PKA mediates constitutive activation of CFTR in human sweat duct.
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.
Department of Pediatrics, UCSD School of Medicine, University of California, San Diego, 9500 Gilman Drive 0830, La Jolla, CA 92093, USA. mmr@ucsd.edu
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Reddy
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Quinton
D000328:Adult; D001427:Bacterial Toxins / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D006152:Cyclic GMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D013545:Sweat Glands / Q000378:metabolism*
D001427:Bacterial Toxins; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019319:Okadaic Acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP