Regulation of the mouse epithelial Ca2+ channel TRPV6 by the Ca2+-sensor calmodulin

TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca2+ influx in a variety of epithelial cells. The activity of these Ca2+ channels is tightly controlled by the intracellular Ca2+ concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca2+-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca2+ sensor mediating the regulation of channel activity was investigated. Overexpression of Ca2+-insensitive CaM mutants (CaM1234 and CaM34) significantly reduced the Ca2+ as well as the Na+ current of TRPV6- but not that of TRPV5-expressing HEK293 cells. By combining pull-down assays and co-immunoprecipitations, we demonstrated that CaM binds to both TRPV5 and TRPV6 in a Ca2+-dependent fashion. The binding of CaM to TRPV6 was localized to the transmembrane domain (TRPV6(327-577)) and consensus CaM-binding motifs located in the N (1-5-10 motif, TRPV6(88-97)) and C termini ( 1 - 8- 14 motif, TRPV6(643-656)), suggesting a mechanism of regulation involving multiple interaction sites. Subsequently, chimeric TRPV6/TRPV5 proteins, in which the N and/or C termini of TRPV6 were substituted by that of TRPV5, were co-expressed with CaM34 in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM34- induced reduction of the Ca2+ and Na+ currents. These results suggest that CaM positively affects TRPV6 activity upon Ca2+ binding to EF-hands 3 and 4, located in the high Ca2+ affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.