Abstract-The Na ϩ /Ca 2ϩ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca 2ϩ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca 2ϩ influx by the NCX (1) is the primary mechanism of Ca 2ϩ entry in purinergically stimulated rat aorta smooth muscle cells and (2)

K. S. Thorneloe and others 70J. Physiol. 541.1

Abstract-The molecular identity of receptor-operated, nonselective cation channels (ROCs) of vascular smooth muscle (VSM) cells is not known for certain. Mammalian homologues of the Drosophila canonical transient receptor potential channels (TRPCs) are possible candidates. This study tested the hypothesis that heteromultimeric TRPC channels contribute to ROC current of A7r5 VSM cells activated by [Arg 8 ]-vasopressin. A7r5 cells expressed transcripts encoding TRPC1, TRPC4␤, TRPC6, and TRPC7. TRPC4, TRPC6, and TRPC7 protein expression was confirmed by immunoblotting and association of TRPC6 with TRPC7, but not TRPC4␤, was detected by coimmunoprecipitation. The amplitude of arginine vasopressin (AVP)-induced ROC current was suppressed by dominant-negative mutant TRPC6 (TRPC6 DN ) but not TRPC5 (TRPC5 DN ) mutant subunit expression. These data indicate a role for TRPC6-and/or TRPC7-containing channels and rule a more complex subunit composition including TRPC1 and TRPC4. Increasing extracellular Ca 2ϩ concentration ([Ca 2ϩ ] o ) from 0.05 to 1 mmol/L suppressed currents owing to native, TRPC7, and heteromultimeric TRPC6-TRPC7 channels, but not TRPC6 current, which was slightly enhanced. The relative changes in native and heteromultimeric TRPC6-TRPC7 current amplitudes for [Ca 2ϩ ] o between Ϸ0.01 and 1 mmol/L were identical, but the changes in homomultimeric TRPC6 and TRPC7 currents were significantly less and greater, respectively, compared with the native channels. Taken together, the data provide biochemical and functional evidence supporting the view that heteromultimeric TRPC6-TRPC7 channels contribute to receptor-activated, nonselective cation channels of A7r5 VSM cells. sensitization of contractile filaments. [1][2][3][4] The depolarization and influx of Ca 2ϩ evoked by GPCRs is attributable in part to the activation of receptor-operated, nonselective cation channels (ROCs) by a signaling pathway involving phospholipase and diacylglycerol (reviewed previously [3][4][5][6] ). The molecular basis of VSM ROCs is not known with certainty, but accumulating evidence suggests that transient receptor potential channel (TRPC) family subunits (TRPC1 to TRPC7) [7][8][9] are likely involved. [3][4][5][6] Moreover, ROCs owing to heterologous expression of TRPC3, TRPC6, or TRPC7 are activated by a similar mechanism involving phospholipase and diacylglycerol. 10 -12 Understanding the molecular basis of VSM ROCs is clearly warranted in light of their important role in control of VSM excitability and contractility 4 -6 and evidence of changes in TRPC expression associated with abnormal contractility and/or VSM cell proliferation. [13][14][15] Identifying the contribution of TRPC subunits to VSM ROCs has been compromised by a lack of specific/selective pharmacological blockers. For this reason, alternative strategies involving antibodies, anti-sense or small interfering RNAs (siRNAs) were used to indicate roles for: (1) TRPC1 as store-operated channels 16 ; (2) TRPC3 as ROCs 17 ; (3) TRPC6 as ROCs and/or mechanosensit...