TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca2+ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca2+ concentrations (Ca-i(2+)) revealed a dose-dependent activation of TRPC5 channels by internal Ca2+ with EC50 of 635.1 and 358.2 nM at negative and positive membrane potentials, respectively. Stepwise increases of Ca-i(2+) induced by photolysis of caged Ca2+ showed that the Ca2+ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 +/- 0.2 ms at Ca-i(2+) below 10 mu M, suggesting that the action of internal Ca2+ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 +/- 0.1 s was also observed at Ca-i(2+) above 10 mu M. In support of a Ca2+-activation mechanism, the thapsigargin-induced release of Ca2+ from internal stores activated TRPC5 channels transiently, and the subsequent Ca2+ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the alpha C-1 and beta(2) subunits of L-type Ca2+ channels, we found that Ca2+ entry through either calcium-release-activated-calcium or voltage-dependent Ca2+ channels is sufficient for TRPC5 channel activation. The Ca2+ entry activated TRPC5 channels under buffering of internal Ca2+ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca2+-activated cation channels that are functionally coupled to Ca2+-selective ion channels through local Ca2+ increases beneath the plasma membrane.