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Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels
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....
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
1999
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230647/ https://www.ncbi.nlm.nih.gov/pubmed/10435999 |
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author | Kanevsky, Max Aldrich, Richard W. |
author_facet | Kanevsky, Max Aldrich, Richard W. |
author_sort | Kanevsky, Max |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2230647 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1999 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22306472008-04-22 Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels Kanevsky, Max Aldrich, Richard W. J Gen Physiol Original Article 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. The Rockefeller University Press 1999-08-01 /pmc/articles/PMC2230647/ /pubmed/10435999 Text en © 1999 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Kanevsky, Max Aldrich, Richard W. Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels |
title | Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels |
title_full | Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels |
title_fullStr | Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels |
title_full_unstemmed | Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels |
title_short | Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels |
title_sort | determinants of voltage-dependent gating and open-state stability in the s5 segment of shaker potassium channels |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230647/ https://www.ncbi.nlm.nih.gov/pubmed/10435999 |
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