Weizhong Zeng

STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (Icrac) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of Icrac and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and Icrac channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8) — both of which may function as Icrac channels or as an Icrac subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and Icrac channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, Icrac and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1D76A yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and Icrac in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, Icrac and TRPC channel activation by STIM1.

STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels

Stromal interacting molecule 1 (STIM1) is a Ca2+ sensor that conveys the Ca2+ load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La3+, suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.

Gating of CFTR by the STAS domain of SLC26 transporters.

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Giand Gq class α subunits. In the present study, we used knockouts of Gq class genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class α subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.

Polarized Expression of Ca2+ Channels in Pancreatic and Salivary Gland Cells CORRELATION WITH INITIATION AND PROPAGATION OF [Ca2+] i WAVES

In polarized epithelial cells [Ca2+] i waves are initiated in discrete regions and propagate through the cytosol. The structural basis for these compartmentalized and coordinated events are not well understood. In the present study we used a combination of [Ca2+] i imaging at high temporal resolution, recording of Ca2+-activated Cl− current, and immunolocalization by confocal microscopy to study the correlation between initiation and propagation of [Ca2+] i waves and localization of Ca2+ release channels in pancreatic acini and submandibular acinar and duct cells. In all cells Ca2+ waves are initiated in the luminal pole and propagate through the cell periphery to the basal pole. All three cell types express the three known inositol 1,4,5-trisphosphate receptors (IP3Rs). Expression of IP3Rs was confined to the area just underneath the luminal and lateral membranes, with no detectable receptors in the basal pole or other regions of the cells. In pancreatic acini and SMG ducts IP3R3 was also found in the nuclear envelope. Expression of ryanodine receptor was detected in submandibular salivary gland cells but not pancreatic acini. Accordingly, cyclic ADP ribose was very effective in mobilizing Ca2+ from internal stores of submandibular salivary gland but not pancreatic acinar cells. Measurement of [Ca2+] i and localization of IP3Rs in the same cells suggests that only a small part of IP3Rs participate in the initiation of the Ca2+ wave, whereas most receptors in the cell periphery probably facilitate the propagation of the Ca2+ wave. The combined results together with our previous studies on this subject lead us to conclude that the internal Ca2+ pool is highly compartmentalized and that compartmentalization is achieved in part by polarized expression of Ca2+ channels.

STIM1 Gates TRPC Channels, but Not Orai1, by Electrostatic Interaction

The receptor-evoked Ca(2+) signal includes activation of the store-operated channels (SOCs) TRPCs and the Orais. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1((639)DD(640)) with the positively charged STIM1((684)KK(685)) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1((684)EE(685))+TRPC1((639)RR(640)), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observed with TRPC3((697)DD(698)). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.

Polarized Expression of Ca2+ Pumps in Pancreatic and Salivary Gland Cells ROLE IN INITIATION AND PROPAGATION OF [Ca2+] i WAVES

The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca2+pumps and characterizing their role in initiation and propagation of Ca2+ waves. Specific and polarized expression of Ca2+ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca2+ release with ionomycin on Ca2+ release evoked by agonists and Ca2+ uptake induced by antagonists. Despite their polarized expression, Ca2+ uptake by SERCA pumps and Ca2+ efflux by PMCA resulted in uniform reduction in [Ca2+] i . Surprisingly, inhibition of the SERCA pumps, but not Ca2+ release by ionomycin, eliminated the distinct initiation sites and propagated Ca2+ waves, leading to a uniform increase in [Ca2+] i . In addition, inhibition of SERCA pumps reduced the rate of Ca2+ release from internal stores. The implication of these findings to rates of Ca2+ diffusion in the cytosol, compartmentalization of Ca2+ signaling complexes, and mechanism of Ca2+ wave propagation are discussed.

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Gα subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellualar loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of α-adrenergic receptor (αAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant αARA293E (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of βAR–αAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, αAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2−/− cells and less sensitive to inhibition by RGS2 in spl−/− cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.

Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3− secretion: relevance to cystic fibrosis

Fluid and HCO3− secretion are vital functions of the pancreatic duct and other secretory epithelia. CFTR and Cl−/HCO3− exchange activity at the luminal membrane are required for these functions. The molecular identity of the Cl−/HCO3− exchangers and their relationship with CFTR in determining fluid and HCO3− secretion are not known. We show here that the Cl−/HCO3− exchanger slc26a6 controls CFTR activity and ductal fluid and HCO3− secretion. Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO3− secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTRinh‐172, knock‐down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6−/− duct cells revealed that deletion of slc26a6 resulted in dis‐regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO3− secretion in vivo.

Membrane-specific Regulation of Cl− Channels by Purinergic Receptors in Rat Submandibular Gland Acinar and Duct Cells

Measurement of [Cl−] i and the Cl− current in the rat salivary submandibular gland (SMG) acinar and duct cells was used to evaluate the role of Cl− channels in the regulation of [Cl−] i during purinergic stimulation. Under resting conditions [Cl−] i averaged 56 ± 8 and 26 ± 7 mm in acinar and duct cells, respectively. In both cells, stimulation with 1 mm ATP resulted in Cl− efflux and subsequent influx. Inhibition of NaKCl2 cotransport had no effect on [Cl−] i changes in duct cells and inhibited only about 50% of Cl− uptake in acinar cells. Accordingly, low levels of expression of NaKCl2 cotransporter protein were found in duct cells. Acinar cells expressed high levels of the cotransporter. Measurement of Cl− current under selective conditions revealed that acinar and duct cells express at least five distinct Cl− channels; a ClCO-like, volume-sensitive, inward rectifying, Ca2+-activated and CFTR-like Cl− currents. ATP acting on both cell types activated at least two channels, the Ca2+-activated Cl−channel and a Ca2+-independent glibenclamide-sensitive Cl−-current, possibly cystic fibrosis transmembrane regulator (CFTR). Of the many nucleotides tested only 2′-3′-benzoylbenzoyl (Bz)-ATP and UTP activated Cl−channels in SMG cells. Despite their relative potency in increasing [Ca2+] i , BzATP in both SMG cell types largely activated the Ca2+-independent, glibenclamide-sensitive Cl− current, whereas UTP activated only the Ca2+-dependent Cl− current. We interpret this to suggest that BzATP and UTP largely activate Cl− channels residing in the membrane expressing the receptor for the active nucleotide. The present studies reveal a potentially new mechanism for transcellular Cl− transport in a CFTR-expressing tissue, the SMG. Coordinated action of the P2z (luminal) and P2u (basolateral) receptors can mediate part of the transcellular Cl− transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid.