Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin.
TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca(2+) influx in a variety of epithelial cells. The activity of these Ca(2+) channels is tightly controlled by the intracellular Ca(2+) concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca(2+)-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca(2+) sensor mediating the regulation of channel activity was investigated. Overexpression of Ca(2+)-insensitive CaM mutants (CaM(1234) and CaM(34)) significantly reduced the Ca(2+) 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 Ca(2+)-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 CaM(34) in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM(34)-induced reduction of the Ca(2+) and Na(+) currents. These results suggest that CaM positively affects TRPV6 activity upon Ca(2+) binding to EF-hands 3 and 4, located in the high Ca(2+) affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.
Lambers
Tim T
TT
Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands.
Weidema
A Freek
AF
Nilius
Bernd
B
Hoenderop
Joost G J
JG
Bindels
René J M
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D050916:TRPV Cation Channels; D014981:Xenopus
D015220:Calcium Channels; D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C120776:TRPV6 channel; D002118:Calcium