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 language | English (US) |
|---|---|
| Pages (from-to) | 4441-4446 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 101 |
| Issue number | 13 |
| DOIs | |
| State | Published - Mar 30 2004 |
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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 journal › Article
}
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.
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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 -