Functional analysis of acid-activated Cl- channels: Properties and mechanisms of regulation

Valeria Capurro, Ambra Gianotti, Emanuela Caci, Roberto Ravazzolo, Luis J V Galietta, Olga Zegarra-Moran

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Cl- channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acidactivated Cl- current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK- 293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH 5.15), permeability sequence (SCN- > I- > Br- > Cl- > gluconate), voltage dependence and sensitivity to blockers of ICl(H)were identical in all cells. These findings suggest a common molecular basis for ICl(H). Weanalysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR,we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca2+, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Cl- channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.

Original languageEnglish
Pages (from-to)105-114
Number of pages10
JournalBBA - Biomembranes
Issue numberPart A
DOIs
Publication statusPublished - 2015

Fingerprint

Functional analysis
Chemical activation
Phosphatidylinositol 3-Kinase
Dynamins
Phosphorylation
Acids
Genistein
Protein-Tyrosine Kinases
Proteins
Phosphotransferases
Genes
Adenosine Triphosphate
Cells
HEK293 Cells
Electric potential
Gene Silencing
Endocytosis
Neuroblastoma
Permeability
Epithelial Cells

Keywords

  • DIDS
  • Dynamin
  • Patch-clamp
  • pH
  • Phosphatidylinositide-3-kinase
  • Tyrosine-protein kinase

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics
  • Medicine(all)

Cite this

Functional analysis of acid-activated Cl- channels : Properties and mechanisms of regulation. / Capurro, Valeria; Gianotti, Ambra; Caci, Emanuela; Ravazzolo, Roberto; Galietta, Luis J V; Zegarra-Moran, Olga.

In: BBA - Biomembranes, No. Part A, 2015, p. 105-114.

Research output: Contribution to journalArticle

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T2 - Properties and mechanisms of regulation

AU - Capurro, Valeria

AU - Gianotti, Ambra

AU - Caci, Emanuela

AU - Ravazzolo, Roberto

AU - Galietta, Luis J V

AU - Zegarra-Moran, Olga

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N2 - Cl- channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acidactivated Cl- current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK- 293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH 5.15), permeability sequence (SCN- > I- > Br- > Cl- > gluconate), voltage dependence and sensitivity to blockers of ICl(H)were identical in all cells. These findings suggest a common molecular basis for ICl(H). Weanalysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR,we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca2+, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Cl- channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.

AB - Cl- channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acidactivated Cl- current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK- 293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH 5.15), permeability sequence (SCN- > I- > Br- > Cl- > gluconate), voltage dependence and sensitivity to blockers of ICl(H)were identical in all cells. These findings suggest a common molecular basis for ICl(H). Weanalysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR,we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca2+, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Cl- channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.

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KW - Tyrosine-protein kinase

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