ENaC in Renal Cortical Collecting Duct Principal Cells Is Stimulated by Increasing Intracellular Ca2+ in The Basal Compartment of The Cell Via a Process Involving Src but not CAMK II

Abstract

We have recently shown that intracellular Ca2+ ([Ca2+]i) in renal cortical collecting duct (CCD) principal cells is compartmentalized and that this compartmentalization is maintained by mitochondria which localize in two separate areas beneath the apical and basolateral membranes and sequester Ca2+ to greatly reduce the rate of cytosolic Ca2+ diffusion. When [Ca2+]i is increased in the cytosolic space adjacent to the apical plasma membrane, ENaC is inhibited, but when [Ca2+]i is increased in the cytosolic space adjacent to the basolateral membrane, ENaC activity is stimulated. While the pathways mediating the effects of apical [Ca2+]i are well characterized, the pathways that transduce the basolateral pathways are unknown. We tested which signaling molecules may be mediating these effects. We used a combination of transepithelial current measurements and single channel patch clamping to identify these molecules. Inhibitors of common Ca2+ ‐sensitive signaling molecules were used in combination with either ionomycin or methyl‐thio‐ATP applied basolaterally to increase [Ca2+]i. Since some basolaterally expressed receptor tyrosine kinases stimulate ENaC via activation of Src and since Src is a Ca2+ ‐sensitive protein, we first tested whether basolateral [Ca2+]i stimulates ENaC via a Src‐mediated pathway. Basolaterally applied ionomycin (15 μM) or methyl‐thio‐ATP (50 μM), causes a two‐fold increase in ENaC open probability (N≥6; P<0.05 per group), but has no effect in the presence of the Src inhibitor PP2 (12 μM; N=6; P>0.5). We also considered another Ca2+ ‐dependent that might have stimulated ENaC. Hypotonicity can stimulate ENaC via activation of calmodulin‐dependent protein kinase II (CAMK II). Using concentrations of the CAMKII inhibitor KN93 from 1–100μM, we found no effect of this drug on amiloride‐sensitive transepithelial current induced by basolateral [Ca2+]i. Overall, we conclude that Src mediates the stimulatory effects of basolateral [Ca2+]i on ENaC but that CAMKII likely does not. Whether this occurs via cross talk with receptor tyrosine kinase signaling pathways remains to be seen.Support or Funding InformationNIH R37‐DK037963 to DCE, NIH R01‐DK100582 to HM and AHA 13POST16820072 to TLT