TRPC channels function independently of STIM1 and Orai1

Abstract

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor and calcium channel, respectively, for Ca 2+ -release activated Ca 2+ (CRAC) channels, channels underlying store-operated Ca 2+ entry (SOCE). In addition, these proteins have been suggested to function in signalling and constructing other channels with biophysical properties distinct from the CRAC channels. Using the human kidney cell line, HEK293, we examined the hypothesis that STIM1 can interact with and regulate members of a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signalling of TRPC channels. Specifically, Ca 2+ entry seen after carbachol treatment in cells transiently expressing TRPC1, TRPC3, TRPC5 or TRPC6 was not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to one published report. We previously reported in stable TRPC7 cells a Ca 2+ entry which was dependent on TRPC7 and appeared store-operated. However, we show here that this TRPC7-mediated entry was also not dependent on either STIM1 or Orai1, as determined by RNA interference (RNAi) and expression of a constitutively active mutant of STIM1. Further, we determined that this entry was not actually store-operated, but instead TRPC7 activity which appears to be regulated by SERCA. Importantly, endogenous TRPC activity was also not regulated by STIM1. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents associated with TRPC6 activity were not affected by RNAi knockdown of STIM1, while SOCE was largely inhibited. Finally, disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of I CRAC . Also, STIM1 punctae were found to localize in regions distinct from lipid rafts. This suggests that TRPC signalling and STIM1/Orai1 signalling occur in distinct plasma membrane domains . Thus, TRPC channels appear to be activated by mechanisms dependent on phospholipase C which do not involve the Ca 2+ sensor, STIM1.

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

“…TRPC1 is also implicated in store-operated calcium entry (25,26), in some cases with ORAI1 (44, 45), so we assessed its potential role in lactation. However, there are contradictory reports of the role of TRPC1 in storeoperated calcium entry (46,47) and our studies show that Trpc1 is unlikely to be associated with the Orai1-mediated calcium influx occurring during lactation, as there was a significant decline in its levels during lactation.…”

TRPC channels as STIM1-regulated SOCs

“…This may explain a recent report claiming that TRPC3 and TRPC6 expressed in HEK cells do not function as SOCs 19 and that knockdown of TRPC6 does not affect SOC activity in proliferative vascular smooth muscle cells. 20 when expressed at high levels, both channels showed spontaneous activity that was independent of STIM1.…”

“…14,[16][17][18] The expression studies generated the most controversy since the findings were not uniform, with several groups reporting that the expressed TRPC channels function as SOCs, 14,[16][17][18] while others using the same expression system concluded that the same TRPC channels do not function as SOCs. 19,20 Several of these differences could be traced to expression level of the channels 16,21 and most likely because several of the TRPC channels can function both in SOC and non-SOC modes (see below).…”

Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1

“…Several studies show that association between TRPC1 and STIM1 increases following store depletion (6,16,19,20), although a recent study was unable to demonstrate this (21). While the ezrin/radixin/moesin (ERM) domain of STIM1 appears to bind to TRPC1, the C-terminal 684 KK 685 residues are involved in gating the channel via electrostatic interaction with TRPC1( 639 DD 640 ) (8,13).…”