Regulation of epithelial chloride channels by protein phosphatase.
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 beta 1) were open greater than 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 beta 1 was 42 pS between -60 and +20 mV with a 300/50 mM KCl gradient. The second channel (G beta 2) 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 beta 2 was observed once, but G beta 1 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 beta 1 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 beta 1 of SRG can be studied in both patch-clamp and bilayer preparations and that G beta 1 is regulated by reversible phosphorylation by cAMP-PK and dephosphorylation by a protein phosphatase.
La
B Q
BQ
Department of Medicine, University of Texas Medical School, Houston 77225.
Carosi
S L
SL
Valentich
J
J
Shenolikar
S
S
Sansom
S C
SC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D004284:Dogfish; D004988:Ethers, Cyclic / Q000494:pharmacology; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism / Q000494:pharmacology; D012491:Salt Gland / Q000502:physiology
D018118:Chloride Channels; D002712:Chlorides; D004988:Ethers, Cyclic; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D005576:Colforsin; D019319:Okadaic Acid; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases