Abstract
Flow-stimulated net K secretion (J(K)) in the cortical collecting duct (CCD) is mediated by an iberiotoxin (IBX)-sensitive BK channel, and requires an increase in intracellular Ca2+ concentration ([Ca2+](i)). The alpha-subunit of the reconstituted BK channel is phosphorylated by PKA and PKC. To test whether the BK channel in the native CCD is regulated by these kinases, J(K) and net Na absorption (J(Na)) were measured at slow (approximately 1) and fast (approximately 5 nl x min(-1) x mm(-1)) flow rates in rabbit CCDs microperfused in the presence of mPKI, an inhibitor of PKA; calphostin C, which inhibits diacylglycerol binding proteins, including PKC; or bisindolylmaleimide (BIM) and Gö6976, inhibitors of classic and novel PKC isoforms, added to luminal (L) and/or basolateral (B) solutions. L but not B mPKI increased J(K) in CCDs perfused at a slow flow rate; a subsequent increase in flow rate augmented J(K) modestly. B mPKI alone or with L inhibitor abolished flow stimulation of J(K). Similarly, L calphostin C increased J(K) in CCDs perfused at slow flow rates, as did calphostin C in both L and B solutions. The observation that IBX inhibited the L mPKI- and calphostin C-mediated increases in J(K) at slow flow rates implicated the BK channel in this K flux, a notion suggested by patch-clamp analysis of principal cells. The kinase inhibited by calphostin C was not PKC as L and/or B BIM and Gö6976 failed to enhance J(K) at the slow flow rate. However, addition of these PKC inhibitors to the B solution alone or with L inhibitor blocked flow stimulation of J(K). Interpretation of these results in light of the effects of these inhibitors on the flow-induced elevation of [Ca2+](i) suggests that the principal cell apical BK channel is tonically inhibited by PKA and that flow stimulation of J(K) in the CCD is PKA and PKC dependent. The specific targets of the kinases remain to be identified.