Mechanoregulation of BK channel activity in the mammalian cortical collecting duct: Role of protein kinases A and C

Flow-stimulated net K secretion (JK) 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 α-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 (∼1) and fast (∼5 nl·min^-1 · 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.