Damodaran Narayanan

Isoform-Selective Physical Coupling of TRPC3 Channels to IP3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Rationale: Inositol 1,4,5-trisphosphate (IP3)-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Objectives: Study mechanisms by which IP3Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. Methods and Results: Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C–coupled receptor agonist, elevated immuno-FRET between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP3R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP3-induced nonselective cation current (ICat) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP3R1 and TRPC3, IP3-induced ICat activation, and ET-1 and IP3-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP3R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP3R N-terminal region that can interact with TRPC channels activated ICat. A TRPC3 CIRB domain peptide attenuated IP3- and ET-1–induced ICat activation and vasoconstriction. Conclusions: IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to ICat activation and vasoconstriction. Close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction.

Caveolin-1 Assembles Type 1 Inositol 1,4,5-Trisphosphate Receptors and Canonical Transient Receptor Potential 3 Channels into a Functional Signaling Complex in Arterial Smooth Muscle Cells

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (ICat) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP3R1 and TRPC3 channels to communicate locally are unclear. Caveolae are plasma membrane microdomains that can compartmentalize proteins. Here, we tested the hypothesis that caveolae and specifically caveolin-1 (cav-1), a caveolae scaffolding protein, facilitate functional IP3R1 to TRPC3 coupling in smooth muscle cells of resistance-size cerebral arteries. Methyl-β-cyclodextrin (MβCD), which disassembles caveolae, reduced IP3-induced ICat activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP3-induced vasoconstriction, but did not alter TRPC3 and IP3R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82–101) also attenuated IP3-induced ICat activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP3R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP3R1 are spatially co-localized in arterial smooth muscle cells. IP3R1 and TRPC3 channel spatial localization was disrupted by MβCD and a CSD peptide. Cholesterol replenishment re-established IP3R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP3R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP3-induced physical coupling of these proteins, leading to ICat generation and vasoconstriction.

Smooth Muscle Cell α2δ-1 Subunits Are Essential for Vasoregulation by CaV1.2 Channels

Rationale: Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore-forming &agr;1 and auxiliary &agr;2&dgr; and &bgr; subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of &agr;2&dgr; subunits. Objective: We investigated the functional significance of &agr;2&dgr; subunits in myocytes of resistance-size (100 to 200 &mgr;m diameter) cerebral arteries. Methods and Results: &agr;2&dgr;-1 was the only &agr;2&dgr; isoform expressed in cerebral artery myocytes. Pregabalin, an &agr;2&dgr;-1/-2 ligand, and an &agr;2&dgr;-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of CaV1.2 &agr;1 and &agr;2&dgr;-1 subunits were present in the arterial myocyte plasma membrane. &agr;2&dgr;-1 knockdown using short hairpin RNA reduced plasma membrane-localized CaV1.2 &agr;1 subunits, caused a corresponding elevation in cytosolic CaV1.2 &agr;1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction (“myogenic tone”), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total &agr;2&dgr;-1 or CaV1.2 &agr;1 proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone. Conclusions: &agr;2&dgr;-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 &agr;1 subunits. &agr;2&dgr;-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 &agr;1 subunits, leading to vasodilation. These data identify &agr;2&dgr;-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.

Dynamic regulation of β1 subunit trafficking controls vascular contractility

Significance Plasma membrane ion channels composed of pore-forming and auxiliary subunits regulate physiological functions in virtually all cell types. A conventional view is that ion channels assemble with their auxiliary subunits prior to surface trafficking of the multiprotein complex. Arterial myocytes express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that modulate contractility and blood pressure and flow. The data here show that although most BKα subunits are plasma membrane-resident, β1 subunits are primarily intracellular in arterial myocytes. Nitric oxide, an important vasodilator, stimulates rapid surface trafficking of β1 subunits, which associate with, and activate, BK channels, leading to vasodilation. Thus, we show that rapid auxiliary subunit trafficking is a unique mechanism to control functional surface ion channel activity. Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca2+ sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

Mitochondria Control Functional CaV1.2 Expression in Smooth Muscle Cells of Cerebral Arteries

Rationale: Physiological functions of mitochondria in contractile arterial myocytes are poorly understood. Mitochondria can uptake calcium (Ca2+), but intracellular Ca2+ signals that regulate mitochondrial Ca2+ concentration ([Ca2+]mito) and physiological functions of changes in [Ca2+]mito in arterial myocytes are unclear. Objective: To identify Ca2+ signals that regulate [Ca2+]mito, examine the significance of changes in [Ca2+]mito, and test the hypothesis that [Ca2+]mito controls functional ion channel transcription in myocytes of resistance-size cerebral arteries. Methods and Results: Endothelin (ET)-1 activated Ca2+ waves and elevated global Ca2+ concentration ([Ca2+]i) via inositol 1,4,5-trisphosphate receptor (IP3R) activation. IP3R-mediated sarcoplasmic reticulum (SR) Ca2+ release increased [Ca2+]mito and induced mitochondrial depolarization, which stimulated mitochondrial reactive oxygen species (mitoROS) generation that elevated cytosolic ROS. In contrast, a global [Ca2+]i elevation did not alter [Ca2+]mito, mitochondrial potential, or mitoROS generation. ET-1 stimulated nuclear translocation of nuclear factor (NF)-&kgr;B p50 subunit and ET-1–induced IP3R-mediated mitoROS elevated NF-&kgr;B–dependent transcriptional activity. ET-1 elevated voltage-dependent Ca2+ (CaV1.2) channel expression, leading to an increase in both pressure (myogenic tone)– and depolarization-induced vasoconstriction. Baseline CaV1.2 expression and the ET-1–induced elevation in CaV1.2 expression were both reduced by IP3R inhibition, mitochondrial electron transport chain block, antioxidant treatment, and NF-&kgr;B subunit knockdown, leading to vasodilation. Conclusions: IP3R-mediated SR Ca2+ release elevates [Ca2+]mito, which induces mitoROS generation. MitoROS activate NF-&kgr;B, which stimulates CaV1.2 channel transcription. Thus, mitochondria sense IP3R-mediated SR Ca2+ release to control NF-&kgr;B–dependent CaV1.2 channel expression in arterial myocytes, thereby modulating arterial contractility.

Smooth muscle cell transient receptor potential polycystin-2 (TRPP2) channels contribute to the myogenic response in cerebral arteries

•  Intravascular pressure is reported to activate several mechanosensitive ion channels, leading to smooth muscle cell (SMC) depolarization, voltage‐dependent Ca2+ channel activation and vasoconstriction; a process known as the ‘myogenic response’. •  Polycystin‐1 and ‐2 (TRPP1 and ‐2) have been shown to differentially regulate the mesenteric artery myogenic response, with TRPP2 expression attenuating vasoconstriction. •  We show that TRPP2 is the major TRPP isoform expressed and that TRPP2 is located primarily in the plasma membrane in cerebral artery SMCs. •  Selective TRPP2 knockdown reduced swelling‐induced non‐selective cation currents (ICat) in SMCs and myogenic tone in cerebral arteries. •  These data indicate that TRPP2 activation contributes to the cerebral artery myogenic response and suggest that TRPP2 performs differential functions in different vascular beds.

Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to <or=100 pS when Cs(+) was replaced with Ca(2+). The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NP(o))-voltage curve was bell shaped, with NP(o) drastically decreasing when voltage was switched from -30 to -40 mV. NP(o) increased when intracellular calcium (Ca(2+)(i)) was raised within 0.1-100 microM to abruptly diminish with higher Ca(2+)(i). Thus maximal activity occurred within the Ca(2+)(i) range found in rat cerebral artery myocytes under physiological conditions. NP(o) was reduced by ruthenium red (80 muM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 microM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 muM), resistant to dantrolene (100 muM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

CaV1.3 Channels and Intracellular Calcium Mediate Osmotic Stress-induced N-terminal c-Jun Kinase Activation and Disruption of Tight Junctions in Caco-2 Cell Monolayers

We investigated the role of a Ca2+ channel and intracellular calcium concentration ([Ca2+]i) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-mediated intracellular Ca2+ depletion. Depletion of extracellular Ca2+ at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca2+ depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca2+ depletion partially reduced osmotic stress-induced rise in [Ca2+]i, whereas thapsigargin and extracellular Ca2+ depletion together resulted in almost complete loss of rise in [Ca2+]i. L-type Ca2+ channel blockers (isradipine and diltiazem) or knockdown of the CaV1.3 channel abrogated [Ca2+]i rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca2+ depletion, thapsigargin, or CaV1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca2+ depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that CaV1.3 and rise in [Ca2+]i play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca2+]i mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca2+ also contributes to osmotic stress-induced tight junction disruption.

Transcriptional Upregulation of α2δ-1 Elevates Arterial Smooth Muscle Cell Voltage-Dependent Ca2+ Channel Surface Expression and Cerebrovascular Constriction in Genetic Hypertension

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes composed of a pore-forming CaV1.2&agr;1 with auxiliary &agr;2&dgr; and &bgr; subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of &agr;2&dgr; subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in &agr;2&dgr;-1 and CaV1.2&agr;1 mRNA and protein in cerebral artery myocytes, with &agr;2&dgr;-1 increasing more than CaV1.2&agr;1. Other &agr;2&dgr; isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized &agr;2&dgr;-1 and CaV1.2&agr;1 proteins, surface &agr;2&dgr;-1:CaV1.2&agr;1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an &agr;2&dgr;-1 ligand, did not alter &agr;2&dgr;-1 or CaV1.2&agr;1 total protein but normalized &agr;2&dgr;-1 and CaV1.2&agr;1 surface expression, surface &agr;2&dgr;-1:CaV1.2&agr;1, CaV1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in &agr;2&dgr;-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that &agr;2&dgr;-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

Angiotensin II stimulates internalization and degradation of arterial myocyte plasma membrane BK channels to induce vasoconstriction.

Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated K(+) (BK) channel α and auxiliary β1 subunits that modulate arterial contractility. In arterial myocytes, β1 subunits are stored within highly mobile rab11A-positive recycling endosomes. In contrast, BKα subunits are primarily plasma membrane-localized. Trafficking pathways for BKα and whether physiological stimuli that regulate arterial contractility alter BKα localization in arterial myocytes are unclear. Here, using biotinylation, immunofluorescence resonance energy transfer (immunoFRET) microscopy, and RNAi-mediated knockdown, we demonstrate that rab4A-positive early endosomes traffic BKα to the plasma membrane in myocytes of resistance-size cerebral arteries. Angiotensin II (ANG II), a vasoconstrictor, reduced both surface and total BKα, an effect blocked by bisindolylmaleimide-II, concanavalin A, and dynasore, protein kinase C (PKC), internalization, and endocytosis inhibitors, respectively. In contrast, ANG II did not reduce BKα mRNA, and sodium nitroprusside, a nitric oxide donor, did not alter surface BKα protein over the same time course. MG132 and bafilomycin A, proteasomal and lysosomal inhibitors, respectively, also inhibited the ANG II-induced reduction in surface and total BKα, resulting in intracellular BKα accumulation. ANG II-mediated BK channel degradation reduced BK currents in isolated myocytes and functional responses to iberiotoxin, a BK channel blocker, and NS1619, a BK activator, in pressurized (60 mmHg) cerebral arteries. These data indicate that rab4A-positive early endosomes traffic BKα to the plasma membrane in arterial myocytes. We also show that ANG II stimulates PKC-dependent BKα internalization and degradation. These data describe a unique mechanism by which ANG II inhibits arterial myocyte BK currents, by reducing surface channel number, to induce vasoconstriction.