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

Key Points: In vascular smooth muscle cells (VSMCs), activation of Ca -permeable store-operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca 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 (PIP ). We investigated the identity of the PKC isoform involved in activating TRPC1-based SOCs in rat mesenteric artery VSMCs.

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 Ca 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 Ca sensor stromal interaction molecule 1 (STIM1) following store depletion.

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

Ca-permeable store-operated channels (SOCs) mediate Ca 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 Ca 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.

Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.

Key Points: Depletion of Ca stores activates store-operated channels (SOCs), which mediate Ca 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)β1/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 depletion induces Gαq-mediated PLCβ1 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-β-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.

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 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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.