Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca2+-activated Cl− (ClCa) channels in native cells. The molecular identity of proteins that generate ClCa currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate ClCa currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl− currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B–TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell ClCa currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca2+ dependence, current-voltage relationship linearization by an elevation in intracellular Ca2+ concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl− concentration, and a negative reversal potential shift when substituting extracellular I− for Cl−. A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC ClCa and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC ClCa currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate ClCa currents in cerebral artery SMCs.
- arterial smooth muscle
- transmembrane protein 16A
ca2+-activated cl− (ClCa) currents have been identified in a variety of different cell types, including vascular smooth muscle cells (SMCs) (2, 12, 16, 22, 24, 30, 32). ClCa currents in vascular SMCs can be subdivided into “classic” ClCa currents and “cGMP-dependent” ClCa currents (21, 28). cGMP-dependent ClCa currents are present in SMCs of some, but not all, arterial beds and exhibit a linear current-voltage (I-V) relationship (27). In contrast, classic ClCa currents are outwardly rectifying at low intracellular Ca2+ concentration ([Ca2+]i) (22, 23). An elevation in [Ca2+]i activates classic ClCa currents and reduces outward rectification, leading to I-V relationship linearization (22). Bestrophins, CLCs, and a human homolog of tweety-3 have been proposed as candidate classic ClCa channels, although several properties of these channels are dissimilar to those of native ClCa channels (13, 17, 23, 31, 35).
Recently, transmembrane protein (TMEM)16A has been identified as a candidate ClCa channel that displays similar characteristics to classic ClCa currents (5, 33, 38). TMEM16A belongs to a family of 10 proteins referred to as TMEM16A–TMEM16H, TMEM16J, and TMEM16K (14, 38). TMEM16A contains a putative structure of eight transmembrane domains, intracellular amino- and carboxy-termini, and a pore-forming region between transmembrane domains 5 and 6 (38). Recent evidence has suggested that mouse conduit artery SMCs and cultured rat pulmonary artery SMCs express multiple TMEM16 channel members that may generate ClCa currents in these cells (8, 26). However, the molecular identity of channels that generate ClCa currents in SMCs of resistance-size arteries that regulate blood pressure and regional flow is unclear. Furthermore, whether cerebral artery SMCs express ClCa channels and generate ClCa currents is controversial, with both positive and negative results being reported (6, 27, 36).
The goal of this study was to examine TMEM16 family member expression and cellular localization and to characterize ClCa currents in arterial SMCs of resistance-size rat cerebral arteries. Here, we demonstrate that isolated pure cerebral artery SMCs express TMEM16A channels but do not express TMEM16B–TMEM16H, TMEM16J, and TMEM16K channels. Arterial biotinylation and immunofluorescence experiments indicate that TMEM16A protein is located predominantly within the arterial SMC plasma membrane. Cerebral artery SMC ClCa currents display properties that are similar to recombinant TMEM16A channels, including Ca2+ sensitivity and Ca2+ dependence of current rectification. TMEM16A knockdown and inhibition using a pore-targeting TMEM16A antibody inhibits arterial SMC ClCa currents. These data indicate that TMEM16A channels generate plasma membrane ClCa currents in cerebral artery SMCs.
MATERIALS AND METHODS
Tissue and cell preparation.
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center. Male Sprague-Dawley rats (6–8 wk) were euthanized by an intraperitoneal injection of pentobarbital sodium (150 mg/kg). Only male rats were used to avoid potential confounding effects of the estrus cycle on ion channel expression and regulation. The brain was removed and placed into physiological saline solution of the following composition (in mM): 112 NaCl, 4.8 KCl, 24 NaHCO3, 1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 10 glucose, which was gassed with 21% O2-5% CO2-74% N2 to pH 7.4. Resistance-size (∼200-μm diameter) cerebral (posterior cerebral, cerebellar, and middle cerebral) arteries were dissected from the brain and used for experimentation. SMCs were isolated from cerebral arteries as previously described (18).
Isolated cerebral artery SMCs were manually selected using an enlarged patch pipette under a microscope. Total RNA was extracted from preparations of between 100 and 500 SMCs using the Absolutely RNA Nanoprep kit (Stratagene). Total RNA was also extracted from the rat brain using TRIzol (Invitrogen). First-strand cDNA was synthesized from 1–5 ng total RNA using Protoscript M-MULV (New England Biolabs). To identify the presence of TMEM16 family members, PCR was performed on first-strand cDNA using TMEM16 variant-specific primers (Table 1) (26). The PCR conditions used were as follows: an initial 2-min denaturation at 94°C followed by 40 cycles of denaturation (94°C for 30 s), annealing (56°C for 30 s), and extension (72°C for 1 min). PCR products were separated on a 1.5% agarose-TEA gel.
Cell culture and transfection.
Human embryonic kidney (HEK)-293 cells were maintained in DMEM (Cellgro) supplemented with 10% FBS and 1% penicillin-streptomycin (Sigma) in an incubator (21% O2-5% CO2) at 37°C. HEK-293 cells were transfected with 1) both pTriEx-4 (Novagen) encoding full-length recombinant TMEM16A (a kind gift from Dr. A. P. Naren, Department of Physiology, University of Tennessee) and pEGFP-N3, a vector encoding recombinant enhanced green fluorescent protein (EGFP) or 2) pEGFP-N3 alone (control). Transfected cells were identified for electrophysiology using EGFP epifluorescence. Electrophysiology and Western blot experiments were performed 36–72 h after HEK-293 cell transfection.
Western blot analysis.
Cerebral arteries were homogenized using Laemmli sample buffer (2.5% SDS, 10% glycerol, 0.01% bromphenol blue, and 5% β-mercaptoethanol in 100 mM Tris·HCl; pH 6.8) and centrifuged at 6,000 g for 10 min to remove cellular debris. Proteins (40 μg/lane) were separated on a 7.5% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were then incubated with rabbit monoclonal anti-TMEM16A (1:500 dilution, Abcam) and, when appropriate, mouse monoclonal anti-actin (1:5,000 dilution, Chemicon) antibodies overnight at 4°C in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk. Proteins were visualized using horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution, Pierce) and a chemiluminescent detection kit (Pierce). Band intensity was quantified by digital densitometry using Quantity One software (Bio-Rad). TMEM16A band intensity was normalized to that of actin.
Surface proteins were biotinylated in intact cerebral arteries as previously described (1, 3, 4, 7). Briefly, arteries were incubated in a 1 mg/ml mixture each of EZ-Link sulfo-NHS-LC-LC-biotin and Maleimide-PEG2-biotin reagents (Pierce) in PBS (Invitrogen) at room temperature for 1 h. Unbound biotin was removed by washing with 100 mM glycine-PBS. For total protein determination, biotinylated arteries were homogenized in lysis buffer consisting of 50 mM Tris·HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% SDS. Total protein was quantified to allow normalization for monomeric avidin (Pierce) pulldown of biotinylated surface proteins. After pulldown, the supernatant contained the nonbiotinylated, cytosolic protein fraction. Biotinylated surface proteins bound to the avidin beads were eluted by boiling in Laemmli buffer containing 5% β-mercaptoethanol. Western blot analysis was used to identify surface and cytosolic proteins. Surface and cytosolic proteins were calculated as a percentage of total protein.
Immunofluorescence and confocal microscopy.
Isolated SMCs were fixed and prepared as previously described (39). Immunofluorescence was also performed on cells using the same protocol except that cells were not permeabilized with Triton X-100. Cells were incubated with monoclonal anti-rabbit TMEM16A antibody (1:500) at 4°C overnight followed by Alexa fluor-488-tagged secondary antibody for 2 h at room temperature. Denatured TMEM16A antibody (boiled for 15 min at 98°C) was used as a control. Immunofluorescence was collected using a Zeiss LSM5 laser-scanning confocal microscope. Alexa fluor-488 was excited with 488-nm light, and emitted light of >510 nm was collected.
Patch-clamp electrophysiology was performed using isolated cerebral artery SMCs or HEK-293 cells. Membrane currents were recorded using an Axopatch 200B amplifier equipped with a CV 203BU headstage, Digidata 1332A, and Clampex 8.2 (Molecular Devices). Pipettes were pulled from borosilicate glass, heat polished to 1–3 MΩ, and waxed to reduce capacitance. Currents were filtered at 1 kHz using a low-pass Bessel filter and digitized at 5 kHz. The pipette solution contained (in mM) 126 CsCl, 10 HEPES, 10 d-glucose, 1 EGTA, 1 MgATP, 0.2 GTP·Na, and 40 sucrose, and pH was adjusted to 7.2 with CsOH. Total MgCl2 and CaCl2 (in mM) were adjusted to 1 mM free Mg2+ and 200 nM or 1 μM free Ca2+, respectively. Free Mg2+ and Ca2+ were calculated using WebmaxC Standard (http://www.stanford.edu/∼cpatton/webmaxcS.htm). The bath solution contained (in mM) 126 N-methyl-d-glutamate chloride (NMDG-Cl), 10 HEPES, 10 d-glucose, 2 CaCl2, 1.2 MgCl2, and 40 sucrose, and pH was adjusted to 7.4 with HCl. To study anion permeability, extracellular Cl− was replaced with I−. To minimize junction potential, the reference Ag/AgCl electrode was immersed in a solution of 3 mM KCl continuous with an agar bridge (4% agar in 3 mM KCl). TMEM16A currents were measured by applying1-s voltage steps between −80 and +100 mV in 20-mV increments using an interpulse holding potential of −40 mV. Currents were normalized to membrane capacitance. Rabbit monoclonal anti-TMEM16A antibody (1:100 dilution, Abcam) was introduced directly into the experimental chamber. Boiled (15 min at 98°C) denatured TMEM16A antibody served as a control for active antibody. The relative anion permeability ratio of I− to Cl− (PI/PCl) was calculated using the calculated shift in reversal potential (Erev) and the constant field equation as follows: PI/PCl = [Cl−]o[eΔErev(zF/RT)]/[I−]o, where [Cl−]o and [I−]o are the extracellular Cl− and I− concentrations, respectively, z is charge, F is Faraday's constant, R is the gas constant, and T is temperature. zF/RT was −0.039 at 25°C.
TMEM16A channel knockdown.
Three small interfering (si)RNA sequences targeting TMEM16A or negative control siRNA (Invitrogen) were inserted into cerebral arteries using reverse permeabilization as previously described (1, 3, 4, 25, 39). Arteries were then maintained in serum-free DMEM-F-12 media supplemented with 1% penicillin-streptomycin (Sigma) for 4 days at 37°C in a sterile incubator (21% O2-5% CO2). Western blot analysis was used to compare the effect of TMEM16A siRNA and control siRNA on protein expression. Band intensities of proteins from arteries treated with either TMEM16A siRNA or control siRNA were compared on the same membranes.
Results are expressed as means ± SE. GraphPad Instat software was used for statistical analysis. Significance was determined using Student's t-tests for paired or unpaired data or ANOVA with a Bonferroni post hoc test for multiple data sets. P < 0.05 was considered statistically significant. Where P > 0.05, power analysis was performed to verify that sample size gave a value of >0.8.
TMEM16A channels are expressed in arterial SMCs of resistance-size rat cerebral arteries.
To investigate the expression of TMEM16A–TMEM16H, TMEM16J, and TMEM16K channel family members, RT-PCR was performed on RNA extracted from populations (∼100–500) of manually selected isolated rat cerebral artery SMCs and on the whole brain. Isolated arterial SMC cDNA amplified transcripts for myosin heavy chain 11 (Myh11), a SMC-specific marker, but did not amplify markers for endothelial cells [platelet-endothelial cell adhesion molecule-1 (PECAM-1)] or astrocytes [aquaporin-4 (AQP-4)], potential contaminating vascular wall cell types (Fig. 1A). In contrast, rat brain cDNA amplified transcripts for SMC-specific (Myh11), endothelial cell-specific (PECAM-1), and astrocyte-specific (AQP-4) markers (Fig. 1A). RT-PCR on pure arterial SMC cDNA amplified transcripts for TMEM16A channels but did not amplify transcripts for TMEM16B–TMEM16H, TMEM16J, or TMEM16K channels (Fig. 1A). In contrast, whole rat brain cDNA amplified transcripts for all 10 TMEM16 family members, including 2 transcripts each for TMEM16F and TMEM16K (Fig. 1A). In summary, these data indicate that cerebral artery SMCs contain message for TMEM16A but not TMEM16B–TMEM16H, TMEM16J, or TMEM16K family members.
Western blot analysis was used to measure TMEM16A expression in cerebral arteries and HEK-239 cells transfected with a vector encoding recombinant TMEM16A. We used a monoclonal antibody raised against one intracellular and one extracellular TMEM16A epitope. The extracellular epitope is located in the loop between transmembrane domains 5 and 6 near the predicted pore (38). The TMEM16A antibody detected a protein band of ∼115 kDa, the predicted molecular mass of TMEM16A channels, in cerebral artery lysate (Fig. 1B). To determine the identity of this protein band, Western blot analysis was performed using recombinant TMEM16A. The same monoclonal antibody detected a similar size protein in lysates from HEK-239 cells expressing TMEM16A (Fig. 1C). In contrast, TMEM16A protein was absent in control HEK-293 cells (Fig. 1C). These data indicate that TMEM16A channels are expressed in arterial SMCs of resistance-size rat cerebral arteries.
TMEM16A channels are primarily located within the cerebral artery SMC plasma membrane.
To examine TMEM16A channel localization in arterial SMCs, surface biotinylation was performed in intact cerebral arteries, as we have previously done (1, 3, 4, 7). Arterial surface biotinylation followed by Western blot analysis indicated that ∼81% of TMEM16A is located within the plasma membrane (Fig. 2, A and B). Immunofluorescence was performed on fixed permeabilized and nonpermeabilized arterial SMCs exposed to the same TMEM16A antibody followed by Alexa fluor-488-tagged secondary antibodies. In permeabilized cells, confocal imaging revealed the predominant localization of TMEM16A in the arterial SMC plasma membrane (Fig. 2C). In nonpermeabilized cells, the TMEM16A antibody labeled the plasma membrane, consistent with recognition of an extracellular epitope (Fig. 2C). No fluorescence was observed in cells exposed to boiled denatured TMEM16A antibody followed by Alexa fluor-488-tagged secondary antibody (Fig. 2C). Collectively, these data indicate that TMEM16A channels are primarily located in the arterial SMC plasma membrane.
TMEM16A contributes to native ClCa currents in arterial SMCs.
To investigate whether TMEM16A channels contribute to ClCa currents in cerebral artery SMCs, patch-clamp electrophysiology was performed. Experiments were carried out using a pipette solution primarily containing CsCl and a bath solution primarily containing NMDG-Cl to limit cation currents. Whole cell Cl− currents were measured in both arterial SMCs and HEK-293 cells expressing recombinant TMEM16A channels. With 200 nM free Ca2+ in the pipette solution, outwardly rectifying Cl− currents were measured in arterial SMCs (Fig. 3, A and B). Elevating the pipette free Ca2+ concentration from 200 nM to 1 μM increased Cl− current density and reduced outward rectification in SMCs, reducing the mean rectification index (I80/I−80) from 4.0 to 1.0 (Fig. 3, A and B). Elevating [Ca2+]i from 200 nM to 1 μM also increased the proportion of peak ClCa current activated immediately after the voltage step from 0.61 ± 0.04 (n = 7) to 0.78 ± 0.06 (n = 5, at +100 mV). Cl− currents in either 200 nM or 1 μM Ca2+ were recorded as long as the patch could be maintained (up to 30 min), indicating that rundown was minimal. To quantify rundown, current density was compared 2 min (to allow pipette solution dialysis with the cytosol) and 18 min after patch formation. The data indicated that with 200 nM and 1 μM [Ca2+]i, currents were 94.4 ± 8.6% (n = 6) and 97.6 ± 2.0% (n = 6) of control after this 16-min time period. These data indicate that ClCa currents do not run down.
Next, we investigated the contribution of Cl−to Ca2+-activated membrane currents in arterial SMCs. Reduction of extracellular Cl− in the NMDG-Cl bath solution from 132 to 46 mM by equimolar replacement with aspartate shifted Erev in the same cells from −3.2 ± 4.5 to 27.2 ± 8.9 mV, or by 30.4 mV (Fig. 4). The calculated shift in Cl− equilibrium potential in this experiment is 27.5 mV, consistent with currents being primarily due to Cl−. Equimolar replacement of 132 mM external Cl− with I− induced an approximately −21.2-mV hyperpolarizing shift in Erev, indicating a PI/PCl of 2.28 (Fig. 4B).
Extracellular application of the TMEM16A antibody used for biochemistry experiments reduced mean arterial SMC ClCa currents from ∼47.5 to 14.6 pA/pF, or by ∼69%, at +100 mV and from approximately −25.9 to −8.3 pA/pF, or by ∼68%, at −80 mV (Fig. 3, B and D). Next, the sensitivity of recombinant TMEM16A channels to the antibody was determined. HEK-293 cells transfected only with a vector encoding EGFP displayed small currents (Fig. 3C). In contrast, HEK-293 cells additionally transfected with the vector encoding TMEM16A channels generated large Cl− currents. The TMEM16A antibody reduced Cl− currents generated by recombinant TMEM16A channels from ∼64.8 to 19.0 pA/pF, or by ∼70%, at +100 mV and from ∼−43.5 to −10.5 pA/pF, or by ∼76%, at −80 mV. In contrast, boiled denatured TMEM16A antibody did not alter arterial SMC ClCa or recombinant TMEM16A current density (Fig. 3, B and D). These data indicate that TMEM16A channels contribute to native ClCa currents in cerebral artery SMCs.
TMEM16A knockdown attenuates ClCa currents in arterial SMCs.
To further examine the molecular identity of channels that generate ClCa currents in cerebral artery SMCs, RNA inhibition was used. siRNA targeting TMEM16A was inserted into cerebral arteries using reverse permeabilization followed by 4-day culture in serum-free DMEM. Western blot analysis indicated that TMEM16A siRNA reduced arterial TMEM16A protein to ∼62% of control siRNA (Fig. 5, A and B). In contrast, TMEM16A siRNA did not alter expression of an ∼75-kDa protein also detected by the antibody (92.4 ± 20.3% of control siRNA, n = 5 each for control and TMEM16A siRNA; Fig. 5A, see also Fig. 1B). Patch-clamp electrophysiology was performed on SMCs isolated from arteries treated with TMEM16A siRNA or control siRNA. TMEM16A knockdown reduced baseline ClCa current density by ∼68% in isolated SMCs (Fig. 5D). These data further indicate that TMEM16A channels generate ClCa currents in cerebral artery SMCs.
Here, we investigated TMEM16 family member expression and characterized ClCa currents in SMCs of resistance-size rat cerebral arteries. We show for the first time, that TMEM16A message and protein are expressed in cerebral artery SMCs. In contrast, TMEM16B– TMEM16H, TMEM16J, and TMEM16K family members are not present. Our findings indicate that TMEM16A protein is primarily located within the arterial SMC plasma membrane. Cerebral artery SMC ClCa currents exhibited properties similar to those of recombinant TMEM16A currents, including outward rectification at low [Ca2+]i. An elevation in [Ca2+]i activated ClCa currents and linearized the I-V relationship in cerebral artery SMCs. A TMEM16A antibody inhibited both arterial SMC ClCa and recombinant TMEM16A currents. Similarly, TMEM16A siRNA reduced both arterial TMEM16A protein and SMC ClCa currents. These data indicate that TMEM16A channels are expressed in SMCs of resistance-size cerebral arteries and generate plasma membrane ClCa currents.
ClCa currents have been described in SMCs from a variety of anatomically different blood vessels, including conduit vessels and pulmonary, retinal, cerebral, and coronary arteries (2, 6, 23, 24, 27, 29). However, the molecular identity of proteins that generate ClCa currents in SMCs of resistance-size arteries was unclear. Three recent studies have described TMEM16A expression in the vasculature. Davis et al. (8) identified TMEM16A and TMEM16B message and TMEM16A protein in lysates from whole murine conduit arteries, which contain multiple cells types, including SMCs and endothelial cells. Immunofluorescence identified TMEM16A channels in murine conduit artery SMCs (8). Sones et al. (34) identified a 160-kDa protein using TMEM16A antibodies from murine whole portal vein, although the antibodies used were not validated. Manoury et al. (26) amplified transcripts for TMEM16A, TMEM16B, TMEM16D, TMEM16E, TMEM16F, and TMEM16K in cultured rat pulmonary artery SMCs. Here, we demonstrate that pure, noncultured SMCs from resistance-size cerebral arteries contain message only for TMEM16A channels. We identified TMEM16A protein in cerebral arteries using Western blot analysis. Immunofluorescence also identified TMEM16A expression in isolated arterial SMCs. Therefore, our data indicate that rat cerebral artery SMCs express only TMEM16A channels. Differences in our data and those of previous studies may be due to the size of arteries studied, the anatomic location of the vasculature, the use of pure SMCs versus the whole vasculature, the use of cell culture, and/or species (8, 26, 34).
TMEM16F and TMEM16K primers each amplified two different size transcripts from whole brain lysates. To date, only one rat sequence has been published for TMEM16F and one for mouse TMEM16K. In contrast, five human splice variants each have been reported for TMEM16F and TMEM16K. Alignment of our primer sequences revealed that they span splice sites in human TMEM16F and TMEM16K variants that are sufficient to account for the difference in the size of the two transcripts for TMEM16F and TMEM16K shown in Fig. 1A. These data suggest that rat TMEM16F and TMEM16K are spliced similarly to those in humans at the locations spanned by our primers.
Conceivably, cerebral artery SMC TMEM16A channels may have been present primarily intracellularly and contributed little to plasma membrane Cl− conductance. To determine the membrane to intracellular distribution of endogenous vascular TMEM16A channels, we performed arterial surface biotinylation. We (1, 3, 4, 7) previously developed this technique and have used it to measure the cellular localization of Cav1.2, transient receptor potential (TRP)C3, and TRPM4 channels as well as α2δ-1 subunits in cerebral artery SMCs. Our data indicate that >80% of TMEM16A is present within the plasma membrane. The remaining intracellular TMEM16A may be present within the Golgi apparatus and be available for membrane trafficking or be present on intracellular organelle membranes. The biotinylation data were supported by immunofluorescence evidence showing that TMEM16A channels were present primarily within the plasma membrane of isolated cerebral artery SMCs. In contrast, in conduit vessels and pulmonary artery SMCs, immunofluorescence experiments indicated that TMEM16A channels were located primarily intracellularly with limited plasma membrane localization (8). These data indicate that TMEM16A channels are present primarily in the cerebral artery SMC plasma membrane and, therefore, should contribute to ClCa currents. Conceivably, in SMCs of conduit vessels and pulmonary arteries, TMEM16A channels may be located mostly intracellularly (8).
The biophysical properties, voltage dependence, and regulation by niflumic acid, a nonspecific ClCa channel blocker, of murine conduit vessel SMC ClCa currents and recombinant TMEM16A currents are similar (8). Here, we compared the voltage and Ca2+ dependence of cerebral artery SMC Cl− currents with those of recombinant TMEM16A channels. Pipette and bath solutions used were Na+ and K+ free to isolate Cl− currents and block cation flux during experimentation. Data indicated that with 200 nM free [Ca2+]i, cerebral artery SMCs generate outwardly rectifying Cl− currents consistent with the properties of recombinant TMEM16A currents (5, 33, 38). An elevation in free [Ca2+]i to 1 μM activated cerebral artery SMC ClCa currents and reduced outward rectification, consistent with recombinant TMEM16A currents (5, 33, 38). With 1 μM free [Ca2+]i in the pipette solution, reducing extracellular Cl− induced a Nerstian shift in current Erev, indicating that currents were primarily due to Cl−. Substitution of I− for Cl− induced a negative shift in Erev similar to that described in rabbit pulmonary artery SMCs (30). ClCa channels in SMCs are more permeant to I− than Cl−, and the relative permeability value for I− of 2.28 determined here is consistent with that observed for TMEM16A channels (38). ClCa currents in cerebral artery SMCs exhibited properties similar to those described in other SMCs, including anion permeability, Ca2+ sensitivity, Ca2+-dependent regulation of current rectification, activation kinetics, and negligible rundown (23). The bath solution we used contained NMDG-Cl and the pipette solution contained Cs+ to inhibit K+ currents. Several TRP channels are expressed in cerebral artery SMCs, including TRPC3, TRPC6, and TRPM4 (9, 37). Conceivably, Cs+ efflux through nonselective cation channels could have contributed to outward currents. TRPM4 channels are low-affinity Ca2+-activated proteins with half-maximal activation at 200 μM [Ca2+]i in cerebral artery SMCs (11). Over the [Ca2+]is used here (200 nM–1 μM), TRPM4 channels would not be active. Furthermore, ClCa currents were measured for up to 30 min with no rundown, a time point well beyond which TRPM4 currents fully run down when using the whole cell configuration (∼20 s) (10). Therefore, TRPM4 channels are unlikely to have contributed to the Ca2+-activated currents described here. These data indicate that ClCa currents in cerebral artery SMCs demonstrate properties similar to recombinant TMEM16A channels.
A lack of specific pharmacological modulators has limited studies attempting to determine the molecularly identity of native ClCa channels. Cl− channel blockers not only inhibit multiple different Cl− channels but can also modify the activity of cation channels, including large-conductance Ca2+-activated K+ channels (15). Our study examined ClCa current regulation using the same TMEM16A antibody used for Western blot analysis and immunofluorescence experiments. This antibody was raised to two epitopes, one of which is an extracellular epitope located in the loop between transmembrane domains 5 and 6, the predicted pore-forming region (38). Therefore, we hypothesized that this antibody would be a TMEM16A channel inhibitor. Extracellular application of the TMEM16A antibody inhibited both isolated arterial SMC ClCa and recombinant TMEM16A currents. As a complimentary approach, we used RNA inhibition, which similarly reduced TMEM16A protein in cerebral arteries and whole cell ClCa currents in SMCs isolated from siRNA-treated arteries. TMEM16A siRNA also reduced ClCa currents in cultured rat pulmonary artery SMCs, although it was not confirmed that this approach reduced TMEM16A protein (26). These data indicate that TMEM16A channels contribute to ClCa currents in cerebral artery SMCs.
Cl− is the most abundant intracellular anion in vascular SMCs, with an intracellular Cl− concentration of ∼50 mM (23). The estimated Erev for Cl− is approximately −30 to −20 mV (19). The physiological voltage range of rat cerebral arteries from fully dilated to fully constricted occurs between membrane potentials of approximately −60 and −20 mV, which elevates global arterial wall [Ca2+]i from ∼100 to 350 nM (20). With physiological ionic gradients, Cl− channel activation results in Cl− efflux and arterial myocyte depolarization. TMEM16A channel activation would lead to membrane depolarization, voltage-dependent Ca2+ channel activation, and vasoconstriction. Therefore, SMC TMEM16A channels, by controlling Cl− flux, would regulate arterial diameter. Future studies will aim to identify intracellular signals that activate TMEM16A channels in cerebral artery SMCs and the functional significance of such modulation.
In conclusion, we demonstrate that TMEM16A is the only TMEM16 family member expressed in cerebral artery SMCs. We show that TMEM16A channels are present primarily in the plasma membrane and that TMEM16A channels generate ClCa currents in cerebral artery SMCs. Identifying TMEM16A channels in these cells will ultimately lead to a better understanding of the mechanisms that control cerebrovascular contractility and regional brain blood flow.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-67061, HL-094378, and HL-110347 (to J. H. Jaggar) and K01-HL-096411 (to A. Adebiyi).
No conflicts of interest, financial or otherwise, are declared by the author(s).
The authors thank Dr. A.P. Naren for the vector encoding recombinant TMEM16A channels.
- Copyright © 2011 the American Physiological Society