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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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Model predictions for the pulse duration dependence of the OFF gating charge movement. Experimental records of wf (left), wfF401L (center), and wfF401A (right) gating currents obtained at −50 and 0 mV are shown as in Fig. 12. The fits of the corresponding models are shown overlaid (thin lines). Modifications of the models needed for these simulations were as follows (s−1): for wf, β0 = 153, δ0 = 14, and λ0 = 113 at 0 mV; for wfF401L, α0 = 1,150, δ0 = 11.5, and λ0 = 39 at −50 mV and β0 = 136 and δ0 = 11.5 at 0 mV; and for wfF401A, α0 = 1,200 and λ0 = 8,000 at both −50 and 0 mV.
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Figure 16: Model predictions for the pulse duration dependence of the OFF gating charge movement. Experimental records of wf (left), wfF401L (center), and wfF401A (right) gating currents obtained at −50 and 0 mV are shown as in Fig. 12. The fits of the corresponding models are shown overlaid (thin lines). Modifications of the models needed for these simulations were as follows (s−1): for wf, β0 = 153, δ0 = 14, and λ0 = 113 at 0 mV; for wfF401L, α0 = 1,150, δ0 = 11.5, and λ0 = 39 at −50 mV and β0 = 136 and δ0 = 11.5 at 0 mV; and for wfF401A, α0 = 1,200 and λ0 = 8,000 at both −50 and 0 mV.

Mentions: Inspection of the time course of the gating charge return as a function of pulse duration (Fig. 12) reveals important differences among the three channel species. Model fits to these data, shown in Fig. 16, indicate that our simulations are adequate to describe the time course of IgOFF for a variety of pulse durations at −50 and 0 mV. In particular, the observed slow decay of OFF gating currents observed in wfF401L at −50 mV at longer pulse durations as the consequence of significant open probability of the channels at that voltage is observed in simulated traces. The complex waveform of wf IgOFF after 0-mV pulses lasting ∼10 ms deviates somewhat from the predictions of our model and would probably be better fit with the introduction of additional steps in the activation pathway (see above). The model predicts that the gating charge return will be rapid and independent of pulse duration in wfF401A, which is clearly a feature of our data.


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)

Model predictions for the pulse duration dependence of the OFF gating charge movement. Experimental records of wf (left), wfF401L (center), and wfF401A (right) gating currents obtained at −50 and 0 mV are shown as in Fig. 12. The fits of the corresponding models are shown overlaid (thin lines). Modifications of the models needed for these simulations were as follows (s−1): for wf, β0 = 153, δ0 = 14, and λ0 = 113 at 0 mV; for wfF401L, α0 = 1,150, δ0 = 11.5, and λ0 = 39 at −50 mV and β0 = 136 and δ0 = 11.5 at 0 mV; and for wfF401A, α0 = 1,200 and λ0 = 8,000 at both −50 and 0 mV.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 16: Model predictions for the pulse duration dependence of the OFF gating charge movement. Experimental records of wf (left), wfF401L (center), and wfF401A (right) gating currents obtained at −50 and 0 mV are shown as in Fig. 12. The fits of the corresponding models are shown overlaid (thin lines). Modifications of the models needed for these simulations were as follows (s−1): for wf, β0 = 153, δ0 = 14, and λ0 = 113 at 0 mV; for wfF401L, α0 = 1,150, δ0 = 11.5, and λ0 = 39 at −50 mV and β0 = 136 and δ0 = 11.5 at 0 mV; and for wfF401A, α0 = 1,200 and λ0 = 8,000 at both −50 and 0 mV.
Mentions: Inspection of the time course of the gating charge return as a function of pulse duration (Fig. 12) reveals important differences among the three channel species. Model fits to these data, shown in Fig. 16, indicate that our simulations are adequate to describe the time course of IgOFF for a variety of pulse durations at −50 and 0 mV. In particular, the observed slow decay of OFF gating currents observed in wfF401L at −50 mV at longer pulse durations as the consequence of significant open probability of the channels at that voltage is observed in simulated traces. The complex waveform of wf IgOFF after 0-mV pulses lasting ∼10 ms deviates somewhat from the predictions of our model and would probably be better fit with the introduction of additional steps in the activation pathway (see above). The model predicts that the gating charge return will be rapid and independent of pulse duration in wfF401A, which is clearly a feature of our data.

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