Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries

Abstract

Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca²⁺-activated Cl⁻ channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca²⁺ signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl⁻ currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca²⁺ chelator that abolishes local Ca²⁺ signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K⁺) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca²⁺ signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction.

arterial smooth muscle cells (myocytes) express several different classes of ion channels that regulate membrane potential and intracellular Ca²⁺ concentration ([Ca²⁺]_i) to control contractility (28). These proteins include channels that are permeant to K⁺, Ca²⁺, and Cl⁻, as well as nonselective cation channels (4, 12, 14, 19). An understanding of the signaling processes that control the activity of these ion channels provides a better understanding of mechanisms that regulate arterial contractility. Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a recently described Ca²⁺-activated Cl⁻ (Cl_Ca) channel expressed in arterial myocytes that regulates membrane potential and contractility (4). Mechanisms that control ANO1 activity in arterial myocytes are poorly understood but important to determine given the functional significance of this protein in the vasculature.

ANO1 channel message and protein have been described in the vasculature, including rat cerebral, pulmonary, and carotid arteries, murine portal vein, retinal and skeletal muscle arterioles, and cultured rat pulmonary artery myocytes (7, 15, 23, 30). Several lines of evidence indicate that ANO1 channels generate Cl_Ca currents in arterial myocytes. Recombinant and myocyte Cl_Ca currents exhibit similar Ca²⁺ dependence and current-voltage relationship linearization by an elevation in [Ca²⁺]_i (6, 23, 30). ANO1 knockdown reduced Cl_Ca current density in rat cerebral and mesenteric arteries and cultured pulmonary artery myocytes (23, 30). T16Ainh-A01, an ANO1 inhibitor, relaxed methoxamine-contracted murine and human blood vessels (8). Selective ANO1 knockdown attenuated intravascular pressure-induced cerebral artery depolarization and vasoconstriction (5). Cell-specific knockout of ANO1 reduced Cl_Ca currents in aortic and cerebral arteriole myocytes, agonist-induced vasoconstriction in retinal and skeletal muscle arterioles, and systemic blood pressure and attenuated hypertension in mice (15). These studies indicate that ANO1 channels generate functional Cl_Ca currents in arterial myocytes.

Ca²⁺ is a principal intracellular activator of ANO1 channels, but the source(s) of Ca²⁺ that activate ANO1 and ion channels that generate these intracellular Ca²⁺ signals is unclear (4). Intracellular Ca²⁺ signals can arise from the opening of plasma membrane Ca²⁺-permeant channels, including voltage-dependent Ca²⁺ and transient receptor potential (TRP) channels and intracellular Ca²⁺ release from sarcoplasmic reticulum (SR) ryanodine-sensitive Ca²⁺ release channels and inositol trisphosphate receptors (IP₃Rs) (12, 14, 17, 26). In cerebral artery myocytes, ANO1 channels are activated by local Ca²⁺ influx through yet unidentified plasma membrane nonselective cation channels (5). Arterial myocytes are proposed to express nonselective cation channels, including multiple members of the TRP family (12). The goal of this study was to identify plasma membrane nonselective cation channels that control ANO1 currents in cerebral artery myocytes.

Here, we show that ANO1 and canonical TRP 6 (TRPC6) channels colocalize in close spatial proximity in the plasma membrane in cerebral artery myocytes. Hyp9, a selective TRPC6 channel activator, stimulated ANO1 currents that were blocked by BAPTA, a fast Ca²⁺ chelator that abolishes local Ca²⁺ signals. Hyp9 also constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01, an ANO1 inhibitor. These data indicate that TRPC6 and ANO1 channels are spatially localized in the cerebral artery myocyte plasma membrane and that TRPC6 channel activation leads to local Ca²⁺ signaling that stimulates ANO1 currents, leading to vasoconstriction.

MATERIALS AND METHODS

Tissue preparation and smooth muscle cell isolation.

Animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of Tennessee Health Science Center. Male Sprague-Dawley rats (6–8 wk old) were euthanized by pentobarbital sodium injection (150 mg/kg ip). The brain was removed, and resistance-size cerebral arteries (posterior cerebral, cerebellar, and middle cerebral, <200 μm diameter) were harvested and maintained in ice-cold (4°C), oxygenated (21% O₂-5% CO₂-74% N₂) physiological saline solution (PSS) containing (in mmol/l) 112 NaCl, 4.8 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 1.8 CaCl₂, 1.2 MgSO₄, and 10 d-glucose (pH 7.4). A HEPES-buffered solution (in mmol/l: 55 NaCl, 5.6 KCl, 10 HEPES, 80 sodium glutamate, 2 MgCl₂, and 10 d-glucose, pH 7.4) was used to dissociate individual myocytes from cerebral arteries, as previously described (5). Cells were maintained at 4°C and used for experimentation within 8 h.

Coimmunoprecipitation.

For each experiment, ice-cold radioimmunoprecipitation buffer was used to harvest lysate from cerebral arteries pooled from two to six rats. Coimmunoprecipitation (co-IP) was performed using the Catch and Release v2.0 co-IP kit (Millipore) according to the manufacturer's instructions. Briefly, arterial lysate was incubated with rabbit IgG or rabbit anti-ANO1 polyclonal antibody (Abcam), antibody-affinity ligand, and capture resin in the column provided. The column was then centrifuged and washed, and bound proteins were released using denaturing buffer. Boiled eluate was run on a SDS-polyacrylamide gel, and protein samples were analyzed by Western blotting.

Protein analysis and Western blotting.

Intact rat cerebral arteries were homogenized using Laemmli buffer (2.5% SDS, 10% glycerol, 0.01% bromphenol blue, and 5% β-mercaptoethanol in 100 mmol/l Tris·HCl, pH 6.8) and centrifuged at 6,000 g for 10 min to remove cellular debris. Proteins (50 μg/lane) were separated on 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Blots were physically cut between 75 and 150 kDa to permit probing for different proteins. The higher-molecular-mass portion was probed for melastatin TRP 4 (TRPM4) and IP₃R1. The lower-molecular-mass blot was probed for ANO1, TRPC3, TRPC6, and actin. Membranes were incubated with primary antibodies (1:250 dilution) overnight at 4°C in Tris-buffered saline with 0.1% Tween 20 and 5% nonfat dry milk. Primary antibodies were as follows: rabbit anti-ANO1 (Sigma-Aldrich), goat anti-TRPC6 (Santa Cruz Biotechnology), rabbit anti-TRPC3 (Alomone Labs), rabbit anti-TRPM4 (Thermo Fisher Scientific), mouse anti-IP₃R1 (University of California, Davis), and mouse anti-actin (EMD Millipore). Bands on Western blots were visualized using horseradish peroxidase-conjugated secondary antibodies and a West Pico chemiluminescence kit (Thermo Fisher Scientific, Rockford, IL) and a Kodak Image F-Pro system. Protein band intensities were determined using Quantity One software (Bio-Rad). In knockdown experiments, for quantification of total protein levels, band intensities were first normalized to actin and then to appropriate control samples.

Immunofluorescence resonance energy transfer and confocal imaging.

Isolated arterial myocytes were fixed in paraformaldehyde and then permeabilized with 0.1% Triton X-100 for 1 min at room temperature. After 1 h of incubation in phosphate-buffered saline (PBS) containing 5% BSA, cells were treated overnight at 4°C with rabbit anti ANO1 (Sigma-Aldrich) and either goat anti-TRPC6 (Santa Cruz Biotechnology), goat anti-TRPM4 (Santa Cruz Biotechnology), or mouse anti-IP₃R1 (NeuroMab) antibody at a dilution of 1:100 in PBS containing 0.5% BSA. Cells were washed with PBS and then incubated for 1 h at 37°C with secondary antibodies: Alexa 488-conjugated chicken anti-rabbit for ANO1, Alexa 546-conjugated donkey anti-mouse for IP₃R1, and Alexa 546-conjugated donkey-anti goat for TRPC6 and TRPM4 (Life Technologies) at a dilution of 1:100 in PBS containing 0.5% BSA. After the cells were washed and mounted, fluorescence images were acquired using a laser-scanning confocal microscope (LSM Pascal, Zeiss). Images were acquired using a z-resolution of ∼0.8 μm. Alexa 488 and Alexa 546 were excited at 488 and 543 nm and emission was collected at 505–530 and >560 nm, respectively. Percentage of weighted colocalization was calculated using Pascal system-embedded software. For normalized Förster resonance energy transfer (N-FRET) analysis, images were background-subtracted and N-FRET was calculated on a pixel-by-pixel basis for the entire image and in regions of interest using the Xia method and Zeiss LSM FRET Macro tool version 2.5, as previously described (20).

Arterial electroporation and protein knockdown.

Three small interference RNA (siRNA) sequences targeting ANO1 or a scrambled (scrm) control (Invitrogen) were inserted into myocytes of intact arterial segments using an electroporator (model CUY21Vivo-SQ, BEX) (5). Transfected arteries were maintained in serum-free DMEM-Ham's F12 medium supplemented with 1% penicillin-streptomycin (Sigma-Aldrich) for 3 days under standard conditions (21% O₂-5% CO₂-74% N₂ at 37°C) and then used in Western blot and patch-clamp electrophysiology experiments. Band intensities of proteins from arteries transfected with ANO1 siRNA and scrm siRNA were compared on the same membrane.

Patch-clamp electrophysiology.

Patch-clamp electrophysiology was performed using myocytes isolated from scrm siRNA- or ANO1 siRNA-transfected arteries. Membrane currents were recorded using an Axopatch 200B amplifier equipped with a CV 203BU headstage, Digidata 1332A, and Clampex 10.3 (Molecular Devices). Pipettes were pulled from borosilicate glass, heat-polished to 1–3 MΩ, and waxed to reduce capacitance. The pipette solution contained (in mmol/l) 140 CsCl, 10 HEPES, 10 d-glucose, 1 EGTA or 1 BAPTA, 1 MgATP, and 0.2 NaGTP (pH 7.2). Total MgCl₂ and CaCl₂ were adjusted to give a final free Mg²⁺ of 1 mmol/l and a free Ca²⁺ of 200 nmol/l. Free Mg²⁺ and Ca²⁺ were calculated using WebmaxC Standard (http://www.stanford.edu/∼cpatton/webmaxcS.htm) and confirmed using a Ca²⁺-sensitive and reference electrode (Corning, Acton, MA). Bath solution contained (in mmol/l) 140 NMDG-Cl, 10 d-glucose, 10 HEPES, 2 CaCl₂, and 1 MgCl₂ (pH 7.4). Isolated cells were voltage-clamped and maintained at a holding potential of −40 mV. Whole cell Cl⁻ currents were measured by application of 1-s voltage steps to between −80 and +100 mV in 20-mV increments. Currents were filtered at 1 kHz, digitized at 5 kHz, and normalized to membrane capacitance. Pharmacological agents were perfused into the experimental chamber. All electrical recordings were performed at room temperature (22°C).

Pressurized artery diameter measurements.

Experiments were performed using fresh isolated middle cerebral arteries and PSS gassed with 21% O₂-5% CO₂-74% N₂ (pH 7.4). The endothelium was denuded by introduction of an air bubble into the lumen for ∼1 min and then washed with PSS.

Endothelium-denuded artery segments 1–2 mm long were cannulated at each end in a perfusion chamber (Living Systems Instrumentation) that was maintained at 37°C and continuously perfused with PSS. Intravascular pressure was altered using an attached reservoir and monitored using a pressure transducer. Arterial diameter was measured at 1 Hz using a CCD camera attached to a Nikon TS100-F microscope and the automatic edge detection function of IonWizard software (Ionoptix, Milton, MA). Luminal flow was absent during experiments. Arteries that did not dilate to carbachol (10 μM), an endothelium-dependent vasodilator, were used for all experiments.

Statistical analysis.

OriginLab and GraphPad InStat software were used for statistical analyses. Data are expressed as means ± SE. Student's t-test was used to compare paired and unpaired data from two populations, and ANOVA with Bonferroni's post hoc test was used for multiple group comparisons. P < 0.05 was considered significant. Power analysis was performed on all data where P > 0.05 to verify that sample size was sufficient to give a power value of >0.8.

RESULTS

ANO1 and TRPC6 channels are spatially colocalized in resistance-size cerebral arteries.

Ion channels that associate with ANO1 in resistance-size (∼200 μm) cerebral arteries were identified using co-IP. An anti-ANO1 antibody pulled down ANO1 together with a strong signal for TRPC6 channels in rat cerebral artery lysate. Blots were also probed for TRPC3, TRPM4, and IP₃R1, the principal functional IP₃R isoform in cerebral artery myocytes (35). A very faint IP₃R1 band was observed, but TRPC3 and TRPM4 were absent in the ANO1 immunoprecipitate. All proteins were absent in lysate exposed to rabbit IgG (Fig. 1A). These data suggest that ANO1 and TRPC6 channels are located in a macromolecular complex that does not contain TRPC3, TRPM4, or IP₃R1 in cerebral arteries.

Fig. 1.

Anoctamin 1 (ANO1) and canonical transient receptor potential (TRPC) 6 (TRPC6) channels coimmunoprecipitate (co-IP) and localize in close spatial proximity in cerebral artery myocytes. A: co-IP was performed using rabbit anti-ANO1 or rabbit IgG polyclonal antibodies. Blots were probed for ANO1, TRPC6, TRPC3, melastatin transient receptor potential (TRPM) 4 (TRPM4), or inositol trisphosphate receptor 1 (IP₃R1). Data are representative of 2 separate co-IP experiments performed using cerebral arteries from a total of 8 rats. B: confocal images illustrating donor, acceptor, overlay, and normalized Förster resonance energy transfer (N-FRET) for primary antibodies for ANO1, TRPC6, TRPM4, and IP₃R1. Scale bars = 10 μm. C: mean data for ANO1-TRPC6 (n = 7), ANO1-TRPM4 (n = 6), and ANO1-IP₃R1 (n = 9). *P < 0.05 vs. ANO1-TRPM4. #P < 0.05 vs. ANO1-IP₃R1.

Immuno-FRET microscopy was used to examine spatial proximity of ANO1 to TRPC6 in isolated cerebral artery myocytes. ANO1 and TRPC6 immunofluorescence exhibited surface localization and generated a strong immuno-FRET signal that was primarily present on the plasma membrane in myocytes (Fig. 1B). The mean N-FRET signal between ANO1- and TRPC6-tagged secondary antibodies was ∼23.3%, suggesting close spatial proximity of these proteins (Fig. 1C). In contrast, ANO1- and TRPM4-bound secondary antibodies produced a weak FRET signal of ∼8.4% (Fig. 1, B and C). The FRET signal between ANO1- and IP₃R1-bound secondary antibodies was similarly weak at ∼7.2% (Fig. 1C). Given that the Förster coefficient for the Alexa 488-Alexa 546 FRET pair used in each of these experiments is ∼6.3 nm, the data suggest that ANO1 and TRPC6 are located in close spatial proximity in the plasma membrane of arterial myocytes.

TRPC6 channels stimulate ANO1 currents in arterial myocytes.

Using patch-clamp electrophysiology, we tested the hypothesis that TRPC6 channels stimulate ANO1 currents in arterial myocytes. Solutions were designed to isolate Cl⁻ currents and to abolish K⁺ and Na⁺ currents. The bath and pipette solutions contained 2 mM and 200 nM free Ca²⁺, respectively, creating a driving force for Ca²⁺ influx at voltages negative to +117 mV, the reversal potential for Ca²⁺.

Hyp9 is a stable derivative of hyperforin, a selective TRPC6 channel activator (21, 22). Hyp9 increased mean Cl⁻ currents from ∼2.2 to 12.3 pA/pF at +100 mV and from approximately −0.7 to −5.6 pA/pF at −80 mV, or ∼5.7- and 8.2-fold, respectively. Hyp9 also reduced the Cl⁻ current rectification index (I₈₀/I₋₈₀) from 2.34 ± 0.31 to 1.44 ± 0.10, a shift toward current-voltage relationship linearity consistent with ANO1 activation by intracellular Ca²⁺ (P < 0.05; Fig. 2). T16Ainh-A01, an ANO1 inhibitor, did not alter control currents but reduced Hyp9-activated Cl⁻ currents from ∼12.3 to 6.0 pA/pF at +100 mV and from −5.6 to −2.5 pA/pF at −80 mV, or ∼51% and 55%, respectively (Fig. 2). T16Ainh-A01 did not alter the current rectification index (I₈₀/I₋₈₀ = 1.52 ± 0.08) when applied in the presence of Hyp9 (P > 0.05; Fig. 2). These data suggest that TRPC6 channel activation stimulates ANO1 currents in arterial myocytes.

Fig. 2.

Hyp9, a TRPC6 channel activator, stimulates ANO1 Cl⁻ currents in cerebral artery myocytes. A: representative whole cell Cl⁻ currents recorded from the same myocyte in control, Hyp9 (10 μM), and Hyp9 (10 μM) + T16Ainh-A01 (10 μM) with a 200 nM free Ca²⁺- and EGTA-containing pipette solution. B: mean current-voltage (I-V) relationships (n = 6 for each condition, except T16Ainh-A01 alone, where n = 3). *P < 0.05 Hyp9 vs. control. #P < 0.05 Hyp9 vs. Hyp9 + T16Ainh-A01.

RNA interference was used to further examine the hypothesis that TRPC6 channels couple to ANO1 in arterial myocytes. ANO1 siRNA reduced arterial ANO1 protein to ∼55% of scrm siRNA controls (Fig. 3). In contrast, ANO1 siRNA did not alter TRPC6 protein (Fig. 3). Patch-clamp electrophysiology was performed on myocytes isolated from arteries treated with ANO1 siRNA or scrm siRNA. Hyp9 stimulated Cl⁻ currents in control siRNA-treated myocytes that were inhibited by T16Ainh-A01, similar to data obtained in fresh myocytes not treated with siRNA (Fig. 2 and Fig. 4, A and C). ANO1 siRNA did not alter baseline Cl⁻ currents but reduced Hyp9 activation of Cl⁻ currents (Fig. 4, A and B). Specifically, Hyp9 increased Cl⁻ currents from ∼2.2 to 9.5 pA/pF, or 4.3-fold, in control myocytes and from ∼1.8 to 3.9 pA/pF, or ∼2.1-fold, in ANO1 siRNA-treated myocytes (at +100 mV; Fig. 4). Mean Hyp9-activated currents in ANO1 siRNA-treated myocytes were ∼41% of those in controls (at +100 mV; Fig. 4, A and B). Similarly, T16Ainh-A01 reduced Cl⁻ currents by ∼5.1 pA/pF in controls and by ∼2.7 pA/pF in ANO1 siRNA-treated myocytes, or ∼48% (Fig. 4, A and B). These data indicate that TRPC6 channel activation stimulates ANO1 currents in cerebral artery myocytes.

Fig. 3.

ANO1 siRNA reduces ANO1, but not TRPC6, protein. A: Western blot illustrating ANO1 (100 kDa), TRPC6 (100 kDa), and actin (37 kDa) in cerebral arteries treated with scrambled (scrm) and ANO1 siRNA. B: mean data (n = 4 for each). *P < 0.05 vs. scrm.

Fig. 4.

ANO1 knockdown inhibits Hyp9-activated Cl⁻ currents. A: representative whole cell Cl⁻ currents recorded in the same scrm siRNA-treated myocyte in control, Hyp9 (10 μM), and Hyp9 (10 μM) + T16Ainh-A01 (10 μM). B: whole cell Cl⁻ currents in the same ANO1 siRNA-treated myocyte in control, Hyp9 (10 μM), and Hyp9 (10 μM) + T16Ainh-A01 (10 μM). C: mean current-voltage (I-V) relationships in scrm-treated myocytes (n = 7 for each condition). *P < 0.05 vs. control. #P < 0.05 vs. Hyp9 + T16Ainh-A01. D: mean I-V relationships in ANO1-treated myocytes (n = 5 for each). *P < 0.05, Hyp9 vs. control. #P < 0.05, Hyp9 vs. Hyp9 + T16Ainh-A01.

TRPC6 channels stimulate ANO1 currents via local Ca²⁺ signaling.

Next, we examined the mechanism by which TRPC6 channels activate ANO1 currents by studying the significance of local or global Ca²⁺ signaling. Replacement of pipette solution EGTA with BAPTA, a fast Ca²⁺ chelator, did not alter control Cl⁻ currents (Fig. 5). With a BAPTA-containing solution, Hyp9 did not activate Cl⁻ currents, nor did T16Ainh-A01 alter currents when applied in the presence of Hyp9 (Fig. 5). These data suggest that TRPC6 channel activation leads to extracellular Ca²⁺ influx and generation of a local intracellular Ca²⁺ signal that stimulates ANO1 channels in arterial myocytes.

Fig. 5.

BAPTA, a fast Ca²⁺ chelator, inhibits Hyp9-activated ANO1 currents. A: whole cell ANO1 currents recorded in a myocyte in control, Hyp9 (10 μM), and Hyp9 (10 μM) + T16Ainh-A01 (10 μM) with a 200 nM free Ca²⁺- and BAPTA-containing pipette solution. B: mean current-voltage (I-V) relationships of ANO1 currents recorded using a BAPTA-containing pipette solution (n = 5 for each group).

TRPC6 and ANO1 channels are functionally coupled in pressurized cerebral arteries.

To investigate physiological functions of TRPC6-ANO1 coupling in myocytes, cerebral artery contractility was measured. Cerebral arteries were cannulated in an experimental chamber and pressurized to 60 mmHg, a physiological intravascular pressure, to stimulate the development of myogenic tone. Changes in diameter were measured in response to Hyp9, T16Ainh-A01, and 60 mM K⁺, which stimulates arterial depolarization and, thus, vasoconstriction through voltage-dependent Ca²⁺ channel activation. Hyp9 stimulated a mean vasoconstriction of ∼11.1 μm, whereas T16Ainh-A01 produced mean vasodilation of ∼16.0 μm (Fig. 6, A and B). T16Ainh-A01 reduced Hyp-induced vasoconstriction to ∼6.2 μm, or ∼44.1% (Fig. 6, A and B). In contrast, T16Ainh-A01 did not alter vasoconstriction to 60 mM K⁺ (Fig. 6, C and D). These data suggest that TRPC6 channels stimulate vasoconstriction through ANO1 channel activation.

Fig. 6.

T16inh-A01 decreases Hyp9-induced vasoconstriction in pressurized cerebral arteries. A: representative traces illustrating Hyp9 (3 μM)-induced vasoconstriction in control and in the presence of T16Ainh-A01 (10 μM) in the same pressurized (60 mmHg) artery. B: mean data (n = 6 for each condition). *P < 0.05 vs. control. #P < 0.05, T16inh-A01 vs. Hyp9. C: representative traces illustrating 60 K⁺-induced vasoconstriction alone and in the presence of T16Ainh-A01 in the same pressurized (60 mmHg) artery. D: mean data (n = 4 for each). *P < 0.05 vs. before application of 60 K⁺.

DISCUSSION

Here, we show that ANO1 and TRPC6 channels are coupled in arterial myocytes and that this interaction stimulates vasoconstriction. Data indicate that ANO1 and TRPC6 proteins co-IP and localize in close spatial proximity in the plasma membrane. In contrast, ANO1 is not located in the same macromolecular complex as TRPC3, TRPM4, or IP₃R1, nor are these proteins in close proximity. Hyp9, a TRPC6 channel activator, stimulated Cl⁻ currents that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of EGTA with BAPTA, a fast Ca²⁺ chelator that blocks local Ca²⁺ signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. These data indicate that TRPC6 channel activation generates a local Ca²⁺ signal that stimulates nearby ANO1 channels in cerebral artery myocytes, leading to vasoconstriction.

Our co-IP experiments indicate that ANO1 and TRPC6 channels are present in the same macromolecular complex. The Förster coefficient for the 488-546-nm fluorophore pair used in the FRET experiments is 6.4 nm, indicating that ANO1 and TRPC6 are localized in close spatial proximity in arterial myocytes. Peripheral localization of the FRET signal and previous evidence that >80% of ANO1 is present in the plasma membrane indicate that the ANO1-TRPC6 complex is primarily at the cell surface in cerebral artery myocytes (30). In contrast, TRPC3, TRPM4, and IP₃R1 did not co-IP with ANO1 channels, and FRET experiments indicate that TRPM4 and IP₃R1 are not spatially localized near ANO1. These data suggest that surface TRPC3 and TRPM4 channels do not signal locally to ANO1 channels in arterial myocytes. SR IP₃R1 and plasma membrane ANO1 channels would not need to be present in the same macromolecular complex to signal locally through Ca²⁺, as they reside within different membranes. However, IP₃R1 and TRPC3 are located in a macromolecular signaling complex that bridges the SR and plasma membrane in arterial myocytes, and this complex does not contain TRPC6 or TRPM4 (3). Plasma membrane large-conductance Ca²⁺-activated (BK_Ca) channels are also located within the same complex as IP₃R1 in cerebral artery myocytes (36). Regardless, SR IP₃R1 channels would not constitute a plasma membrane Ca²⁺ influx pathway for ANO1 activation. These data suggest that multiple spatially distinct and exclusive local signaling complexes exist in arterial myocytes: one containing TRPC6 and ANO1, another containing IP₃R1, TRPC3, and BK_Ca, and yet another containing TRPM4 channels.

TRPC6 and TRPC3 channels are permeant to Na⁺ and Ca²⁺, IP₃R1 is a Ca²⁺-selective channel, and TRPM4 is a Na⁺-selective channel (12, 26). Data supporting signaling from TRPC6 to ANO1 include Hyp9 activation of Cl⁻ currents and blockade of Hyp9 activation of Cl⁻ currents by both T16Ainh-A01 and selective ANO1 knockdown in myocytes. Hyp9 is a stable derivative of hyperforin, a selective TRPC6 channel activator that does not stimulate TRPC1, TRPC3, TRPC4, TRPC5, TRPC7, TRPM6, TRPM8, TRPA1, or TRPV1 (22). Equimolar substitution of EGTA for BAPTA inhibited this signaling pathway, indicating that TRPC6 stimulates ANO1 currents through local Ca²⁺ signaling. T16Ainh-A01 did not alter the rectification index of Hyp9-activated currents, suggesting that this blocker does not directly interfere with the mechanism by which Ca²⁺ activates ANO1. ANO1 siRNA reduced ANO1 protein by approximately half, which is consistent with Hyp9 activation of a smaller current in arteries treated with siRNA. The concentration (10 μM) of T16Ainh-A01 used is maximal for ANO1 current inhibition in intestinal epithelial cells (25) but did not abolish ANO1 currents in the present study. There are several explanations for these findings, including 1) expression in arterial myocytes of an ANO1 variant that is less sensitive to T16Ainh-A01 and 2) Hyp9 activation of another local Cl_Ca channel that is not blocked by T16Ainh-A01. A slight shift in the activation kinetics of ANO1 currents in scrm siRNA-treated myocytes was observed in the presence of T16Ainh-A01. One explanation is that an alteration of ANO1 splice variation occurs when arteries are placed in serum-free culture for the period of time required to knock down ANO1. Testing this hypothesis was beyond the scope of the current investigation.

ANO1 channels are sensitive to [Ca²⁺]_i (100 nM–2 μM) higher than those that occur globally (100–300 nM) under physiological conditions in arterial myocytes, further supporting a local Ca²⁺-signaling mechanism of ANO1 activation (4, 5, 17, 29). The absence of IP₃R1 from the protein complex containing ANO1 is consistent with knowledge that IP₃R-mediated intracellular Ca²⁺ release produces little or no vasoconstriction in the cerebral artery, mesenteric artery, and urinary bladder myocytes (24, 27, 33). Rather, caveolin-1 colocalizes SR IP₃R1 and plasma membrane TRPC3 channels in close spatial proximity, enabling IP₃-induced physical coupling of these proteins, leading to a plasma membrane nonselective cation current (1–3). This pathway is activated by endothelin-1, a vasoconstrictor, does not require intracellular Ca²⁺ release, and leads to membrane depolarization, voltage-dependent Ca²⁺ channel activation, and vasoconstriction (2). In contrast, IP₃R-mediated Ca²⁺ release stimulates ANO1 in nociceptive sensory neurons and interstitial cells of Cajal of the small intestine (18, 37). TRPM4-mediated Na⁺ influx would not be expected to stimulate ANO1, which is a Ca²⁺-activated channel (12). The absence of TRPC3 and TRPM4 from protein complexes containing ANO1 suggests that ion channel spatial organization in myocytes is more structured than previously thought and raises the possibility that multiple different local signaling domains exist in the plasma membrane.

ANO1 channel inhibition produces vasodilation and attenuates agonist-induced vasoconstriction in several different vascular preparations, including rat cerebral arteries, rabbit pulmonary arteries, and mouse skeletal muscle arteries and aorta (5, 8, 13, 15). Intravascular pressure also activates ANO1 channels in rat cerebral artery myocytes, leading to membrane depolarization and vasoconstriction, termed “myogenic tone” (5). Here, we show that T16Ainh-A01, a pharmacological ANO1 inhibitor, is a pressurized endothelium-denuded cerebral vasodilator. This finding supports previous evidence using knockdown that ANO1 activation contributes to myogenic vasoconstriction, which is a myocyte-specific response (5). T16Ainh-A01 did not alter depolarization-induced vasoconstriction, supporting selectivity of this inhibitor for ANO1. TRPC6 channels are activated by G protein-coupled receptors and intravascular pressure in myocytes, including those from cerebral arteries and portal vein (16, 32). It also appears that we have shown, for the first time, that Hyp9, a selective pharmacological TRPC6 channel activator, stimulates vasoconstriction in endothelium-denuded resistance-size cerebral arteries. Hyperforin, an unstable precursor of Hyp9, promotes contraction in wild-type, but not TRPC6 knockout, mouse aorta (11). Thus, overlap exists in physiological mechanisms proposed to stimulate both ANO1 and TRPC6 channels and functional responses in vascular myocytes. We provide the first evidence that these channels communicate locally and that ANO1 activity is controlled by nearby TRPC6 channels in cerebral artery myocytes. Although we have established a local and functional signaling relationship between TRPC6 and ANO1 channels, future studies are required to examine physiological stimuli that act through this mechanism in arterial myocytes. Physiological stimuli, including intravascular pressure and vasoconstrictor agonists, activate multiple mechanisms in addition to those involving TRPC6 and ANO1 that would need to be taken into account (9). TRPC6 and ANO1 may not only interact through local Ca²⁺ signaling, but they may independently contribute to functional responses; such an investigation would require careful experimental dissection of each component. There are no selective TRPC6 channel inhibitors, a factor that has hindered functional studies of these channels. Furthermore, manipulation of TRPC6 expression may lead to modified levels of other ion channels in myocytes, creating additional technical challenges. For example, TRPC6 gene knockout in mice increased vascular contractility and elevated blood pressure due to compensatory upregulation of TRPC3 in arterial myocytes (10). TRPC6 and ANO1 have also been associated with vascular diseases, including systemic and pulmonary hypertension (13, 15, 31, 34, 38). Future studies should test the hypothesis that signaling between TRPC6 and ANO1 is modified in disease.

In summary, we demonstrate that TRPC6 and ANO1 channels are spatially localized in the cerebral artery myocyte plasma membrane and that TRPC6 channel activation leads to local Ca²⁺ signaling that stimulates ANO1 currents, leading to vasoconstriction.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-67061 and HL-110347 to J. H. Jaggar and an American Heart Association Scientist Development Grant to M. D. Leo.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Q.W, D.N., K.P.K., M.D.L. and J.H.J. developed the concept and designed the research; Q.W, D.N., K.P.K. and M.D.L. performed the experiments; Q.W, D.N., K.P.K. and M.D.L. analyzed the data; Q.W, D.N., K.P.K., M.D.L. and J.H.J. interpreted the results of the experiments; Q.W, D.N., K.P.K. and M.D.L. prepared the figures; Q.W and J.H.J. drafted the manuscript; Q.W, D.N., K.P.K., M.D.L. and J.H.J. approved the final version of the manuscript.