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.

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.

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.

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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 storeoperated 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؉ C alcium signals, which can be induced by a variety of stimuli, control a myriad of functions in the body. In the liver, bile secretion, glucose production, and permeability of tight junctions are all regulated by calcium signals. The liver is mainly composed of hepatocytes, multifunctional cells involved in the regulation of a number of critical homeostatic hormone-controlled pathways. In hepatocytes, Ca 2ϩ signaling occurs in the form of baseline oscillations that stem from periodic openings of Ca 2ϩ channels in the membrane of the endoplasmic reticulum (ER). 1,2 These oscillations are usually repetitive spikes separated by intervals that can range from a few milliseconds to a few minutes, depending on the type of agonist and its concentration. Moreover, when oscillations become spatially organized, intercellular calcium waves occur, representing an efficient form of communication between cells through which physiological responses can be coordinated. 3,4 Like most receptor-activated calcium signals, the generation and maintenance of oscillations involves two components-the release of internally stored calcium from the ER and the entry of extracellular calcium to maintain adequate Ca 2ϩ stores in the ER. This second component often involves a process known as capacitative calcium entry or store-operated calcium entry (SOCE), occurring through channels located in the plasma membrane. 5,6 The best characterized of these store-operated channels is the Ca 2ϩ -release-activated Ca 2ϩ (CRAC) channel. 6-8 The