Apical localization of a functional TRPC3/TRPC6-Ca2+-signaling complex in polarized epithelial cells. Role in apical Ca2+ influx

Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 was localized apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 was localized basolaterally, whereas TRPC6 was detected in both locations. Localization of Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cbeta1 and -beta2 was also predominantly apical. TRPC3 co-immunoprecipitated with endogenous TRPC6, phospholipase Cbetas, Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake was higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux were also higher in TRPC3-expressing cells. Consistent with this, OAG induced [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that was blocked by EGTA addition to the apical medium. Most importantly, (i) TRPC3 was detected in the apical region of rat submandibular gland ducts, whereas TRPC6 was present in apical as well as basolateral regions of ducts and acini; and (ii) OAG stimulated Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion.     

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.     

Involvement of apical P2Y2 receptor-regulated CFTR activity in muscarinic stimulation of Cl(-) reabsorption in rat submandibular gland

Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl(-) channels in Ca(2+)-activated Cl(-) reabsorption in the ductal system of the rat submandibular gland. Here, we address the mechanism by which basal CFTR activation contributes to the transepithelial Cl(-) movement evoked by muscarinic stimulation. The Cl(-) concentration ([Cl(-)]) was increased in the final saliva from rat submandibular gland during pilocarpine stimulation when a small interfering RNA for CFTR or a specific CFTR inhibitor, CFTR(inh)-172, was injected retrogradely into the gland's own duct, indicating that basal CFTR activation is involved in Cl(-) reabsorption. Systemically administered propranolol failed to alter the [Cl(-)], suggesting little involvement of a beta-adrenergic pathway in the Cl(-) movement that occurs through basal CFTR activation. Intraductal injection of suramin (a nonspecific P2-receptor antagonist) increased the salivary [Cl(-)], indicating the existence of endogenous purinergic activation. Upon separate intraductal injection, ATP and a P2Y(2)-receptor agonist, UTP, decreased the salivary [Cl(-)] almost equipotently. CFTR(inh)-172 and suramin each prevented these effects, whereas 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a P2X(7) agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y(2) receptors on the luminal surface of epithelial cells in the striated ducts of this gland. These results raise the possibility that muscarinic stimulation-induced Cl(-) reabsorption occurs through basal CFTR activity and that this is regulated by P2Y(2) receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.     

Inhibition of erythropoiesis by Smad6 in human cord blood hematopoietic stem cells

There is increasing evidence that mesangial cells are important targets of chronic hypoxia injury. Impaired Ca(2+) signaling has been found in mesangial cells (MCs) subjected to chronic hypoxia. However, the mechanisms underlying this phenomenon have not yet been defined. In the present study, we found that chronic hypoxia enhanced the expression of TRPC6 and TRPC6-dependent Ca(2+) entry, and TRPC6 knockdown inhibited the chronic hypoxia-induced increase in [Ca(2+)]i, suggesting that TRPC6-mediated Ca(2+) entry is responsible for the elevated [Ca(2+)]i induced by chronic hypoxia in MCs. In addition, TRPC6 knockdown attenuated chronic hypoxia-induced actin assembly and actin reorganization. We concluded that the upregulation of TRPC6 is involved in the Ca(2+) signaling and actin assembly in human MCs after chronic hypoxia. These findings provide new insight into the mechanisms underlying the cellular response of MCs to hypoxia.     

The dynamics of luminal depletion and the stochastic gating of Ca2+-activated Ca2+ channels and release sites

Single channel models of intracellular calcium (Ca(2+)) channels such as the 1,4,5-trisphosphate receptor and ryanodine receptor often assume that Ca(2+)-dependent transitions are mediated by constant background cytosolic [Ca(2+)]. This assumption neglects the fact that Ca(2+) released by open channels may influence subsequent gating through the processes of Ca(2+)-activation or inactivation. Similarly, the influence of the dynamics of luminal depletion on the stochastic gating of intracellular Ca(2+) channels is often neglected, in spite of the fact that the sarco/endoplasmic reticulum [Ca(2+)] near the luminal face of intracellular Ca(2+) channels influences the driving force for Ca(2+), the rate of Ca(2+) release, and the magnitude and time course of the consequent increase in cytosolic domain [Ca(2+)]. Here we analyze how the steady-state open probability of several minimal Ca(2+)-regulated Ca(2+) channel models depends on the conductance of the channel and the time constants for the relaxation of elevated cytosolic [Ca(2+)] and depleted luminal [Ca(2+)] to the bulk [Ca(2+)] of both compartments. Our approach includes Monte Carlo simulation as well as numerical solution of a system of advection-reaction equations for the multivariate probability density of elevated cytosolic [Ca(2+)] and depleted luminal [Ca(2+)] conditioned on each state of the stochastically gating channel. Both methods are subsequently used to study the role of luminal depletion in the dynamics of Ca(2+) puff/spark termination in release sites composed of Ca(2+) channels that are activated, but not inactivated, by cytosolic Ca(2+). The probability density approach shows that such minimal Ca(2+) release site models may exhibit puff/spark-like dynamics in either of two distinct parameter regimes. In one case, puffs/spark termination is due to the process of stochastic attrition and facilitated by rapid Ca(2+) domain collapse [cf. DeRemigio, H., Smith, G., 2005. The dynamics of stochastic attrition viewed as an absorption time on a terminating Markov chain. Cell Calcium 38, 73-86]. In the second case, puff/spark termination is promoted by the local depletion of luminal Ca(2+).     

Enhancement of barrier function by overexpression of claudin-4 in tight junctions of submandibular gland cells

In salivary glands, primary saliva is produced by acini and is modified by the reabsorption and secretion of ions in the ducts. Thus, the permeability of intercellular junctions in the ducts is considered to be lower than in the acini. We have examined the relationship between the expressed claudin isotypes and the barrier functions of tight junctions in a submandibular gland epithelial cell line, SMIE. SMIE cells were originally derived from rat submandibular duct cells, but their barrier functions are not as efficient as those of Madin-Darby canine kidney cells. Large molecules, such as 70-kDa dextran, diffuse across the monolayers, although E-cadherin and occludin, adherens junction and tight junction proteins, respectively, are expressed in SMIE cells. Claudin-3 protein has also been detected, but the expression level of claudin-3 mRNA is much lower than in the original submandibular glands. Other claudins including claudin-4 (originally expressed in the duct cells) have not been detected. Because of the limited expression of claudins, SMIE cells are suitable for studying the role(s) of claudins. To examine the function of claudin-4 in submandibular glands, we have overexpressed green fluorescence protein (GFP)-fused claudin-4 in SMIE cells. Cells that express GFP-fused claudin-4 have a higher transepithelial electrical resistance and a lower permeability of 70-kDa dextran, although the expression levels of occludin and claudin-3 are hardly affected. Therefore, claudin-4 plays a role in the regulation of the barrier function of tight junctions in submandibular glands. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (16591868), by a Nihon University Multidisciplinary Research Grant for 2006 and 2007, and by a Grant-in-Aid for a 2003 Multidisciplinary Research Project from MEXT.     

Asymmetrical, agonist-induced fluctuations in local extracellular [Ca(2+)] in intact polarized epithelia.

We recently proposed that extracellular Ca(2+) ions participate in a novel form of intercellular communication involving the extracellular Ca(2+)-sensing receptor (CaR). Here, using Ca(2+)-selective microelectrodes, we directly measured the profile of agonist-induced [Ca(2+)]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid-secreting cells. The Ca(2+)-mobilizing agonist carbachol elicited a transient, La(3+)-sensitive decrease in basolateral [Ca(2+)] (average approximately 250 microM, but as large as 530 microM). Conversely, carbachol evoked an HgCl2-sensitive increase in [Ca(2+)] (average approximately 400 microM, but as large as 520 microM) in the lumen of single gastric glands. Both responses were significantly reduced by pre-treatment with sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump inhibitors or with the intracellular Ca(2+) chelator BAPTA-AM. Immunofluorescence experiments demonstrated an asymmetric localization of plasma membrane Ca(2+) ATPase (PMCA), which appeared to be partially co-localized with CaR and the gastric H(+)/K(+)-ATPase in the apical membrane of the acid-secreting cells. Our data indicate that agonist stimulation results in local fluctuations in [Ca(2+)]ext that would be sufficient to modulate the activity of the CaR on neighboring cells.     

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.     

Transient receptor potential canonical channels are required for in vitro endothelial tube formation

In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca(2+) oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells.     

Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae

In response to heat-stable enterotoxin of Vibrio cholerae non-O1, the initial rise of cytosolic Ca(2+) occurred with activation of IP(3). Chelation of extracellular Ca(2+) with EGTA and suspension of cells in Ca(2+) free buffer both demonstrated the involvement of internal stores in the rise of [Ca(2+)]i. Cells pretreated with dantrolene resulted in decrease of [Ca(2+)]i response which suggested that the rise of intracellular level of Ca(2+) was mostly due to the mobilization from IP(3) sensitive stores. When the cytosolic Ca(2+) was chelated by loading the cells with BAPTA, NAG-ST could not induce Ca(2+) entry to the cell as assessed by Mn(2+) quenching of fura-2 fluorescence which suggested that calcium influx across the plasma membrane depends upon initial rise of this bivalent cation that maintained the sustained phase of [Ca(2+)]i response. Addition of toxin to the fura-2-loaded cells, preincubated with lanthanum chloride, resulted in reduction of [Ca(2+)]i level with a short duration of irregular sustained phase further suggesting that the influx of Ca(2+) across the plasma membrane might be through the calcium channel.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

STIM1-dependent and STIM1-independent function of transient receptor potential canonical (TRPC) channels tunes their store-operated mode

Ca(2+) influx by store-operated Ca(2+) channels is a key component of the receptor-evoked Ca(2+) signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca(2+) influx. Recent studies have revealed how STIM1 activates TRPC1 in response to store depletion; however, the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. Here, we established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). Significantly, WT but not mutant TRPC channels were inhibited by scavenging STIM1 with Orai1(R91W), indicating the STIM1 dependence and independence of WT and mutant TRPC channels, respectively. Importantly, mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca(2+) influx.     

Inositol 1,4,5-trisphosphate-independent calcium signalling by platelet-derived growth factor in the human SH-SY5Y neuroblastoma cell.

In adherent SH-SY5Y human neuroblastoma cells, activation of G-protein-coupled muscarinic M3 receptors evoked a biphasic elevation of both intracellular [Ca(2+)] ([Ca(2+)]i) and inositol-1,4,5-trisphosphate (D-Ins(1,4,5)P3) mass. In both cases, temporal profiles consisted of rapid transient elevations followed by a decline to a lower, yet sustained level. In contrast, platelet-derived growth factor (PDGF), a receptor tyrosine kinase agonist acting via PDGF receptor b chains in these cells, elicited a slow and transient elevation of [Ca(2+)]i that returned to basal levels within 5 to 10 min with no evidence of inositol phosphate generation. Full responses for either receptor type required intracellular and extracellular Ca(2+) and mobilization of a shared thapsigargin-sensitive intracellular Ca(2+) store. Strategies that affected the ability of D-Ins(1,4,5)P3 to interact with the Ins(1,4,5)P3-receptor demonstrated an Ins(1,4,5)P3-dependency of the muscarinic receptor-mediated elevation of [Ca(2+)]i but showed that PDGF-mediated elevations of [Ca(2+)]i are Ins(1,4,5)P3-independent in these cells.     

Na+-Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na⁺-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.     

Activation of Transient Receptor Potential Canonical 3 (TRPC3)-mediated Ca2+ Entry by A1 Adenosine Receptor in Cardiomyocytes Disturbs Atrioventricular Conduction

Although the activation of the A(1)-subtype of the adenosine receptors (A(1)AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A(1)AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A(1)AR by CCPA induced sarcolemmal Ca(2+) entry. However, A(1)AR stimulation did not induce Ca(2+) release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A(1)AR-enhanced Ca(2+) entry. Ca(2+) entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A(1)AR-enhanced Ca(2+) entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A(1)AR-induced conduction disturbances in the embryonic heart. Our data showing that A(1)AR activation subtly mediates a proarrhythmic Ca(2+) entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca(2+) entry and cardiac function are altered. Thus, the A(1)AR-TRPC3 axis may represent a potential therapeutic target.     

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.     

Impaired Orai1-mediated resting Ca2+ entry reduces the cytosolic [Ca2+] and sarcoplasmic reticulum Ca2+ loading in quiescent junctophilin 1 knock-out myotubes

In the absence of store depletion, plasmalemmal Ca(2+) permeability in resting muscle is very low, and its contribution in the maintenance of Ca(2+) homeostasis at rest has not been studied in detail. Junctophilin 1 knock-out myotubes (JP1 KO) have a severe reduction in store-operated Ca(2+) entry, presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T-tubule junction, leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of the Orai1-mediated Ca(2+) entry pathway on overall Ca(2+) homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca(2+) entry, [Ca(2+)](rest), and intracellular Ca(2+) content compared with WT myotubes and unlike WT myotubes, are refractory to BTP2, a Ca(2+) entry blocker. JP1 KO myotubes show down-regulation of Orai1 and Stim1 proteins, suggesting that this pathway may be important in the control of resting Ca(2+) homeostasis. WT myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca(2+) homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and -3 but overexpress TRPC4 and -6; on the other hand, the TRPC expression profile in Orai1(E190Q) myotubes was comparable with WT. These data suggest that an important fraction of resting plasmalemmal Ca(2+) permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca(2+)](rest) and resting Ca(2+) stores and that this pathway is defective in JP1 KO myotubes.     

The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system

We previously reported that phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a lipid product of phosphoinositide 3-kinase (PI3K), induced Ca(2+) influx via a noncapacitative pathway in platelets, Jurkat T cells, and RBL-2H3 mast cells. The identity of this Ca(2+) influx system, however, remains unclear. Here, we investigate a potential link between PIP(3)-sensitive Ca(2+) entry and the canonical transient receptor potential (TRPC) channels by developing stable human embryonic kidney (HEK) 293 cell lines expressing TRPC1, TRPC3, TRPC5, and TRPC6. Two lines of evidence support TRPC6 as a putative target by which PIP(3) induces Ca(2+) influx. First, Fura-2 fluorometric Ca(2+) analysis shows the ability of PIP(3) to selectively stimulate [Ca(2+)](i) increase in TRPC6-expressing cells. Second, pull-down analysis indicates specific interactions between biotin-PIP(3) and TRPC6 protein. Our data indicate that PIP(3) activates store-independent Ca(2+) entry in TRPC6 cells via a nonselective cation channel. Although the activating effect of PIP(3) on TRPC6 is reminiscent to that of 1-oleoyl-2-acetyl-sn-glycerol, this activation is not attributable to the diacylglycerol substructure of PIP(3) since other phosphoinositides failed to trigger Ca(2+) responses. The PIP(3)-activated Ca(2+) entry is inhibited by known TRPC6 inhibitors such as Gd(3+) and SKF96365 and is independent of IP(3) production. Furthermore, we demonstrated that TRPC6 overexpression or antisense downregulation significantly alters the amplitude of PIP(3)- and anti-CD3-activated Ca(2+) responses in Jurkat T cells. Consequently, the link between TRPC6 and PIP(3)-mediated Ca(2+) entry provides a framework to account for an intimate relationship between PI3K and PLCgamma in initiating Ca(2+) response to agonist stimulation in T lymphocytes.     

Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).     

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects.