Role of calmodulin and myosin light chain kinase in the activation of carbachol-activated cationic current in murine ileal myocytes

We investigated the effect of calmodulin (CaM) and myosin light chain kinase (MLCK) on murine ileal myocytes using the whole-cell patch-clamp technique. Under the voltage clamp, at the holding potential of -60 mV, 50 micromol/L carbachol (CCh) induced inward currents (I CCh), and spontaneous decay of I CCh occurred. The peak inward currents induced by the repetitive application of CCh (50 micromol/L) tended to decrease in amplitude. Intracellular application of 0.2 mmol/L guanosine 5'-O-(gamma-thio)triphosphate (GTP gammaS) from the patch electrode induced an inward current at a holding potential of -60m V, and the peak inward currents induced by the repetitive application of Cs tended to decrease slightly in amplitude. The amplitude of I CCh was reduced by pretreatment either with W-7, trifluoroperazine, W-5, and melittin (CaM inhibitors) or with ML-7 and ML-9 (selective MLCK inhibitors), and the inhibitory effects were reversible. However, when we pretreated with 50 micromol/L W-7 or 5 micromol/L ML-7 on GTP gammaS-induced inward currents, almost no inhibition was observed in the inward currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTP gammaS-induced currents. We conclude that CaM and MLCK modulate the activation process of I CCh in murine ileal myocytes and suggest that the classical type transient receptor potential (TRPC) channel 5 might be a candidate for nonselective cationic currents (NSCC) activated by muscarinic stimulation in gastrointestinal smooth muscle cells.     

The Transient Receptor Potential Protein Homologue TRPC4/5 as a Candidate for the Nonselective Cationic Channel Activated by Muscarinic Stimulation in the Murine Stomach

The transient receptor potential protein homologue TRPC5 was reported as a molecular identity for the muscarinic receptor-activated nonselective cationic channel (NSCC) in the murine stomach smooth muscle. The canonical, or classical, transient receptor potential proteins, TRPC4 and TRPC5, were suggested as members of the same subfamily of TRPC channels and to be coexpressed as a heteromultimer of both TRPC as well as a homotetramer of each TRPC protein. Thus, we investigated whether the TRPC4 channel is also responsible for the NSCC activated by acetylcholine (ACh) or carbachol (CCh) using electrophysiological techniques. The TRPC channels were expressed in HEK293 cells. When murine TRPC4 channels (mTRPC4) were expressed, the current–voltage relationship of mTRPC4 was also similar to that recorded in native murine gastric myocytes or mTRPC5-expressing HEK cells. With 0.2 mM GTPγS in the pipette solution, the currents in mTRPC4-expressing cells were activated transiently like those in NSCC in the murine stomach and the expressed mTRPC5. The currents recorded in mTRPC4-expressing cells were inhibited by 1 mM La³⁺ and 100 μM flufenamate. The currents recorded in mTRPC4-expressing cells depended on the extracellular calcium concentration. From the above results, we suggest that mTRPC4/5 might be candidates for the NSCC activated by ACh or CCh in the murine stomach.     

Desensitization of canonical transient receptor potential channel 5 by protein kinase C

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channel in mammalian cells. TRPC5 is desensitized rapidly after activation by G protein-coupled receptor. Herein we report our investigation into the desensitization of mTRPC5 and localization of the molecular determinants of this desensitization using mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 microM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). Increased EGTA or omission of MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 microM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19-36), inhibited this desensitization of TRPC5 activated by 100 microM CCh. When TRPC5 current was activated by intracellular GTPgammaS, PKC inhibitors prevented TRPC5 desensitization and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and suggest that threonine at residue 972 of mouse TRPC5 might be required for its phosphorylation by PKC.     

Functional characteristics of TRPC4 channels expressed in HEK 293 cells

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by GTPγS was not desensitized. TPRC4 activation by GTPγS was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK _a of 7.3. Finally, TPRC4 activation by GTPγS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles. These authors contributed equally to this work.     

The specific activation of TRPC4 by Gi protein subtype

The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Especially, TRPC4 has the similar properties to Ca²⁺-permeable nonselective cation channels (NSCCs) activated by muscarinic stimulation in visceral smooth muscles. In visceral smooth muscles, NSCCs activated by muscarinic stimulation were blocked by anti-Gαi/o antibodies. However, there is still no report which Gα proteins are involved in the activation process of TRPC4. Among Gα proteins, only Gαi protein can activate TRPC4 channel. The activation effect of Gαi was specific for TRPC4 because Gαi has no activation effect on TRPC5, TRPC6 and TRPV6. Coexpression with muscarinic receptor M2 induced TRPC4 current activation by muscarinic stimulation with carbachol, which was inhibited by pertussis toxin. These results suggest that Gαi is involved specifically in the activation of TRPC4.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

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.     

Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5

Mammalian transient receptor potential channels (TRPCs) form a family of Ca(2+)-permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca(2+) entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca(2+) store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G(q/11)-coupled receptors or receptor tyrosine kinases induced Mn(2+) entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G(q/11)-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). However, depletion of intracellular Ca(2+) stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at -60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPgammaS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I(CRAC), the most extensively characterized store-operated current.     

Stage Depandent Inhibition of Plasmodiun falcipaium falciparum by Potent Ca2+ and Calmodulin Modulators

The effects Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 μM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 μM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, ditiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythocyte invasion by merozoites. Fluroscence microscopy with the cationic flurescent dye rhodamine 123 revealed that nicardipine. TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although al Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

Stage-dependent inhibition of Plasmodium falciparum by potent Ca2+ and calmodulin modulators

The effects of Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 microM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 microM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, diltiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythrocyte invasion by merozoites. Fluorescence microscopy with the cationic fluorescent dye rhodamine 123 revealed that nicardipine, TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although all Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

Lack of kinase regulation of canonical transient receptor potential 3 (TRPC3) channel-dependent currents in cerebellar Purkinje cells

Canonical transient receptor potential (TRPC) channels are widely expressed in the brain and play several roles in development and normal neuronal function. In the cerebellum, Purkinje cell TRPC3 channels underlie the slow excitatory postsynaptic potential observed after parallel fiber stimulation. In these cells TRPC3 channel opening requires stimulation of metabotropic glutamate receptor 1, activation of which can also lead to the induction of long term depression (LTD), which underlies cerebellar motor learning. LTD induction requires protein kinase C (PKC) and protein kinase G (PKG) activation, and although PKC phosphorylation targets are well established, virtually nothing is known about PKG targets in LTD. Because TRPC3 channels are inhibited after phosphorylation by PKC and PKG in expression systems, we examined whether native TRPC3 channels in Purkinje cells are a target for PKG or PKC, thereby contributing to cerebellar LTD. We find that in Purkinje cells, activation of TRPC3-dependent currents is not inhibited by conventional PKC or PKG to any significant extent and that inhibition of these kinases does not significantly impact on TRPC3-mediated currents either. Based on these and previous findings, we propose that TRPC3-dependent currents may differ significantly in their regulation from those overexpressed in expression systems.     

Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca(2+)-conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (DT40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-beta. We conclude that TRPC3 channels are activated independently of InsP(3)Rs through DAG production resulting from receptor-mediated activation of either PLC-gamma or PLC-beta.     

Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.     

Regulation of Cl- channels by multifunctional CaM kinase.

cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.     

Activation of calcium-dependent kinases and epidermal growth factor receptor regulate muscarinic acetylcholine receptor-mediated MAPK/ERK activation in thyroid epithelial cells

We previously showed that stimulation of muscarinic acetylcholine receptors (mAChR) by carbachol (Cch) caused a time- and dose-dependent increase of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) phosphorylation in thyroid epithelial cells. In this study, we demonstrated that mAChR stimulation also induced a time-dependent increase in the tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), which was prevented by pretreatment of thyroid epithelial cells with the specific Src-family tyrosine kinase inhibitor PP2. Besides, phosphorylation of Pyk2 was attenuated by chelation of extracellular Ca(2+) or inhibition of phospholipase C (PLC), and was evoked by thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor. Incorporation of Pyk2 antisense oligonucleotides in thyroid epithelial cells to down-regulated Pyk2 expression or pretreatment of cells with the Ca(2+)/calmodulin protein kinase II (CaM kinase II) inhibitor KN-62 significantly reduced Cch-induced MAPK/ERK phosphorylation. In addition, Cch-induced MAPK/ERK phosphorylation was partially inhibited by LY294002 and wortmannin, two selective inhibitors of phosphatidylinositol 3-kinase (PI3K), tyrphostin AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, and (-)-perillic acid, a post-translational inhibitor of small G-proteins isoprenylation. Taken together, our data suggest that Pyk2, CaM kinase II and Src-family tyrosine kinases are key molecules for the activation of MAPK/ERK cascade through the EGFR/Ras/Raf pathway in thyroid epithelial cells in response to mAChR stimulation.     

Glutamate induces focal adhesion kinase tyrosine phosphorylation and actin rearrangement in heterologous mGluR1-expressing CHO cells via calcium/calmodulin signaling

Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) stimulate phospholipase C (PLC) and lead to mobilization of intracellular Ca(2+) and activation of protein kinase C (PKC). In this investigation, using heterologous receptor-expressing Chinese hamster ovary (CHO) cells, we showed that stimulation of mGluR1 or mGluR5 with glutamate rapidly increases tyrosine phosphorylation of focal adhesion kinase (FAK) (maximum at 1-3 min) in a dose-dependent manner (half-maximal responses at approximately 2 microM). In mGluR1-expressing cells, the glutamate-induced increase of FAK tyrosine phosphorylation was blocked by not only the PLC inhibitor, U73122, but also depletion of intracellular Ca(2+) and effectively abrogated by calmodulin (CaM) inhibitors, calmidazolium and fluphenazine. However, neither the PKC inhibitor, GF109203X, nor the CaM kinase II inhibitor, KN-62, inhibited glutamate-stimulated FAK tyrosine phosphorylation. Stimulation of mGluR1 caused a marked increase in actin stress fiber formation. Importantly, this actin rearrangement was prevented by the CaM inhibitor, but not by the PKC inhibitor and is thus in a good agreement with the signaling cascade of the mGluR1-FAK pathway. These results suggest that the Ca(2+)/CaM signaling and its downstream FAK tyrosine phosphorylation play an important role in cellular function of mGluR1.     

Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.     

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).     

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.     

Involvement of the phospholipase C beta1 pathway in desensitization of the carbachol-activated nonselective cationic current in murine gastric myocytes

In murine gastrointestinal myocytes muscarinic stimulation activates nonselective cation channels via a G-protein and Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to murine gastric myocytes held at -60 mV, using the whole-cell patch-clamp method. The properties of the inward cationic currents were similar to those of the nonselective cation channels activated by muscarinic stimulation in other gastrointestinal smooth muscle cells. CCh-induced ICCh and spontaneous decay of ICCh (desensitization of ICCh) occurred. Unlike the situation in guinea pig gastric myocytes, desensitization was not affected by varying [EGTA]i. Pretreatment with the PLC inhibitor (U73122) blocked the activation of ICCh, and desensitization of ICCh was attenuated in PLC beta1 knock-out mice. These results suggest that the desensitization of ICCh in murine gastric myocytes is not due to a pathway dependent on intracellular Ca2+ but to the PLC beta1 pathway.