Jian Shi

Potent vasorelaxant activity of the TMEM16A inhibitor T16Ainh‐A01

T16Ainh‐A01 is a recently identified inhibitor of the calcium‐activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16Ainh‐A01 for inhibition of calcium‐activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.

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)P3) and diacylglycerol (DAG) on TRPC6‐like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P3 at 10 μm increased 3‐fold TRPC6‐like activity induced by 10 μm 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), a DAG analogue. Ins(1,4,5)P3 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 μm reduced phosphatidylinositol 4,5‐bisphosphate (PIP2) association with TRPC6 and TRPC7, and produced a 40‐fold increase in OAG‐induced TRPC6/C7 activity. Anti‐PIP2 antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8‐PIP2, a water‐soluble PIP2 analogue, inhibited OAG‐induced TRPC6/C7 activity with an IC50 of 0.74 μm. Ins(1,4,5)P3 rescued OAG‐induced TRPC6/C7 activity from inhibition by diC8‐PIP2 in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P3 receptor antagonist heparin. In contrast, Ins(1,4,5)P3 did not overcome diC8‐PIP2‐induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6‐Tri‐O‐butyryl‐Ins(1,4,5)P3/AM (6‐Ins(1,4,5)P3), a cell‐permeant analogue of Ins(1,4,5)P3, at 10 μm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP2, but not TRPC6 and PIP2. In contrast, 10 μm OAG reduced association between TRPC6 and PIP2, but not between TRPC7 and PIP2. The present work provides the first evidence that Ins(1,4,5)P3 modulates native TRPC channel activity through removal of the inhibitory action of PIP2 from TRPC7 subunits.

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

Ca2+‐permeable cation channels consisting of canonical transient receptor potential 1 (TRPC1) proteins mediate Ca2+ 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 Ca2+ 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 Ca2+ 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 (PIP2) and phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) 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, PIP2, and PIP3. TRPC5 channels in TRPC1−/−VSMCs were activated by increasing intracellular Ca2+ concentrations ([Ca2+]i), whereas increasing [Ca2+]i had no effect in WT VSMCs. We conclude that agents that deplete intracellular Ca2+ 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, PIP2, and PIP3.Shi, J., Ju, M., Abramowitz, J., Large, W. A., Birnbaumer, L., Albert, A. P. 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. FASEB J. 26, 409–419 (2012). www.fasebj.org

TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+‐ and PKC‐dependent mechanism in native vascular myocytes

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca2+ buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15–45 pS TRPC6 channels in the same outside‐out patches. However, increasing Ang II to 100 nm abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti‐TRPC1 and anti‐TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six‐fold TRPC6 channel activity evoked by 1–100 nm Ang II in outside‐out patches. Bath application of T1E3, an anti‐TRPC1 antibody raised against an extracellular epitope, also increased Ang II‐evoked TRPC6 channel activity. With high intracellular Ca2+ buffering conditions (10 mm BAPTA), 10 nm Ang II‐induced TRPC6 channel activity was increased by about five‐fold compared to channel activity with low Ca2+ buffering. In addition, increasing intracellular Ca2+ levels ([Ca2+]i) at the cytosolic surface inhibited 10 nm Ang II‐evoked TRPC6 channel activity in inside‐out patches. Moreover, in zero external Ca2+ (0 [Ca2+]o) 100 nm Ang II induced TRPC6 channel activity in outside‐out patches. Pre‐treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1–100 nm Ang II and blocked the inhibitory action of [Ca2+]i on TRPC6 channel activity. Co‐immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca2+]o and the anti‐TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5‐mediated Ca2+ influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor‐activated TRPC channels expressed in the same native VSMCs.

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) Ca2+‐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.—Shi, J., Birnbaumer, L., Large, W. A., and Albert, A. P. Myristoylated alanine‐rich C kinase substrate coordinates native TRPC1 channel activation by phosphatidylinositol 4,5‐bisphosphate and protein kinase C in vascular smooth muscle. FASEB J. 28, 244–255 (2014). www.fasebj.org

Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels

Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K+ channels in these responses. In wire myography studies, increasing [Ca2 +]o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca2 +]o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca2 +]o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca2 +-activated K+ channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca2 +]o-induced vasorelaxations. Increasing [Ca2 +]o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations.

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

In vascular smooth muscle cells (VSMCs), stimulation of TRPC1-based SOCs mediate Ca2+ entry pathways which 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 Gαq/PLCβ1 activities and PKC phosphorylation, but it is unclear how store depletion stimulates this gating pathway. The present work examines this issue by focusing on the role of STIM1, an endo/sarcoplasmic reticulum Ca2+ sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 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 PLCβ1 activity measured with the fluorescent PIP2/InsP3 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 PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, 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. Our findings 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 PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating. n nThis article is protected by copyright. All rights reservedDepletion 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)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‐operated TRPC1 channel and PLCβ1 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 PLCβ1, 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/PLCβ1/PKC activity to induce channel gating.

Store‐operated STIM1 translocation and interaction with TRPC1 at the plasma membrane stimulates PLC activity to induce channel gating in vascular smooth muscle cells

In vascular smooth muscle cells (VSMCs), stimulation of TRPC1-based SOCs mediate Ca2+ entry pathways which 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 Gαq/PLCβ1 activities and PKC phosphorylation, but it is unclear how store depletion stimulates this gating pathway. The present work examines this issue by focusing on the role of STIM1, an endo/sarcoplasmic reticulum Ca2+ sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 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 PLCβ1 activity measured with the fluorescent PIP2/InsP3 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 PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, 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. Our findings 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 PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating. n nThis article is protected by copyright. All rights reservedDepletion 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)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‐operated TRPC1 channel and PLCβ1 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 PLCβ1, 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/PLCβ1/PKC activity to induce channel gating.

Store depletion induces Gαq-mediated PLCβ1 activity to stimulate TRPC1 channels in vascular smooth muscle cells

Depletion of sarcoplasmic reticulum (SR) Ca2+ 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 TRPC 1 SOCs, and U73122 and GF109203X also reduced store‐operated PKC‐dependent phosphorylation of TRPC1 proteins. Three distinct SR Ca2+ 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 trans‐locations of the fluorescent biosensor GFP‐PLCδ1‐PH from the cell membrane to the cytosol, which were inhibited by U73122. Knockdown of PLCβ1 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 TRPC 1 and Gαq, and TRPC 1 andPLCβ1. We propose a novel activation mechanism for TRPC 1 SOCs in VSMCs, in which store depletion induces formation of TRPC1‐Gαq‐PLCβ1 complexes that lead to PKC stimulation and channel gating.—Shi, J., Miralles, F., Birnbaumer, L., Large, W. A., Albert, A. P. Store depletion induces Gαq‐mediated PLCβ1 activity to stimulate TRPC1 channels in vascular smooth muscle cells. FASEB J. 30, 702‐715 (2016). www.fasebj.org

Pharmacological profile of phosphatidylinositol 3‐kinases and related phosphatidylinositols mediating endothelinA receptor‐operated native TRPC channels in rabbit coronary artery myocytes

BACKGROUND AND PURPOSE EndothelinA (ETA) receptor‐operated canonical transient receptor potential (TRPC) channels mediate Ca2+ influx pathways, which are important in coronary artery function. Biochemical pathways linking ETA receptor stimulation to TRPC channel opening are unknown. We investigated the involvement of phosphatidylinositol 3‐kinases (PI3K) in ETA receptor activation of native heteromeric TRPC1/C5/C6 and TRPC3/C7 channels in rabbit coronary artery vascular smooth muscle cells (VSMCs).