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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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The model accounts for the sigmoidicity in wt and F401 mutants. The activation families for the three models, with the relevant transitions shown in the box, were scaled in time and amplitude as described in the text to assess the dependence of sigmoidal delay on pulse voltage. Current families were simulated at voltages shown. Compare these results with the experimental data in Fig. 10.
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Figure 20: The model accounts for the sigmoidicity in wt and F401 mutants. The activation families for the three models, with the relevant transitions shown in the box, were scaled in time and amplitude as described in the text to assess the dependence of sigmoidal delay on pulse voltage. Current families were simulated at voltages shown. Compare these results with the experimental data in Fig. 10.

Mentions: Sigmoidal activation kinetics are a cardinal feature of Shaker channel gating and, as shown in Fig. 10, they remain present in the F401 mutants. The amount of sigmoidicity, as defined earlier, and the way it varies with pulse potential is a sensitive means to assess the presence of a slow first reverse transition from the open state (Zagotta et al. 1994a). In Fig. 20, the models for the wt, F401L, and F401A that differ mainly in the rate of that transition are used to generate activation families that are scaled in amplitude and time, as described in the discussion of Fig. 10. The relative spacing of these traces gives a measure of sigmoidicity over the voltage ranges of −35 to +55 mV (wt model), −55 to +45 mV (F401L model), and −40 to +120 mV (F401A model). As in the experimental records, F401L and wt model simulations show the progression from lower to asymptotically higher sigmoidicity, which is a prediction for a forward biased cooperative step with the slow rate of leaving the open state. F401A simulations display nearly identical sigmoidicity over a wide voltage range characteristic of this mutant's currents.


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)

The model accounts for the sigmoidicity in wt and F401 mutants. The activation families for the three models, with the relevant transitions shown in the box, were scaled in time and amplitude as described in the text to assess the dependence of sigmoidal delay on pulse voltage. Current families were simulated at voltages shown. Compare these results with the experimental data in Fig. 10.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 20: The model accounts for the sigmoidicity in wt and F401 mutants. The activation families for the three models, with the relevant transitions shown in the box, were scaled in time and amplitude as described in the text to assess the dependence of sigmoidal delay on pulse voltage. Current families were simulated at voltages shown. Compare these results with the experimental data in Fig. 10.
Mentions: Sigmoidal activation kinetics are a cardinal feature of Shaker channel gating and, as shown in Fig. 10, they remain present in the F401 mutants. The amount of sigmoidicity, as defined earlier, and the way it varies with pulse potential is a sensitive means to assess the presence of a slow first reverse transition from the open state (Zagotta et al. 1994a). In Fig. 20, the models for the wt, F401L, and F401A that differ mainly in the rate of that transition are used to generate activation families that are scaled in amplitude and time, as described in the discussion of Fig. 10. The relative spacing of these traces gives a measure of sigmoidicity over the voltage ranges of −35 to +55 mV (wt model), −55 to +45 mV (F401L model), and −40 to +120 mV (F401A model). As in the experimental records, F401L and wt model simulations show the progression from lower to asymptotically higher sigmoidicity, which is a prediction for a forward biased cooperative step with the slow rate of leaving the open state. F401A simulations display nearly identical sigmoidicity over a wide voltage range characteristic of this mutant's currents.

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