Lambers Tim T, Weidema A Freek, Nilius Bernd, Hoenderop Joost G J, Bindels René J M
Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands.
J Biol Chem. 2004 Jul 9;279(28):28855-61. doi: 10.1074/jbc.M313637200. Epub 2004 Apr 30.
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.
瞬时受体电位（TRP）通道超家族的成员TRPV5和TRPV6可促进多种上皮细胞中的Ca(2+)内流。这些Ca(2+)通道的活性受到通道口附近细胞内Ca(2+)浓度的严格控制。然而，TRPV5/TRPV6的Ca(2+)依赖性活性的分子机制仍然未知。在此，研究了钙调蛋白（CaM）作为介导通道活性调节的Ca(2+)传感器的假定作用。Ca(2+)不敏感的CaM突变体（CaM(1234)和CaM(34)）的过表达显著降低了表达TRPV6的HEK293细胞的Ca(2+)以及Na(+)电流，但未降低表达TRPV5的HEK293细胞的电流。通过结合下拉实验和免疫共沉淀，我们证明CaM以Ca(2+)依赖性方式与TRPV5和TRPV6结合。CaM与TRPV6的结合定位于跨膜结构域（TRPV6(327 - 577)）以及位于N端（1 - 5 - 10基序，TRPV6(88 - 97)）和C端（1 - 8 - 14基序，TRPV6(643 - 656)）的共有CaM结合基序，提示了一种涉及多个相互作用位点的调节机制。随后，将TRPV6的N端和/或C端被TRPV5的相应部分替换的嵌合TRPV6/TRPV5蛋白与CaM(34)在HEK293细胞中共同表达。用TRPV5的N端和/或C端替换TRPV6的相应部分并不影响CaM(34)诱导的Ca(2+)和Na(+)电流的降低。这些结果表明，Ca(2+)与位于高Ca(2+)亲和力CaM C端的EF手3和4结合后，CaM对TRPV6活性产生正向影响；这一过程涉及TRPV6的N端、C端和跨膜结构域。
