ncbi.nlm.nih.gov/gene/7225) have been implicated as candidates for cardiac SACNS. TRPC1:
Analysis of mRNA expression suggested that TRPC1 is present in the human heart. 48 Using immuno-histochemical labelling and confocal imaging, TRPC1 protein was found price WAY-100635 to colocalise with phalloidin stain in rat ventricular myocytes. 42 This suggests that TRPC1 may be located in T-tubules and is consistent with the hypothesised spatial distribution of endogenous SACNS in adult ventricular cardiomyocytes. Mechanosensitivity of TRPC1 was first noted by Maroto et al. 49 in Xenopus oocytes. In their experiments, ISAC,NS was measured after membrane protein fractionation and reconstitution of individual proteins in liposomes. A particularly mechanosensitive fraction was found to contain an 80-kDa protein which was immunoreactive to TRPC1 antibody, indicating the presence of a TRPC1 homologue. Further expression of the human TRPC1 (hTRPC1) isoform in Xenopus oocytes and Chinese hamster ovary (CHO) K1 cells increased ISAC,NS tenfold, whereas microinjection of antisense hTRPC1 RNA greatly reduced ISAC,NS in both cell types. Since publication, these findings have been challenged by several studies, including one by some of the authors of the original report. They found that transfection of hTRPC1 into COS cells (a fibroblast-related cell line, originally derived from kidney tissue of monkey) had no discernible effect, while transfection
of a different putative SAC (the SACK TREK-1; see below), induced an increase by three orders of magnitude in mechanosensitive whole-cell currents. This result puts into question the significance of the less pronounced (ten-fold) increase seen in the earlier experiments. 50 The authors of the later study found limited ion channel expression at the sarcolemma, which is in agreement with a more recent report showing predominantly intracellular expression of transfected TRPC1 in a skeletal muscle cell line, unless co-expressed with Cav3 (http://www.ncbi.nlm.nih.gov/gene/859). 51 Thus, even if TRPC1 is successfully transfected, it may require associated
molecular machinery for a correct subcellular localization and/or proper function. In addition, TRPC1 may require other TRPC isoforms to Anacetrapib form a functional heteromeric channel. 52 The conflicting results reported above highlight problems that can be associated with the use of heterologous expression systems to study cardiac ion channels. Clearly, the intracellular environment of stable cell lines differs significantly from that of cardiomyocytes, while additional transfection with exogenous ion channels can alter the structure and function of recipient cells. 50 Given the dependence of SAC gating properties on micro-mechanical and structural properties of a cell, it is difficult to establish suitable control protocols, 50 or to arrive at definitive conclusions from these experiments.