The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple water and ion transporter and channel proteins. Notably, all the key transporter and channel proteins in this process appear to be activated, or are up-regulated, by an increase in the intracellular Ca2+ concentration ([Ca2+]i). Consequently, salivation occurs in response to agonists that generate an increase in [Ca2+]i. The mechanisms that act to modulate these increases in [Ca2+]i obviously influence the secretion of salivary fluid. Such modulation may involve effects on mechanisms of both Ca2+ release and Ca2+ entry and the resulting spatial and temporal aspects of the [Ca2+]i signal, as well as interactions with other signaling pathways in the cells. The molecular cloning of many of the transporter and regulatory molecules involved in fluid and electrolyte secretion has yielded a better understanding of this process at the cellular level. The subsequent characterization of mice with null mutations in many of these genes has demonstrated the physiological roles of individual proteins. This review focuses on recent developments in determining the molecular identification of the proteins that regulate the fluid secretion process.

Recent studies have indicated a critical role for STIM (stromal interacting molecule) proteins in the regulation of the store-operated mode of receptor-activated Ca 2+ entry. Current models emphasize the role of STIM located in the endoplasmic reticulum membrane, where a Ca 2+ -binding EF-hand domain within the N-terminal of the protein lies within the lumen and is thought to represent the sensor for the depletion of intracellular Ca 2+ stores. Dissociation of Ca 2+from this domain induces the aggregation of STIM to regions of the ER immediately adjacent to the plasma membrane where it acts to regulate the activity of store-operated Ca 2+ channels. However, the possible effects of STIM on other modes of receptor-activated Ca 2+ entry have not been examined. Here we show that STIM1 also regulates the arachidonic-acid-regulated Ca 2+ -selective (ARC) channels -receptor-activated Ca 2+ entry channels whose activation is entirely independent of store depletion. Regulation of the ARC channels by STIM1 does not involve dissociation of Ca 2+ from the EF-hand, or any translocation of STIM1. Instead, a critical role of STIM1 resident in the plasma membrane is indicated. Thus, exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1 inhibits ARC channel activity without significantly affecting the store-operated channels. A similar specific inhibition of the ARC channels is seen in cells expressing a STIM1 construct in which the N -linked glycosylation sites essential for the constitutive cell surface expression of STIM1, were mutated. We conclude that, in contrast to store-operated channels, regulation of ARC channels by STIM1 depends exclusively on the pool of STIM1 constitutively residing in the plasma membrane. These data demonstrate that STIM1 is a more universal regulator of Ca 2+ entry pathways than previously thought, and appears to have multiple modes of action.

The prevailing model of receptor-activated Ca 2ϩ entry in non-excitable cells is the so-called "capacitative" or store-operated model in which the emptying of the agonist-sensitive stores alone is both necessary and sufficient to activate Ca 2ϩ

Agonist-activated Ca2+ entry plays a critical role in Ca 2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca 2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca 2+ release-activated Ca 2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.

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