Obligatory role for PKCδ in PIP2 -mediated activation of store-operated TRPC1 channels in vascular smooth muscle cells.

KEY POINTS: In vascular smooth muscle cells (VSMCs), activation of Ca2+ -permeable store-operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease. Activation of TRPC1-based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). We investigated the identity of the PKC isoform involved in activating TRPC1-based SOCs in rat mesenteric artery VSMCs. TRPC1-based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ-dependent phosphorylation of TRPC1. Furthermore, generation of store-operated interactions between PIP2 and TRPC1 and activation of TRPC1-based SOCs by PIP2 required PKCδ. These findings reveal that PKCδ activity has an obligatory role in activating TRPC1-based SOCs, through regulating PIP2 -mediated channel opening. ABSTRACT: In vascular smooth muscle cells (VMSCs), stimulation of Ca2+ -permeable canonical transient receptor potential channel 1 (TRPC1)-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1-based SOCs are activated. Stimulation of TRPC1-based SOCs requires protein kinase C (PKC) activity, with store-operated PKC-dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Experimental protocols used to activate TRPC1-based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+ -insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1-based SOCs in contractile rat mesenteric artery VSMCs. Store-operated whole-cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1-based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store-operated PIP2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP2 -mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1-based SOCs and highlight that store-operated PKCδ activity is obligatory for channel opening by PIP2 , the probable activating ligand.

Insights into Activation Mechanisms of Store-Operated TRPC1 Channels in Vascular Smooth Muscle.

In vascular smooth muscle cells (VMSCs), the stimulation of store-operated channels (SOCs) mediate Ca2+ influx pathways which regulate important cellular functions including contraction, proliferation, migration, and growth that are associated with the development of vascular diseases. It is therefore important that we understand the biophysical, molecular composition, activation pathways, and physiological significance of SOCs in VSMCs as these maybe future therapeutic targets for conditions such as hypertension and atherosclerosis. Archetypal SOCs called calcium release-activated channels (CRACs) are composed of Orai1 proteins and are stimulated by the endo/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) following store depletion. In contrast, this review focuses on proposals that canonical transient receptor potential (TRPC) channels composed of a heteromeric TRPC1/C5 molecular template, with TRPC1 conferring activation by store depletion, mediate SOCs in native contractile VSMCs. In particular, it summarizes our recent findings which describe a novel activation pathway of these TRPC1-based SOCs, in which protein kinase C (PKC)-dependent TRPC1 phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) are obligatory for channel opening. This PKC- and PIP2-mediated gating mechanism is regulated by the PIP2-binding protein myristoylated alanine-rich C kinase (MARCKS) and is coupled to store depletion by TRPC1-STIM1 interactions which induce Gq/PLCß1 activity. Interestingly, the biophysical properties and activation mechanisms of TRPC1-based SOCs in native contractile VSMCs are unlikely to involve Orai1.

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells.

Ca2+-permeable store-operated channels (SOCs) mediate Ca2+ entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCß1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1-/- mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1-/- VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1-/- VSMCs, store depletion induced PLCß1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1-/- VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCß1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.

Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCß1 to induce TRPC1 channel activation in vascular smooth muscle cells.

KEY POINTS: Depletion of Ca2+ stores activates store-operated channels (SOCs), which mediate Ca2+ entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)ß1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques. Store-operated TRPC1 channel and PLCß1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1-/- cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCß1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCß1/PKC activity to induce channel gating. ABSTRACT: In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)ß1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca2+ sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1-/- VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCß1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1-/- cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCß1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCß1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCß1 activity to induce PKC phosphorylation of TRPC1 and channel gating.

Store depletion induces Gαq-mediated PLCß1 activity to stimulate TRPC1 channels in vascular smooth muscle cells.

Depletion of sarcoplasmic reticulum (SR) Ca(2+) stores activates store-operated channels (SOCs) composed of canonical transient receptor potential (TRPC) 1 proteins in vascular smooth muscle cells (VSMCs), which contribute to important cellular functions. We have previously shown that PKC is obligatory for activation of TRPC1 SOCs in VSMCs, and the present study investigates if the classic phosphoinositol signaling pathway involving Gαq-mediated PLC activity is responsible for driving PKC-dependent channel gating. The G-protein inhibitor GDP-ß-S, anti-Gαq antibodies, the PLC inhibitor U73122, and the PKC inhibitor GF109203X all inhibited activation of TRPC1 SOCs, and U73122 and GF109203X also reduced store-operated PKC-dependent phosphorylation of TRPC1 proteins. Three distinct SR Ca(2+) store-depleting agents, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, cyclopiazonic acid, and N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamineed, induced translocations of the fluorescent biosensor GFP-PLCδ1-PH from the cell membrane to the cytosol, which were inhibited by U73122. Knockdown of PLCß1 with small hairpin RNA reduced both store-operated PLC activity and stimulation of TRPC1 SOCs. Immunoprecipitation studies and proximity ligation assays revealed that store depletion induced interactions between TRPC1 and Gαq, and TRPC1 and PLCß1. We propose a novel activation mechanism for TRPC1 SOCs in VSMCs, in which store depletion induces formation of TRPC1-Gαq-PLCß1 complexes that lead to PKC stimulation and channel gating.

Myristoylated alanine-rich C kinase substrate coordinates native TRPC1 channel activation by phosphatidylinositol 4,5-bisphosphate and protein kinase C in vascular smooth muscle.

Canonical transient receptor potential 1 (TRPC1) Ca(2+)-permeable cation channels contribute to vascular tone and blood vessel remodeling and represent potential therapeutic targets for cardiovascular disease. Protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are obligatory for native TRPC1 channel activation in vascular smooth muscle cells (VSMCs) but how PKC and PI(4,5)P2 act together to induce channel gating remains unresolved. The present study reveals that myristoylated alanine-rich C kinase substrate (MARCKS) protein coordinates activation of TRPC1 channels by PKC and PI(4,5)P2. TRPC1 channels and MARCKS form signaling complexes with PI(4,5)P2 bound to MARCKS; in this configuration TRPC1 channels are closed. Activators of TRPC1 channels induce PKC phosphorylation of TRPC1 proteins, which causes dissociation of TRPC1 subunits from MARCKS and release of PI(4,5)P2 from MARCKS; PI(4,5)P2 subsequently binds to TRPC1 subunits to induce channel opening. Calmodulin acting at, or upstream of, MARCKS is also required for TRPC1 channel opening through a similar gating mechanism involving PKC and PI(4,5)P2. These novel findings show that MARCKS coordinates native TRPC1 channel activation in VSMCs by acting as a reversible PI(4,5)P2 buffer, which is regulated by PKC-mediated TRPC1 phosphorylation. Moreover, our data provide evidence that PI(4,5)P2 is a gating ligand of TRPC1 channels.

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).

Ins(1,4,5)P3 interacts with PIP2 to regulate activation of TRPC6/C7 channels by diacylglycerol in native vascular myocytes.

We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P(3) at 10 microm increased 3-fold TRPC6-like activity induced by 10 microm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P(3) had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 microm reduced phosphatidylinositol 4,5-bisphosphate (PIP(2)) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP(2) antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP(2), a water-soluble PIP(2) analogue, inhibited OAG-induced TRPC6/C7 activity with an IC(50) of 0.74 microm. Ins(1,4,5)P(3) rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP(2) in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P(3) receptor antagonist heparin. In contrast, Ins(1,4,5)P(3) did not overcome diC8-PIP(2)-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P(3)/AM (6-Ins(1,4,5)P(3)), a cell-permeant analogue of Ins(1,4,5)P(3), at 10 microm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP(2), but not TRPC6 and PIP(2). In contrast, 10 microm OAG reduced association between TRPC6 and PIP(2), but not between TRPC7 and PIP(2). The present work provides the first evidence that Ins(1,4,5)P(3) modulates native TRPC channel activity through removal of the inhibitory action of PIP(2) from TRPC7 subunits.

Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes.

We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin-1 (ET-1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET-1 evoked non-selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ET(A) or ET(B) receptor antagonists, respectively BQ-123 and BQ788, when administered separately. However, in the presence of both antagonists, ET-1-evoked channel activity was abolished indicating that both ET(A) and ET(B) receptor stimulation activate this conductance. Stimulation of both ET(A) and ET(B) receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti-TRPC1 antibodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC-dependent mechanism. ET(A) receptor-mediated TRPC1 channel activity was selectively inhibited by phosphoinositol-3-kinase (PI-3-kinase) inhibitors wortmannin (50 nM) and PI-828 and by antibodies raised against phosphoinositol-3,4,5-trisphosphate (PIP(3)), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Moreover, exogenous application of diC8-PIP(3) stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ET(A) receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP(3). In contrast, ET(B) receptor-mediated TRPC1 channel activity was inhibited by the PI-phospholipase C (PI-PLC) inhibitor U73122. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI-PLC, also activated PKC-dependent TRPC1 channel activity. OAG-induced TRPC1 channel activity was inhibited by anti-phosphoinositol-4,5-bisphosphate (PIP(2)) antibodies and high concentrations of wortmannin (20 microM) which depleted tissue PIP(2) levels. In addition exogenous application of diC8-PIP(2) activated PKC-dependent TRPC1 channel activity. These data indicate that stimulation of ET(B) receptors evokes PKC-dependent TRPC1 activity through PI-PLC-mediated generation of DAG and requires a permissive role of PIP(2). In conclusion, we provide the first evidence that stimulation of ET(A) and ET(B) receptors activate native PKC-dependent TRPC1 channels through two distinct phospholipids pathways involving a novel action of PIP(3), in addition to PIP(2), in rabbit coronary artery myocytes.

Identification of canonical transient receptor potential (TRPC) channel proteins in native vascular smooth muscle cells.

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable non-selective cation channels, which on stimulation allow influx of Na(+) and Ca(2+) ions into cells. It is proposed that stimulation of TRPC conductances by neurotransmitters and hormones such as noradrenaline, angiotensin II and endothelin-1 have important functions in vascular smooth muscle cells including vasoconstriction, cell growth and proliferation. Moreover constitutive TRPC activity contributes to setting the resting membrane potential of vascular myocytes. Activation of TRPC channels is thought to provide a direct Ca(2+) influx pathway and evoke indirect Ca(2+) entry by inducing depolarisation and opening of voltage-gated Ca(2+) channels and by stimulating the reverse mode of the Na(+)/Ca(2+) exchanger. Therefore identification of native TRPC channel proteins, which underlie these mechanisms, will provide important information on physiological functioning of vascular tissue and these conductances are pharmacological targets for the prevention of cardiovascular diseases such as hypertension. This review focuses on different experimental approaches that have been used to elucidate the molecular identity of TRPCs in native vascular myocytes. It will discuss the advantages and problems associated with using siRNA and anti-sense technologies in primary cell cultures, cell lines and transgenic mice models. In addition we describe recent work, which combines studies on the effect of anti-TRPC antibodies and pharmacological agents on biophysically characterised single cation channel currents to identify TRPC channel proteins in freshly dispersed vascular myocytes. These data provide strong evidence that native vascular myocytes contain diverse TRPC-mediated channels which are usually composed of complex heterotetrameric structures possessing marked pharmacological differences.

Role of phosphoinositol 4,5-bisphosphate and diacylglycerol in regulating native TRPC channel proteins in vascular smooth muscle.

Stimulation of receptor-operated (ROCs) and store-operated (SOCs) Ca(2+)-permeable cation channels by vasoconstrictors has many important physiological functions in vascular smooth muscle. The present review indicates that ROCs and SOCs with diverse properties in different blood vessels are likely to be explained by composition of different subunits from the canonical transient receptor potential (TRPC) family of cation channel proteins. In addition we illustrate that activation of native TRPC ROCs and SOCs involves different phospholipase-mediated transduction pathways linked to generation of diacylglycerol (DAG). Moreover we describe recent novel data showing that the endogenous phospholipid phosphoinositol 4,5-bisphosphate (PIP(2)) has profound and contrasting actions on TRPC ROCs and SOCs. Optimal activation of a native TRPC6 ROC by angiotensin II (Ang II) requires both depletion of PIP(2) and generation of DAG which leads to stimulation of TRPC6 via a PKC-independent mechanism. The data also indicate that PIP(2) has a marked constitutive inhibitory action of TRPC6 and DAG and PIP(2) are physiological antagonists on TRPC6 ROCs. In contrast PIP(2) stimulates TRPC1 SOCs and has an obligatory role in activation of these channels by store-depletion which requires PKC-dependent phosphorylation of TRPC1 proteins. Finally, we conclude that interactions between PIP(2) bound to TRPC proteins at rest, generation of DAG and PKC-dependent phosphorylation of TRPC proteins have a fundamental role in activation mechanisms of ROCs and SOCs in vascular smooth muscle.

Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes.

In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP(2), a water soluble form of PIP(2), to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP(2)-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP(2) antibodies and by depletion of tissue PIP(2) levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP(2) at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP(2) whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP(2). Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP(2) in SOC activation requires PKC-dependent phosphorylation of TRPC1.

Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes.

The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP(2)) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 microM diC8-PIP(2) in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP(2) also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) with an IC(50) of 7.6 microM. Anti-PIP(2) antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP(2) wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP(2) scavenger poly-L-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP(2) and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP(2). These results provide for the first time compelling evidence that constitutively produced PIP(2) exerts a powerful inhibitory action on native TRPC6 channels.

Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes.

In vascular smooth muscle, store-operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)-induced whole-cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin-1, CPA and the cell-permeant Ca(2+) chelator BAPTA-AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mM [Ca(2+)](o) the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mM [Ca(2+)](o) the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13-dibutyrate (PDBu), the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti-TRPC6 blocked SOCs only in coronary artery and anti-TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

Dual effect of calmodulin on store-operated Ca2+ -permeable cation channels in rabbit portal vein myocytes.

We have previously described a Ca(2+)-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes, which is activated by agents that deplete internal Ca(2+) stores and also by protein kinase C (PKC). In the present study, we investigated the effect of calmodulin (CaM) on store-operated channels (SOCs) with electrophysiological techniques. Bath application of the CaM inhibitor calmidazolium (CMZ) to quiescent cells produced transient activation of SOC activity in cell-attached patches. CMZ produced a dual effect on cyclopiazonic acid (CPA)-evoked SOCs by initially inducing an increase in mean open probability (NP(o)) and subsequently producing a marked inhibition of SOC activity. In contrast, SOCs activated by the cell-permeable Ca(2+) chelator 1,2-bis (2-aminophenoxy)ethane-N-N,N',N'-tetraacetic acid (BAPTA-AM) were inhibited by CMZ. In inside-out patches where SOCs were activated by CPA or the PKC activator phorbol-12,13-dibutyrate (PDBu), bath application of CaM induced an initial inhibition followed by an increase in SOC activity. In contrast, CaM only enhanced BAPTA-AM-evoked SOC activity in inside-out patches. Bath application of CaM to the cytoplasmic surface of quiescent inside-out patches evoked single channel currents with biophysical properties similar to SOCs. The inhibitory action of CaM on PDBu-induced SOC activity was inhibited by the calmodulin-dependent kinase II (CaM kinase II) inhibitor autocamtide-related inhibitory peptide (AIP) but not by inhibitors of calcineurin or myosin light chain kinase (MLCK). In addition, CaM-evoked channel currents were inhibited by coapplication of purified CaM kinase II but not by inhibitors of CaM kinase II, calcineurin or MLCK. With whole-cell and cell-attached recording, bath application of the CaM kinase II inhibitors KN93 and AIP evoked SOCs in unstimulated myocytes. These results indicate that CaM has pronounced dual inhibitory and excitatory actions on SOCs with the inhibitory effect of CaM mediated by CaM kinase II. Moreover, the present work provides strong evidence that CaM has an important role in activating SOCs, possibly through a direct action on channel/associated proteins.

Receptor-operated Ca2(+)-permeable nonselective cation channels in vascular smooth muscle: a physiologic perspective.

This article summarizes the literature on receptor-operated Ca2(+)-permeable nonselective cation channels in vascular smooth muscle cells. One of these conductances, the P2X1 receptor, is a classic ligand-gated channel, but others are likely to be mediated via G-protein-coupled receptors. The most studied receptor-operated channel in vascular myocytes is the norepinephrine-evoked nonselective cation channel in rabbit portal vein myocytes. The data regarding the transduction mechanisms and biophysical properties of whole-cell and single-channel currents in this preparation are described. The channels have a conductance of 20 to 25 pS and complex kinetic behavior with at least two open and two closed states. These channels are activated by norepinephrine and acetylcholine via G-protein-coupled receptors linked to phospholipase C and by diacylglycerol (DAG). The action of DAG occurs by a mechanism independent of protein kinase C, but other kinases may mediate the responses to norepinephrine and DAG. In addition, activation of tyrosine kinases leads to opening of this channel. Other vasoconstrictors, such as endothelin, vasopressin, serotonin, and angiotensin II, open Ca2(+)-permeable nonselective cation channels, but there may be differences between these conductances and the norepinephrine-evoked channels. A homologue of the transient receptor potential protein (TRPC6) is an essential component of the norepinephrine-activated channel in rabbit portal vein, and it is likely that this family of proteins plays an important role in mediating Ca2+ influx in vascular smooth muscle.

Modulation of ICl(Ca) in vascular smooth muscle cells by oxidizing and cysteine-reactive reagents.

The present study investigated the effect of redox agents on Ca2+-activated Cl- currents ( ICl(Ca)) recorded in smooth muscle cells isolated from rabbit portal vein. In perforated-patch experiments on portal vein cells the amplitude of ICl(Ca) evoked by either spontaneous release of Ca2+ from internal stores or Ca2+ influx through voltage-dependent Ca2+ channels was markedly and irreversibly enhanced by the non-specific oxidant, diamide (10-200 microM). Diamide also prolonged the decay of both currents. The reductant dithiothreitol had no effect on control ICl(Ca) but reversed the increase of current amplitude produced by diamide. Diamide also increased global intracellular Ca2+ at rest but had no effect on the time-course of Ca "sparks" determined by confocal microscopy. Diamide and the endogenous oxidant hydrogen peroxide increased the amplitude and prolonged the kinetics of ICl(Ca)evoked by pipette solutions containing free Ca2+ clamped at 500 nM. Similar effects were observed with the hydrophilic thiol-reactants thimerosal and p-chloromercuriphenylsulphonic acid (PCMPS). Therefore, the gating and activation of Ca2+-activated Cl- conductance is sensitive to redox modification.