Mechanism and Molecular Determinant for Regulation of Rabbit Transient Receptor Potential Type 5 (TRPV5) Channel by Extracellular pH

Byung Il Yeh, Tie Jun Sun, Jason Z. Lee, Hsi Hsien Chen, Chou Long Huang

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa ∼6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular acidification Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a "pH sensor," and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.

Original languageEnglish (US)
Pages (from-to)51044-51052
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number51
DOIs
StatePublished - Dec 19 2003

Fingerprint

Acidification
Glutamic Acid
Rabbits
Glutamine
Amino Acids
Protons
pH sensors
Protein Conformation
Mutation
Clamping devices
Sulfhydryl Compounds
Aspartic Acid
Reaction rates
Intestines
Cysteine
Conformations
Rate constants
Substitution reactions
Tissue
Calcium

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism and Molecular Determinant for Regulation of Rabbit Transient Receptor Potential Type 5 (TRPV5) Channel by Extracellular pH. / Yeh, Byung Il; Sun, Tie Jun; Lee, Jason Z.; Chen, Hsi Hsien; Huang, Chou Long.

In: Journal of Biological Chemistry, Vol. 278, No. 51, 19.12.2003, p. 51044-51052.

Research output: Contribution to journalArticle

@article{c0bc24e72aab432cb11fb8ad80b8a43e,
title = "Mechanism and Molecular Determinant for Regulation of Rabbit Transient Receptor Potential Type 5 (TRPV5) Channel by Extracellular pH",
abstract = "The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa ∼6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular acidification Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a {"}pH sensor,{"} and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.",
author = "Yeh, {Byung Il} and Sun, {Tie Jun} and Lee, {Jason Z.} and Chen, {Hsi Hsien} and Huang, {Chou Long}",
year = "2003",
month = "12",
day = "19",
doi = "10.1074/jbc.M306326200",
language = "English (US)",
volume = "278",
pages = "51044--51052",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "51",

}

TY - JOUR

T1 - Mechanism and Molecular Determinant for Regulation of Rabbit Transient Receptor Potential Type 5 (TRPV5) Channel by Extracellular pH

AU - Yeh, Byung Il

AU - Sun, Tie Jun

AU - Lee, Jason Z.

AU - Chen, Hsi Hsien

AU - Huang, Chou Long

PY - 2003/12/19

Y1 - 2003/12/19

N2 - The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa ∼6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular acidification Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a "pH sensor," and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.

AB - The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa ∼6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular acidification Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a "pH sensor," and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.

UR - http://www.scopus.com/inward/record.url?scp=0346435094&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0346435094&partnerID=8YFLogxK

U2 - 10.1074/jbc.M306326200

DO - 10.1074/jbc.M306326200

M3 - Article

VL - 278

SP - 51044

EP - 51052

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 51

ER -