 4.16 e0 and V1/2 = −50.4 mV (S411A); z = 4.10 e0 and V1/2 = −49.6 mV (S412A).
Mentions: F401 is one of five phenylalanines in the Shaker S5 sequence (at positions 401, 402, 404, 410, and 416; see Fig. 1 C). To investigate whether other amino acid substitutions in S5 have similar effects on activation gating, we conducted alanine mutagenesis of the four phenylalanines downstream (towards the carboxyl terminus) of F401 as well as other S5 hydrophobic residues (leucines at positions 396, 398, 399, 403, and 409, and serines at positions 411 and 412), noting that it was an alanine substitution at F401 that resulted in the greatest effects (see below). Only F404A, F416A, L403A, S411A, and S412A gave rise to reliable ionic current expression. The results are shown in Fig. 4. The mutants' steady state activation voltage dependence shows few differences from the wt other than a small 1–10-mV depolarizing shift in most of the G(V) curves. The apparent valence of activation was not altered in any of the mutants. For L403A, these findings confirm earlier observations on channels with intact inactivation (Lopez et al. 1991; McCormack et al. 1991) that this residue, the fifth leucine in a putative heptad motif spanning the S4–S5 regions, plays at most a minor role in voltage-dependent gating. Results from the two serine substitutions imply that removal of the hydroxyl groups from the respective side chains does not alter the activation process.

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