TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.     

Potentiation of TRPC5 by protons

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca(2+)-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La(3+) and Gd(3+). This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC(50) of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H(+) on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H(+) or Gd(3+) that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H(+) indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca(2+) entry and depolarization.     

Functional organization of TRPC-Ca2+ channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1-TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca(2+) influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP(2)) hydrolysis, generation of IP(3) and DAG, and IP(3)-induced Ca(2+) release from the intracellular Ca(2+) store via inositol trisphosphate receptor (IP(3)R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca(2+) entry mechanisms. The former is regulated by the emptying/refilling of internal Ca(2+) store(s) while the latter depends on PIP(2) hydrolysis (due to changes in PIP(2) per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca(2+) entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca(2+)](i) signals which are critical for precise control of downstream cellular functions.     

Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth

Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). Unlike most other TRP-related channels, which are inhibited by La(3+) and Gd(3+), currents through TRPC4 and TRPC5 are potentiated by La(3+). Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La(3+) or Gd(3+), whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La(3+) or Gd(3+) at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La(3+), but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu(543) and Glu(595)/Glu(598), situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu(595)/Glu(598) in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La(3+) and Gd(3+) have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La(3+) at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.     

Differential distribution, clustering, and lateral diffusion of subtypes of the inositol 1,4,5-trisphosphate receptor

Regulation of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) by IP(3) and Ca(2+) allows them to initiate and regeneratively propagate intracellular Ca(2+) signals. The distribution and mobility of IP(3)R determines the spatial organization of these Ca(2+) signals. Until now, there has been no systematic comparison of the distribution and mobility of the three mammalian IP(3)R subtypes in a uniform background. We used confocal microscopy and fluorescence recovery after photobleaching to define these properties for each IP(3)R subtype expressed heterologously in COS-7 cells. IP(3)R1 and IP(3)R3 were uniformly distributed within the membranes of the endoplasmic reticulum (ER), but the distribution of IP(3)R2 was punctate. The mobile fractions (M(f) = 84 ± 2 and 80 ± 2%) and diffusion coefficients (D = 0.018 ± 0.001 and 0.016 ± 0.002 μm(2)/s) of IP(3)R1 and IP(3)R3 were similar. Other ER membrane proteins (ryanodine receptor type 1 and sarco/endoplasmic reticulum Ca(2+)-ATPase type 1) and a luminal protein (enhanced GFP with a KDEL retrieval sequence) had similar mobile fractions, suggesting that IP(3)R1 and IP(3)R3 move freely within an ER that is largely, although not entirely, continuous. IP(3)R2 was less mobile, but IP(3)R2 mobility differed between perinuclear (M(f) = 47 ± 4% and D = 0.004 ± 0.001 μm(2)/s) and near-plasma membrane (M(f) = 64 ± 6% and D = 0.013 ± 0.004 μm(2)/s) regions, whereas IP(3)R3 behaved similarly in both regions. We conclude that IP(3)R1 and IP(3)R3 diffuse freely within a largely continuous ER, but IP(3)R2 is more heterogeneously distributed and less mobile, and its mobility differs between regions of the cell.     

Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.     

Homer 1 mediates store- and inositol 1,4,5-trisphosphate receptor-dependent translocation and retrieval of TRPC3 to the plasma membrane

Store-operated Ca(2+) channels (SOCs) mediate receptor-stimulated Ca(2+) influx. Accumulating evidence indicates that members of the transient receptor potential (TRP) channel family are components of SOCs in mammalian cells. Agonist stimulation activates SOCs and TRP channels directly and by inducing translocation of channels in intracellular vesicles to the plasma membrane (PM). The mechanism of TRP channel translocation in response to store depletion and agonist stimulation is not known. Here we use TRPC3 as a model to show that IP(3) and the scaffold Homer 1 (H1) regulate the rate of translocation and retrieval of TRPC3 from the PM. In resting cells, TRPC3 exists in TRPC3-H1b/c-IP(3)Rs complexes that are located in part at the PM and in part in intracellular vesicles. Binding of IP(3) to the IP(3)Rs dissociates the interaction between IP(3)Rs and H1 but not between H1 and TRPC3 to form IP(3)Rs-TRPC3-H1b/c. TIRFM and biotinylation assays show robust receptor- and store-dependent translocation of the TRPC3 to the PM and their retrieval upon termination of cell stimulation. The translocation requires depletion of stored Ca(2+) and is prevented by inhibition of the IP(3)Rs. In HEK293, dissociating the H1b/c-IP(3)R complex with H1a results in TRPC3 translocation to the PM, where it is spontaneously active. The TRPC3-H1b/c-IP(3)Rs complex is reconstituted by infusing H1c into these cells. Reconstitution is inhibited by IP(3). Deletion of H1 in mice markedly reduces the rates of translocation and retrieval of TRPC3. Conversely, infusion of H1c into H1(-/-) cells eliminates spontaneous channel activity and increases the rate of channel activation by agonist stimulation. The effects of H1c are inhibited by IP(3). These findings together with our earlier studies demonstrating gating of TRPC3 by IP(3)Rs were used to develop a model in which assembly of the TRPC3-H1b/c-IP(3)Rs complexes by H1b/c mediates both the translocation of TRPC3-containing vesicles to the PM and gating of TRPC3 by IP(3)Rs.     

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.     

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.     

TRPC1 proteins confer PKC and phosphoinositol activation on native heteromeric TRPC1/C5 channels in vascular smooth muscle: comparative study of wild-type and TRPC1-/- mice

Ca(2+)-permeable cation channels consisting of canonical transient receptor potential 1 (TRPC1) proteins mediate Ca(2+) influx pathways in vascular smooth muscle cells (VSMCs), which regulate physiological and pathological functions. We investigated properties conferred by TRPC1 proteins to native single TRPC channels in acutely isolated mesenteric artery VSMCs from wild-type (WT) and TRPC1-deficient (TRPC1(-/-)) mice using patch-clamp techniques. In WT VSMCs, the intracellular Ca(2+) store-depleting agents cyclopiazonic acid (CPA) and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) both evoked channel currents, which had unitary conductances of ～2 pS. In TRPC1(-/-) VSMCs, CPA-induced channel currents had 3 subconductance states of 14, 32, and 53 pS. Passive depletion of intracellular Ca(2+) stores activated whole-cell cation currents in WT but not TRPC1(-/-) VSMCs. Differential blocking actions of anti-TRPC antibodies and coimmunoprecipitation studies revealed that CPA induced heteromeric TRPC1/C5 channels in WT VSMCs and TRPC5 channels in TRPC1(-/-) VSMCs. CPA-evoked TRPC1/C5 channel activity was prevented by the protein kinase C (PKC) inhibitor chelerythrine. In addition, the PKC activator phorbol 12,13-dibutyrate (PDBu), a PKC catalytic subunit, and phosphatidylinositol-4,5-bisphosphate (PIP(2)) and phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) activated TRPC1/C5 channel activity, which was prevented by chelerythrine. In contrast, CPA-evoked TRPC5 channel activity was potentiated by chelerythrine, and inhibited by PDBu, PIP(2), and PIP(3). TRPC5 channels in TRPC1(-/-) VSMCs were activated by increasing intracellular Ca(2+) concentrations ([Ca(2+)](i)), whereas increasing [Ca(2+)](i) had no effect in WT VSMCs. We conclude that agents that deplete intracellular Ca(2+) stores activate native heteromeric TRPC1/C5 channels in VSMCs, and that TRPC1 subunits are important in determining unitary conductance and conferring channel activation by PKC, PIP(2), and PIP(3).     

TRPC3 channels colocalize with Na+/Ca2+ exchanger and Na+ pump in axial component of transverse-axial tubular system of rat ventricle

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na(+)-K(+)-ATPase (NKA) pump and the Na(+)/Ca(2+) exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca(2+)-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.     

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.     

Molecular determinants of PKA-dependent inhibition of TRPC5 channel

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gα(s). Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gα(s) [Gα(s) (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gα(s) (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by G(s)/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β(2)-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.     

A crucial role for cAMP and protein kinase A in D1 dopamine receptor regulated intracellular calcium transients

D1-like dopamine receptors stimulate Ca(2+) transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca(2+)(i) following 'priming' of endogenous G(q/11)-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca(2+) transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP(3) production, but pretreatment of cells with the D1R agonist potentiated G(q)-linked receptor agonist mobilization of intracellular Ca(2+) stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca(2+) oscillations in response to G(q/11)-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca(2+) responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP(3)-mediated intracellular Ca(2+) store mobilization.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture

Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca(2+) handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine (Ry)-sensitive Ca(2+) stores, store-operated Ca(2+) entry (SOCE), and receptor-operated Ca(2+) entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca(2+) levels and increased IP(3)-sensitive Ca(2+) store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca(2+) release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP(3) receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP(3)/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP(3) receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca(2+) handling that occurs during vascular growth and remodeling.     

On the role of TRPC1 in control of Ca2+ influx, cell volume, and cell cycle

Ca(+) signaling plays a crucial role in control of cell cycle progression, but the understanding of the dynamics of Ca(2+) influx and release of Ca(2+) from intracellular stores during the cell cycle is far from complete. The aim of the present study was to investigate the role of the free extracellular Ca(2+) concentration ([Ca(2+)](o)) in cell proliferation, the pattern of changes in the free intracellular Ca(2+) concentration ([Ca(2+)](i)) during cell cycle progression, and the role of the transient receptor potential (TRP)C1 in these changes as well as in cell cycle progression and cell volume regulation. In Ehrlich Lettré Ascites (ELA) cells, [Ca(2+)](i) decreased significantly, and the thapsigargin-releasable Ca(2+) pool in the intracellular stores increased in G(1) as compared with G(0). Store-depletion-operated Ca(2+) entry (SOCE) and TRPC1 protein expression level were both higher in G(1) than in G(0) and S phase, in parallel with a more effective volume regulation after swelling [regulatory volume decrease (RVD)] in G(1) as compared with S phase. Furthermore, reduction of [Ca(2+)](o), as well as two unspecific SOCE inhibitors, 2-APB (2-aminoethyldiphenyl borinate) and SKF96365 (1-(β-[3-(4-methoxy-phenyl)propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride), inhibited ELA cell proliferation. Finally, Madin-Darby canine kidney cells in which TRPC1 was stably silenced [TRPC1 knockdown (TRPC1-KD) MDCK] exhibited reduced SOCE, slower RVD, and reduced cell proliferation compared with mock controls. In conclusion, in ELA cells, SOCE and TRPC1 both seem to be upregulated in G(1) as compared with S phase, concomitant with an increased rate of RVD. Furthermore, TRPC1-KD MDCK cells exhibit decreased SOCE, decreased RVD, and decreased proliferation, suggesting that, at least in certain cell types, TRPC1 is regulated during cell cycle progression and is involved in SOCE, RVD, and cell proliferation.     

TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) results from Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through Ca(2+)-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca(2+) entry (CCE) induced by Ca(2+) store depletion represents a major Ca(2+) influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca(2+)-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca(2+) currents and CCE generated by Ca(2+) influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca(2+) channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca(2+)](cyt).