cAMP sensitivity conferred to the epithelial Na+ channel by alpha-subunit cloned from guinea-pig colon

The rate of Na+ (re)absorption across tight epithelia such as in distal kid ney nephron and colon is to a large extent controlled at the level of the e pithelial Na+ channel (ENaC). In kidney, antidiuretic hormone (ADH, vasopre ssin) stimulates the expression/activity of this channel by a cAMP/protein- kinase-A- (PKA-) mediated pathway. However, a clear upregulation of ENaC fu nction by cAMP could not be reproduced with cloned channel subunits in the Xenopus oocyte expression system, suggesting the hypothesis that an additio nal factor is missing. In contrast, we show here that membrane-permeant cAM P can activate ENaC expressed in Xenopus oocytes (3.8-fold) upon replacemen t of the rat alpha-subunit by a new a-subunit cloned from guinea-pig colon (gp alpha). This a-subunit is 76% identical with its rat orthologue origina ting from ADH-insensitive rat colon. The biophysical fingerprints of the hy brid ENaC formed by this guinea-pig alpha-subunit together with rat beta- a nd gamma-subunits are indistinguishable from those of rat ENaC (rENaC). Inj ection of the PKA inhibitor PKI-(6-22)amide into the oocyte had no effect o n the basal activity of rat ENaC but inhibited the activity of gpa-containi ng hybrid ENaC and greatly decreased its stimulation by cAMP. This suggests that, unlike for rat ENaC, tonic PKA activity is required for basal functi on of gpa-containing ENaC and that PKA mediates its cAMP-induced activation . This regulatory behaviour is not common to all ENaCs containing an a-subu nit cloned from an ADH-responsive tissue since xENaC, which was cloned from the ADH-sensitive Xenopus laevis A6 epithelia, is, when expressed in oocyt es, resistant to cAMP, similar to rat ENaC. This study demonstrates that th e PKA sensitivity of ENaC can depend on the nature of the ENaC alpha-sub-un it and raises the possibility that cAMP can stimulate ENaCs by different me chanisms.