Expression of a Diverse Array of Ca 2+ ‐Activated K + Channels are Functionally Coupled to the Mechanosensitive TRPV4 Channel in the Late Distal Tubule

The voltage‐ and Ca2+‐activated, big conductance K+ channel (BK, maxi‐K channel) is known to be expressed in the distal tubule of kidney where it underlies flow‐ and Ca2+‐dependent K+ secretion in the connecting tubule (CNT) and cortical collecting duct (CCD). While the mechanosensitive TRPV4 channel is central for controlling Ca2+ influx, the potential role of other Ca2+‐activated K+ channels, KCa, in regulating K+ secretion in these cells is largely unknown. Using qPCR mRNA analysis and immunoblots of mouse kidney and the CCD cell line, mCCDcl1 (a K+ secreting cell line; Fodstad H et al., Am J Physiol 296:F966–75, 2009) an array of KCa channels was identified. Subsequent immunohistochemical staining for expressed ion channel proteins in CCD, CNT, and the mCCDcl1 cells, revealed expression of TRPV4 and three subfamilies of KCa channels: the small‐conductance SK channels (SK1 and SK3), the intermediate conductance IK channel (IK1), and the BK‐alpha channel. Intracellular Ca2+ imaging of mCCDcl1 cells demonstrated that the KCa channels were functionally regulated by TRPV4 and that each of the KCa channels displayed a potent functional cross‐talk with TRPV4 upon TRPV4 activation (GSK1016790A, 3–10 nM). Indeed, upon KCa channel activation induced by TRPV4, intracellular Ca2+ levels were suppressed upon inhibition of each given KCa channel subtype, SK, IK, BK, by addition of selective K+ channel inhibitors‐‐apamin (300 nM), TRAM‐34 (300 nM), or Iberiotoxin (100 nM), respectively. Further, for mCCDcl1 cells grown on permeable supports, activation of TRPV4 (GSK1016790A) induced a rapid decrease in transepithelial electrical resistance (TEER) that was partially restored by selective inhibition of each KCa subtype, SK, IK, and BK, as above. Immunohistochemcial staining of mouse CCD demonstrated expression of these channels in most cells, although significant differential staining of principle cells (PC) versus intercalated cells (IC) was apparent with the following staining patterns: TRPV4 PC>IC, BKa PC=IC, IK1 PC=IC, SK1 IC>>PC, and SK3 PC>>IC. It is concluded that an array of KCa channels are expressed in CNT and CCD and, like BK‐alpha, are functionally regulated by TRPV4‐mediated Ca2+ influx where strong cross‐talk between the KCa and TRPV4 exists. Further, it is postulated that with the high Ca2+ binding affinity of SK1/3 and IK1, these KCa channels will be activated early on during the initial induction of TRPV4. The observed functional cross‐talk will lead to early elevation in intracellular Ca2+ levels which will enhance activation of the low Ca2+ binding affinity channel, BK, and, in turn, give rise to enhanced BK‐mediated K+ secretion. Whether SK1/3 and IK1 directly participate in K+ secretion is likely, but remains to be verified in future studies. Support or Funding Information NIH R01 DK098401 AHA 14GRNT20380916