Evidence that TRPC3 is a molecular component of the 1alpha,25(OH)2D3-activated capacitative calcium entry (CCE) in muscle and osteoblast cells.
In chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8), 1alpha,25-dihydroxy-Vitamin-D(3) [1alpha,25(OH)(2)D(3)] stimulates release of Ca(2+) from inner stores and extracellular cation influx through both voltage-dependent and capacitative Ca(2+) entry (CCE) channels. We investigated the involvement of TRPC proteins in CCE induced by 1alpha,25(OH)(2)D(3). Two fragments were amplified by RT-PCR, exhibiting >85% sequence homology with human TRPC3. Northern and Western blots employing TRPC3-probes and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein. Both cell types transfected with anti-TRPC3 antisense oligodeoxynucleotides showed reduced CCE and Mn(2+) entry induced by either thapsigargin or 1alpha,25(OH)(2)D(3). In muscle cells, anti-VDR antisense inhibited steroid-induced Ca(2+) and Mn(2+) influx and co-immunoprecipitation of TRPC3 and VDR was observed, suggesting an association between both proteins and a functional role of the receptor in 1alpha,25(OH)(2)D(3) activation of CCE. In osteoblasts, two PCR fragments showing high homology with human INAD-like sequences were obtained. Northern blot and antisense functional assays suggested the involvement of the INAD-like protein in CCE regulation by the hormone. Therefore, we propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)(2)D(3) modulation of CCE in muscle and osteoblastic cells, which seems to implicate VDR-TRPC3 association and the participation of a INAD-like scaffold protein.
Santillán
G
G
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.
Baldi
C
C
Katz
S
S
Vazquez
G
G
Boland
R
R
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002642:Chick Embryo; D017931:DNA Primers; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D051381:Rats; D050052:TRPC Cation Channels
D017931:DNA Primers; D007473:Ion Channels; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D002117:Calcitriol; D002118:Calcium