Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity

Delphine Bichet, Yu-Fung Lin, Christian A. Ibarra, Cindy Shen Huang, B. Alexander Yi, Yuh Nung Jan, Lily Yeh Jan

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K+ channels allow K+ but not the smaller and more abundant Na + ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K + channel signature sequence, approximates the hydration shell of K+ ions. However, the K+ channel signature sequence is not sufficient for K+ selectivity. To identify structural elements important for K+ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K+ selectivity and hence the channel's ability to complement the K+ transport deficiency of Δtrk1Δtrk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K+ medium contain multiple suppressors, each partially restoring K+ selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K+ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.

Original languageEnglish (US)
Pages (from-to)4441-4446
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number13
DOIs
StatePublished - Mar 30 2004

Fingerprint

Potassium Channels
Yeasts
Mutation
G Protein-Coupled Inwardly-Rectifying Potassium Channels
Ion Channel Gating
Ions
Genetic Suppression
Heart Rate
Insulin
Growth

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity. / Bichet, Delphine; Lin, Yu-Fung; Ibarra, Christian A.; Huang, Cindy Shen; Yi, B. Alexander; Jan, Yuh Nung; Jan, Lily Yeh.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 13, 30.03.2004, p. 4441-4446.

Research output: Contribution to journalArticle

Bichet, Delphine ; Lin, Yu-Fung ; Ibarra, Christian A. ; Huang, Cindy Shen ; Yi, B. Alexander ; Jan, Yuh Nung ; Jan, Lily Yeh. / Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity. In: Proceedings of the National Academy of Sciences of the United States of America. 2004 ; Vol. 101, No. 13. pp. 4441-4446.
@article{747657ee5fd5454c90926388136a0e78,
title = "Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity",
abstract = "Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K+ channels allow K+ but not the smaller and more abundant Na + ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K + channel signature sequence, approximates the hydration shell of K+ ions. However, the K+ channel signature sequence is not sufficient for K+ selectivity. To identify structural elements important for K+ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K+ selectivity and hence the channel's ability to complement the K+ transport deficiency of Δtrk1Δtrk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K+ medium contain multiple suppressors, each partially restoring K+ selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K+ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.",
author = "Delphine Bichet and Yu-Fung Lin and Ibarra, {Christian A.} and Huang, {Cindy Shen} and Yi, {B. Alexander} and Jan, {Yuh Nung} and Jan, {Lily Yeh}",
year = "2004",
month = "3",
day = "30",
doi = "10.1073/pnas.0401195101",
language = "English (US)",
volume = "101",
pages = "4441--4446",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "13",

}

TY - JOUR

T1 - Evolving potassium channels by means of yeast selection reveals structural elements important for selectivity

AU - Bichet, Delphine

AU - Lin, Yu-Fung

AU - Ibarra, Christian A.

AU - Huang, Cindy Shen

AU - Yi, B. Alexander

AU - Jan, Yuh Nung

AU - Jan, Lily Yeh

PY - 2004/3/30

Y1 - 2004/3/30

N2 - Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K+ channels allow K+ but not the smaller and more abundant Na + ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K + channel signature sequence, approximates the hydration shell of K+ ions. However, the K+ channel signature sequence is not sufficient for K+ selectivity. To identify structural elements important for K+ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K+ selectivity and hence the channel's ability to complement the K+ transport deficiency of Δtrk1Δtrk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K+ medium contain multiple suppressors, each partially restoring K+ selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K+ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.

AB - Potassium channels are widely distributed. To serve their physiological functions, such as neuronal signaling, control of insulin release, and regulation of heart rate and blood flow, it is essential that K+ channels allow K+ but not the smaller and more abundant Na + ions to go through. The narrowest part of the channel pore, the selectivity filter formed by backbone carbonyls of the GYG-containing K + channel signature sequence, approximates the hydration shell of K+ ions. However, the K+ channel signature sequence is not sufficient for K+ selectivity. To identify structural elements important for K+ selectivity, we randomly mutagenized the G protein-coupled inwardly rectifying potassium channel 3.2 (GIRK2) bearing the S177W mutation on the second transmembrane segment. This mutation confers constitutive channel activity but abolishes K+ selectivity and hence the channel's ability to complement the K+ transport deficiency of Δtrk1Δtrk2 mutant yeast. S177W-containing GIRK2 mutants that support yeast growth in low-K+ medium contain multiple suppressors, each partially restoring K+ selectivity to S177W-containing double mutants. These suppressors include mutations in the first transmembrane segment and the pore helix, likely exerting long-range actions to restore K+ selectivity, as well as a mutation of a second transmembrane segment residue facing the cytoplasmic half of the pore, below the selectivity filter. Some of these suppressors also affected channel gating (channel open time and opening frequency determined in single-channel analyses), revealing intriguing interplay between ion permeation and channel gating.

UR - http://www.scopus.com/inward/record.url?scp=1842533080&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842533080&partnerID=8YFLogxK

U2 - 10.1073/pnas.0401195101

DO - 10.1073/pnas.0401195101

M3 - Article

VL - 101

SP - 4441

EP - 4446

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 13

ER -