Caveolae facilitate TRPV4-mediated Ca²⁺ signaling and the hierarchical activation of Ca²⁺-activated K⁺ channels in K⁺-secreting renal collecting duct cells.

Transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated Ca²⁺ signaling induces early activation of small/intermediate Ca²⁺-activated K⁺ channels, SK3 (KCNN3) and IK1 (KCNN4), which leads to membrane hyperpolarization and enhanced Ca²⁺ influx, which is critical for subsequent activation of the large conductance Ca²⁺-activated K⁺ channel BK (KCNMA1) and K⁺ secretion in kidney cortical collecting duct (CCD) cells. The focus of the present study was to determine if such coordinated hierarchical/sequential activation of these channels in CCD was orchestrated within caveolae, a known microcompartment underlying selective Ca²⁺-signaling events in other cells. In K⁺-secreting mouse principal cell (PC) line, mCCDcl1 cells, knockdown of caveolae caveolin-1 (CAV-1) depressed TRPV4-mediated Ca²⁺ signaling and activation of SK3, intermediate conductance channel (IK1), and BK. Immunofluorescence colocalization analysis and coimmunoprecipitation assays demonstrated direct coupling of TRPV4 with each of the KCa channels in both mCCDcl1 and whole mouse kidney homogenates. Likewise, extending this analysis to CAV-1 demonstrates colocalization and direct coupling of CAV-1 with TRPV4, SK3, IK1, and BK, providing strong support for coupling of the channels in caveolae microdomains. Furthermore, differential expression of CAV-1 along the CCD was apparent where CAV-1 was strongly expressed within and along the cell borders of kidney PCs and intercalated cells (ICs), although significantly less in ICs. It is concluded that caveolae provide a key microdomain in PCs and ICs for coupling of TRPV4 with SK3, IK1, and BK that directly contributes to TRPV4-mediated Ca²⁺ signaling in these domains leading to rapid and sequential coupling of TRPV4-SK3/IK1-BK that may play a central role in mediating Ca²⁺-dependent regulation of BK and K⁺ secretion.

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