TRPC channels function independently of STIM1 and Orai1.

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.

Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes.

UNLABELLED: The process of capacitative or store-operated Ca(2+) entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca(2+) stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca(2+) entry is readily demonstrated with protocols that provide extensive Ca(2+) store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca(2+) influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca(2+) oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca(2+) oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca(2+) entry but only partially reduced the frequency of Ca(2+) oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. CONCLUSION: Our findings establish that store-operated Ca(2+) entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent.

Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP.

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.

Ligament cells stretch-adapted on a microgrooved substrate increase intercellular communication in response to a mechanical stimulus.

An in vitro model was used to investigate the effect of mechanical stimuli on adaptation to load and calcium signaling in aligned medial collateral ligament cells (MCL). This model used a patterned silicone membrane to align the cells parallel with the direction of the microgrooves. Alignment created an architecture that simulated a degree of cell orientation in native ligament tissue. It was hypothesized that aligned ligament cells would be more efficient at calcium wave propagation than cells that were randomly oriented. It was further hypothesized that calcium wave propagation would be greater among cells that were both aligned and subjected to mechanical stretch compared to cells that were aligned but not stretched. Rat MCL cells were loaded with Fura-2AM, a calcium-binding dye, and mechanically indented using a micropipette tip. A ratio-imaging fluorescence technique was used to quantitate the calcium (Ca2+) response. It was concluded that stretching ligament cells prior to stimulation increased their sensitivity to load and their ability to propagate a calcium wave. However, the ability of aligned cells to propagate this wave was not significantly different when compared to nonaligned cells. Treatment of cultures with inhibitors such as apyrase and suramin significantly reduced the number of cells recruited in the calcium response. Hence, it was concluded that ATP released from mechanically stimulated cells was a principal mediator responsible for the rise in intracellular calcium in ligament cells. Further, purinoceptor activation may amplify the signal to alert and recruit more cells in a response to mechanical stimulation.