Regulation of epithelial chloride channels by protein phosphatase

B. Q. La, S. L. Carosi, J. Valentich, S. Shenolikar, S. C. Sansom

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

A combination of planar bilayer and patch-clamp techniques was used to determine whether apical membrane Cl- channels of shark (Squalus acanthias) rectal gland (SRG) were regulated by a phosphorylating and dephosphorylating cycle. In channel reconstitution studies, apical membrane vesicles of SRG were purified, incubated in ATP-Mg2+ and the presence or absence (control) of catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAMP-PK) and incorporated into planar lipid bilayers. In the presence of cAMP-PK, two distinct Cl- channels were found when imposing either 450/50 or 300/50 mM KCl (cis/trans) gradients. The most frequently observed channels (G(β1)) were open >80% at all potentials between -60 and +20 mV (trans ground) and were inactivated by alkaline phosphatase added to the cis chamber. The single-channel conductance of G(β1) was 42 pS between -60 and +20 mV with a 300/50 mM KCl gradient. The second channel (G(β2) was always observed in pairs of 62-pS subchannels and was not affected by alkaline phosphatase, but the open probability increased with depolarizing potentials. G(β2) was observed once, but G(β1) was never observed in the absence of cAMP-PK. In parallel patch-clamp studies of the apical membrane of cultured SRG, a 50-pS channel similar to G(β1) was noted after incubating cells with either forskolin, an activator of adenylate cyclase, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A. It is concluded that G(β1) of SRG can be studied in both patch-clamp and bilayer preparations and that G(β1) is regulated by reversible phosphorylation by cAMP-PK and dephosphorylation by a protein phosphatase.

Original languageEnglish (US)
Pages (from-to)C1217-C1223
JournalAmerican Journal of Physiology - Cell Physiology
Volume260
Issue number6 29/6
DOIs
StatePublished - 1991

Keywords

  • Okadaic acid
  • Patch clamp
  • Planar lipid bilayers
  • Shark rectal gland

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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