Regulation of the mouse epithelial Ca2+ channel TRPV6 by the Ca2+-sensor calmodulin
Regulation of the mouse epithelial Ca2+ channel TRPV6 by the Ca2+-sensor calmodulin
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DOI：
10.1074/jbc.m313637200
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发表时间：
2004-07-09
影响因子：
4.8
通讯作者：
Bindels, RJM
中科院分区：
文献类型：
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作者：
Lambers, TT;Weidema, AF;Bindels, RJM
TRPV5和TRPV6是瞬时受体电位(Trp)通道超家族的成员，它们促进多种上皮细胞的钙内流。这些钙通道的活性受到靠近通道口的细胞内钙离子浓度的严格控制。然而，TRPV5/TRPV6的钙依赖活性的分子机制仍不清楚。在这里，我们研究了钙调素(CaM)作为钙离子感受器在调节通道活动中的作用。钙不敏感的CaM突变体(CaM1234和CaM34)的过表达显著降低了TRPV6细胞的钙电流和钠电流，但对表达TRPV5的HEK293细胞的钙电流和钠电流无明显影响。通过结合下拉试验和免疫共沉淀，我们证明了CaM以钙离子依赖的方式与TRPV5和TRPV6结合。CaM与TRPV6的结合定位于跨膜区(TRPV6(327-577))和位于N(1-5-10基序，TRPV6(88-97))和C末端(1-8-14基序，TRPV6(643-656))的共同CaM结合基序，提示了一种涉及多个相互作用位点的调控机制。随后，将TRPV6/TRPV5嵌合蛋白与CaM34在HEK293细胞中共表达，其中TRPV6的N和/或C端被TRPV5取代。用TRPV5交换TRPV6的N和/或C端并不影响CaM34引起的钙、钠电流的降低。这些结果表明，当钙离子与EF-HAND3和EF-HAND 4结合时，CaM对TRPV6的活性有正向影响，它们位于高钙亲和力的CaM C末端，涉及TRPV6的N和C末端以及跨膜区。
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.