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Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels
Inhibition of N- (Ca(v)2.2) and P/Q-type (Ca(v)2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channe...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2003
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217353/ https://www.ncbi.nlm.nih.gov/pubmed/12771191 http://dx.doi.org/10.1085/jgp.200208770 |
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author | Agler, Heather L. Evans, Jenafer Colecraft, Henry M. Yue, David T. |
author_facet | Agler, Heather L. Evans, Jenafer Colecraft, Henry M. Yue, David T. |
author_sort | Agler, Heather L. |
collection | PubMed |
description | Inhibition of N- (Ca(v)2.2) and P/Q-type (Ca(v)2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gβγ complex (Gβγ). Far less is known about the important interaction sites on Gβγ itself. Here, we developed a novel electrophysiological paradigm, “compound-state willing-reluctant analysis,” to describe Gβγ interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gβγ with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gβ(1), at residues known to interact with Gα. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gβγ for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for “steering” or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel. |
format | Text |
id | pubmed-2217353 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2003 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22173532008-04-16 Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels Agler, Heather L. Evans, Jenafer Colecraft, Henry M. Yue, David T. J Gen Physiol Article Inhibition of N- (Ca(v)2.2) and P/Q-type (Ca(v)2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gβγ complex (Gβγ). Far less is known about the important interaction sites on Gβγ itself. Here, we developed a novel electrophysiological paradigm, “compound-state willing-reluctant analysis,” to describe Gβγ interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gβγ with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gβ(1), at residues known to interact with Gα. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gβγ for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for “steering” or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel. The Rockefeller University Press 2003-06 /pmc/articles/PMC2217353/ /pubmed/12771191 http://dx.doi.org/10.1085/jgp.200208770 Text en Copyright © 2003, 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 | Article Agler, Heather L. Evans, Jenafer Colecraft, Henry M. Yue, David T. Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels |
title | Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels |
title_full | Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels |
title_fullStr | Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels |
title_full_unstemmed | Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels |
title_short | Custom Distinctions in the Interaction of G-protein β Subunits with N-type (Ca(V)2.2) Versus P/Q-type (Ca(V)2.1) Calcium Channels |
title_sort | custom distinctions in the interaction of g-protein β subunits with n-type (ca(v)2.2) versus p/q-type (ca(v)2.1) calcium channels |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217353/ https://www.ncbi.nlm.nih.gov/pubmed/12771191 http://dx.doi.org/10.1085/jgp.200208770 |
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