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Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the result...

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Autores principales: Zhang, Yu-Yang, Robertson, Janice L., Gray, Daniel A., Palmer, Lawrence G.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234023/
https://www.ncbi.nlm.nih.gov/pubmed/15572348
http://dx.doi.org/10.1085/jgp.200409166
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author Zhang, Yu-Yang
Robertson, Janice L.
Gray, Daniel A.
Palmer, Lawrence G.
author_facet Zhang, Yu-Yang
Robertson, Janice L.
Gray, Daniel A.
Palmer, Lawrence G.
author_sort Zhang, Yu-Yang
collection PubMed
description Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport.
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spelling pubmed-22340232008-03-21 Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels Zhang, Yu-Yang Robertson, Janice L. Gray, Daniel A. Palmer, Lawrence G. J Gen Physiol Article Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport. The Rockefeller University Press 2004-12 /pmc/articles/PMC2234023/ /pubmed/15572348 http://dx.doi.org/10.1085/jgp.200409166 Text en Copyright © 2004, 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
Zhang, Yu-Yang
Robertson, Janice L.
Gray, Daniel A.
Palmer, Lawrence G.
Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels
title Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels
title_full Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels
title_fullStr Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels
title_full_unstemmed Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels
title_short Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels
title_sort carboxy-terminal determinants of conductance in inward-rectifier k channels
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234023/
https://www.ncbi.nlm.nih.gov/pubmed/15572348
http://dx.doi.org/10.1085/jgp.200409166
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