[show abstract][hide abstract] ABSTRACT: Although several protein-protein interactions have been reported between transient receptor potential (TRP) channels, they are all known to occur exclusively between members of the same group. The only intergroup interaction described so far is that of TRPP2 and TRPC1; however, the significance of this interaction is unknown. Here, we show that TRPP2 and TRPC1 assemble to form a channel with a unique constellation of new and TRPP2/TRPC1-specific properties. TRPP2/TRPC1 is activated in response to G-protein-coupled receptor activation and shows a pattern of single-channel conductance, amiloride sensitivity and ion permeability distinct from that of TRPP2 or TRPC1 alone. Native TRPP2/TRPC1 activity is shown in kidney cells by complementary gain-of-function and loss-of-function experiments, and its existence under physiological conditions is supported by colocalization at the primary cilium and by co-immunoprecipitation from kidney membranes. Identification of the heteromultimeric TRPP2/TRPC1 channel has implications in mechanosensation and cilium-based Ca(2+) signalling.
[show abstract][hide abstract] ABSTRACT: Depletion of intracellular Ca2+ stores leads to the activation of Ca2+ inflow through store-operated Ca2+ channels. Although the identity of these channels is unknown, there is considerable evidence that the transient receptor potential channel 1 (TRPC1) participates in the formation of these channels. We show that TRPC1 physically interacts with the a-isoform of the inhibitor of the myogenic family (I-mfa), a known inhibitor of basic helix-loop-helix transcription factors, in vitro and in vivo. The interaction is mediated by the C-terminal cytoplasmic tail of TRPC1 and the C-terminal cysteine-rich domain of I-mfa. Using the whole cell configuration of the patch clamp technique, we show that ectopic expression of I-mfa in CHO-K1 cells reduces native store-activated Ca2+ currents, whereas knock-down of endogenous I-mfa in A431 cells by RNA interference enhances these currents. Pipette perfusion of purified recombinant I-mfa rescues the effect of I-mfa knock-down on store-operated conductance. Finally, cell dialysis with a monoclonal antibody specific to TRPC1 results in the suppression of store-activated conductance in cells lacking I-mfa, but not in I-mfa expressing cells. We propose that I-mfa functions as a molecular switch to suppress the store dependence of TRPC1.
Journal of Biological Chemistry 01/2004; 278(52):52763-72. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80 kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92 kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the alpha- and beta-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92 kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120 kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92 kDa or 120 kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated beta-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.