TRPC3-like protein is involved in the capacitative cation entry induced by 1alpha,25-dihydroxy-vitamin D3 in ROS...

In ROS 17/2.8 rat osteoblastic-like cells a capacitative Ca(2+) entry (CCE) pathway operates which is activated by either 1alpha,25-dihydroxy-vitamin D3 (1alpha,25(OH)(2)D3 or thapsigargin (Tpg)-induced depletion of Ca(2+) stores. In view of recent evidence favoring a role for transient receptor potential (TRP) proteins in mediating CCE, we investigated if channels involved in the 1alpha,25(OH)(2)D3-sensitive CCE in rat osteoblasts were related to an endogenous TRP-canonical (TRPC) isoform homologue. By reverse transcription (RT)-PCR using mRNA from ROS 17/2.8 cells and primers based on conserved regions within the mammalian TRPC3/6/7 subfamily, two fragments were amplified of 390 and 201 bp with 100 and 94% sequence identity, respectively, with human TRPC3. Northern blot analysis showed the presence of a 3.5 kb transcript and both immunobloting and immunocytochemistry using a specific anti-TRPC3 antibody confirmed endogenous expression of a TRPC3-like protein ( approximately 110 kDa) with membrane localization. In ROS 17/2.8 cells intranuclearly microinjected with anti-TRPC3 antisense oligodeoxynucleotides (ODN), both the initial rate and magnitude of CCE activated by either 1alpha,25(OH)(2)D3 or Tpg were markedly reduced, whereas no changes were detected in control-injected cells. The present findings constitute the first evidence to date suggesting that an endogenous TRPC3-like protein is functionally involved in the CCE route activated by 1alpha,25(OH)(2)D3 in a secosteroid target cell. We anticipate TRPC3 as a candidate for mediating store-operated non-selective cation entry into osteoblasts.