ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle

Abstract

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to Gq-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca2+ events are modulated by a variety of ion channels including voltage-gated calcium channels (CaV1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca2+ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP3R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle-specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, CaV1.2, and IP3R in the contractile and intracellular Ca2+ signaling properties of mouse PA smooth muscle expressing the Ca2+ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of CaV1.2 or IP3R, or Ca2+ store depletion equally inhibited 5-HT-induced tone and intracellular Ca2+ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both CaV1.2 and IP3R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both CaV1.2 and IP3R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca2+ events supported by a microenvironment comprising ANO1, CaV1.2, and IP3R. In this model, ANO1 and CaV1.2 would indirectly support cyclical Ca2+ release events from IP3R and propagation of intracellular Ca2+ waves.

Figure 1.

Pharmacological block or genetic knockdown of ANO1 produces a similar inhibition of the contraction of mouse pulmonary artery to 5-HT as blocking VGCC or emptying Ca²⁺ stores from the SR. (A) Typical isometric force recordings in response to high K⁺ Krebs (85.4 mM) and increasing cumulative concentrations of 5-HT ranging from 0.01 to 30 μM as indicated by the bars above the traces, in the absence (left) or presence (right) of the ANO1 inhibitor CaCC_Inh-A01, also indicated by a horizontal bar above the trace. (B) Mean cumulative dose–response curves to 5-HT in mouse pulmonary arteries from wild-type C57/BL6 mice in the absence (black circles, Control; n = 14), or presence (blue squares; n = 5) of 1 μM nifedipine to block VGCC, 10 μM CPA to deplete SR Ca²⁺ stores (black diamonds; n = 4), 10 μM CaCC_Inh-A01 (magenta circles; n = 6) to block ANO1, or 1 μM xestospongin C to block IP₃R (magenta triangles; n = 6). Each data point is a mean ± SEM of net contractile force normalized to the second high K⁺–Krebs solution-induced contraction (see examples in A and description in Materials and methods). (C) Mean cumulative dose–response curves to 5-HT in mouse pulmonary arteries from two conditional smooth muscle-specific and inducible ANO1 KO mice (SMC-ANO1-KO), one with floxed ANO1 flanking exons 5 and 6 (SMC-ANO1-KO-^ΔEx5/6), and the other with floxed ANO1 flanking exon 12 (SMC-ANO1-KO-^ΔEx12). The cumulative dose–response relationship to 5-HT in SMC-ANO1-KO-^ΔEx12 injected with safflower oil served as a control (black squares; SMC-ANO1-KO-^ΔEx12-Oil; n = 4) and displayed remarkable similarity to the control curve obtained with wild-type mice (B). SMC-ANO1-KO-^ΔEx12 injected with tamoxifen (TMX) led to a significant reduction in contraction amplitude (upper magenta triangles; SMC-ANO1-KO-^ΔEx12-TMX; n = 5), which was unaffected by exposure to 10 μM CaCC_Inh-A01 (black diamonds; n = 3). The magnitude of the contraction to 5-HT for tamoxifen-injected SMC-ANO1-KO-^ΔEx5/6 mice was also reduced to a similar extent to that exerted by exon 12 deletion (inverted blue triangles; n = 5). (D) Reverse-transcription qPCR demonstrating ANO1 knockdown in aortic smooth muscle but not colonic tissue from both SMC-ANO1-KO mice used in this study (ANO1-KO-^ΔEx5/6 and ANO1-KO-^ΔEx12). Measurements were performed between 50 and 60 d after injection with safflower seed oil (light gray bars) or tamoxifen (open bars). ANO1 expression was normalized to β-actin. Each overlaid data point represents one animal; * and ***, significantly different from control with P < 0.05 and P < 0.001, respectively. (E) Ca²⁺-activated Cl⁻ currents were abolished in PASMCs from SMC-ANO1-KO mice. Top: Typical families of Ca²⁺-activated Cl⁻ currents (I_ClCa) recorded in PASMCs from SMC-ANO1-KO-^ΔEx12 injected with vehicle (oil-injected; traces on the left) or tamoxifen (TMX-injected; traces on the right). Currents evoked with a pipette solution set to 1 μM Ca²⁺ were recorded in response to the voltage-clamp protocol shown below the traces. Bottom: Mean current–voltage relationships for I_ClCa measured at the end of voltage-clamp steps ranging from −100 to +120 mV from a holding potential of −50 mV in PASMCs from SMC-ANO1-KO-^ΔEx12 mice injected with vehicle (SMC-ANO1-KO-Oil; filled circles) or tamoxifen (SMC-ANO1-KO-TMX; open circles). Each data point is a mean ± SEM SMC-ANO1-KO-Oil: N = 2, n = 5; SMC ANO1-KO-TMX: N = 4, n = 8; with N and n representing the number of animals and cells, respectively. For all panels, *** and * indicate a significant difference between means with P < 0.001 and P < 0.05, respectively.

Figure 2.

The ANO1 blocker CaCC_Inh-A01 produced no effect on the high K⁺-mediated contraction of the mouse pulmonary artery. (A) Typical contractile force experiment showing that increasing the concentration of CaCC_Inh-A01 from 1 to 30 μM (progressively thickening black bar shown over the trace) produced no noticeable effect on the contraction (blue trace) elicited by 85.4 mM K⁺–Krebs solution (K⁺ concentration changes are indicated with the black line below the contraction trace). (B) Mean bar graph summarizing the effects of different concentrations of CaCC_Inh-A01 on the contraction elicited by high K⁺–Krebs solution as in A. Contractile force at each concentration of CaCC_Inh-A01 was normalized to the net contraction elicited by high K⁺–Krebs solution obtained during the prior wash with this solution as shown on the left side of A. Each bar represents the mean ± SEM and data points overlaid on each bar represents individual animals (N = 5). (C) Typical contraction experiment showing the potent block produced by the Ca_V1.2 blocker nifedipine (Nif.; 1 μM; top black bar) on the contraction (blue trace) elicited by 85.4 mM K⁺–Krebs solution (bottom black bar). (D) Mean bar graph summarizing the effects of nifedipine on the contraction mediated by high K⁺–Krebs solution as in A. The mean net contractile force measured in the presence of nifedipine was normalized to that measured in the absence of the drug just prior to its addition (blue bar). The bar with nifedipine is a mean ± SEM. Overlaid data points represent different animals (N = 10). (E) Mean cumulative dose–response curves to 5-HT in mouse pulmonary arteries from wild-type C57/BL6 mice in the presence of normal extracellular Ca²⁺ concentration with (magenta circles; 1.8 Ca²⁺ + 10 μM CPA; N = 4) or without (black circles, Control [1.8 Ca²⁺]; N = 14) 10 μM CPA to deplete SR Ca²⁺ stores, or in Ca²⁺-free with (green circles; 0 Ca²⁺ + 10 μM CPA; N = 6) or without (blue circles; 0 Ca²⁺; N = 18) CPA. Each data point is mean ± SEM of net contractile force normalized to the second high K⁺–Krebs solution-induced contraction (see examples in A and description in Materials and methods). The control (1.8 Ca²⁺) and 1.8 Ca²⁺ + CPA curves were reproduced from Fig. 1 B. For all panels, *** indicates a significant difference between means with P < 0.001; n.s.: not significant.

Figure 3.

Ca²⁺ oscillations triggered by 5-HT in individual smooth muscle cells from an intact mouse endothelium-denuded PA are potently inhibited by the inhibition of ANO1. All data were collected from the same PA from a conditional smooth muscle–specific and inducible GCaMP3 mouse injected with tamoxifen to induce Cre expression. (A) Ca²⁺ imaging was performed in the absence of an agonist (Control). The left panel shows one image from a video from which a ST map (middle colored image) was created in the area spanned by the diagonal white line. Fluorescence intensity was measured under the three white lines on the ST map (corresponding to two different cells) and plotted as a function of time as shown on the right. There was no detectable activity in these two cells as well as across the entire field of view of the movie. (B) Same nomenclature as in A except that the preparation was exposed to 1 μM 5-HT for 5 min. A ST map created in the same manner as that in A shows clear evidence of asynchronous Ca²⁺ transients. This is more evident from examining the fluorescence intensity profile of the same two cells analyzed in A, which displayed repetitive Ca²⁺ transient of distinct magnitude and frequency. (C) The nomenclature of this panel is identical to that of B and C, with the exception that the PA was exposed to 10 μM CaCC_Inh-A01 for 10 min while still being incubated with 5-HT. Examination of the ST map reveals little, if any, Ca²⁺ oscillations in the presence of the ANO1 inhibitor; Ca²⁺ transients were no longer apparent in the same two cells analyzed in A and B.

Figure 4.

Examples of asynchronous Ca²⁺ oscillations and waves in PASMCs from intact PAs from SMC-GCaMP3 mice. (A) Examples of ST maps created by analyzing a video recorded from an intact endothelium-denuded PA from a SMC-GCaMP3 mouse exposed to 1 μM 5-HT for 5 min. The left larger image was extracted from a video stack onto which were drawn two white scan lines. The scan lines were oriented along or orthogonally to the main axis of the cells. The two images on the right show ST maps that were created by reporting fluorescence intensity registered under the scan line drawn orthogonally (Transverse Mapping) or along (Longitudinal Mapping) the main axis of the cells, respectively. The top ST map clearly shows a lack of cell-to-cell propagation of Ca²⁺ transients (bright luminous spots) in the transverse direction. Longitudinal mapping also suggests a lack of or poor evidence for propagation from one cell to the next along the main axis of the cells. Ca²⁺ transients propagated along the main axis of the cells (as evidenced from the near vertical streaks of fluorescence) but collapsed at the end of the cells, failing to propagate the signal to neighboring cells (one example highlighted by the yellow box). (B) Two series of images showing the initiation and propagation of two Ca²⁺ waves (top and bottom sets of images) taken at different times from the original movie. The white arrows show trigger sites. The yellow arrows illustrate the direction of propagation of the waves. The ST map at the bottom of the panel was reconstructed from the entire video with time passing from left to right. The white arrows indicate the location of at least three trigger sites that initiated Ca²⁺ waves in this particular cell. The trigger sites randomly changed from either of the cell tips or the middle of the cell. Bidirectional propagation from the middle site is evident from the “V” shape appearance of these particular waves.

Figure 5.

Sample experiment illustrating how ANO1 knockdown exerted a strong inhibition of 5-HT-induced Ca²⁺ oscillations in a PA from a tamoxifen-injected SMC-ANO1-KO-^ΔEx12-GCaMP3 mouse. The top left panel is an image from a video stack recorded in a pulmonary artery from a conditional smooth muscle cell-specific and inducible ANO1 knockout mouse expressing GCaMP3 specifically in smooth muscle cells, which was exposed to 1 μM 5-HT for 5 min. One ST map constructed from the white line crossing the image is shown in the lower left corner and reveals very little activity. The fluorescence intensity profile as a function of time of two cells from the ST map labeled with the letters a and b are shown on the right. Cell 1 displayed no significant Ca²⁺ activity while Cell 2 showed low-frequency Ca²⁺ events.

Figure 6.

Asynchronous Ca²⁺ oscillations evoked by 5-HT require both functional ANO1 and VGCC. Mean data for each of four parameters measured from Ca²⁺ transients elicited by 1 μM 5-HT (5 min) in PA from control SMC-GCaMP3 (light blue bars) or SMC-ANO1-KO-^ΔEx12-GCaMP3 (light gray bars) mice. (A–D) The frequency of Ca²⁺ oscillations (A), peak Ca²⁺ transient amplitude (F/F₀; B), integrated area under the curve (C), and FWHM (D) were measured as shown in the upper right corner. For each dataset, the mean is indicated by a filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells. SMC-GCaMP3 + 5-HT: N = 7, n = 114 for peak, area under the curve, and FWHM, and n = 116 for frequency; SMC-GCaMP3 + 5-HT + CaCC_Inh-A01 (CaCC_Inh): N = 7, n = 15 for peak, area under the curve, and FWHM, and n = 76 for frequency; SMC-GCaMP3 + 5-HT + nifedipine (Nif): N = 2, n = 32 for peak, area under the curve, and FWHM, and n = 47 for frequency; GCaMP3 + 5-HT + CPA: N = 2, n = 29; SMC-ANO1-KO-^ΔEx12-GCaMP3; 5-HT: N = 7, n = 39 for peak, area under the curve, and FWHM, and n = 137 for frequency. For all panels, ***, **, and * indicate a significant difference between means with P < 0.001, P < 0.01, and P < 0.05, respectively.

Figure 7.

Blocking ANO1 or Ca_V1.2 depletes SR Ca²⁺ stores. (A) Typical isometric force recording obtained under control conditions showing the effect of depleting the SR Ca²⁺ stores. After eliciting a sustained contraction with high K⁺ (KCl; 85.4 mM), the preparation was switched to normal Krebs for 20 min, which relaxed the artery to baseline. The solution was subsequently changed to a Ca²⁺-free Krebs solution containing 100 μM EGTA and 10 μM cyclopiazonic acid (0 Ca + CPA), which triggered a transient contraction produced by releasing Ca²⁺ from internal SR Ca²⁺ stores. The area under the curve for the transient contraction highlighted in red was measured and used as an index of the amount of Ca²⁺ stored in the SR. (B and C) Similar experiments to that shown in A, with the exception that each preparation was incubated for 10 min with 1 μM nifedipine or 10 μM CaCC_Inh-A01, respectively, prior to switching to the Ca²⁺-free solution with CPA in the presence of either drug. Both compounds exerted a strong inhibition of the transient contraction elicited by CPA as evident from the much reduced area under the curve shown in red. (D) Pooled data from similar experiments are shown in A–C. For each dataset, the mean is indicated by a filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points of the integrated contraction measured in the presence of 0 Ca + CPA, normalized to the KCl-induced contraction (a.u.: arbitrary unit). Control, N = 5; nifedipine, N = 7; CaCC_Inh-A01, N = 4; * indicates a significant difference between means with P < 0.05. The comparison between the Control and CaCC_Inh-A01 groups was just at the limit of significance as shown.

Figure 8.

ANO1, Ca_V1.2, and IP₃R colocalize in peripheral coupling sites to form signaling complexes. (A and B) Co-IP of Ca_V1.2 or IP₃R with ANO1 from lysates of the pulmonary artery from wild-type mice. Pulldown was carried out with anti-ANO1 antibody and then probed by Western blot with anti-Ca_V1.2, anti-IP₃R, or anti-ANO1 antibodies. Five to six mouse tissues per experiment, each ran in triplicates. (C and D) Freshly isolated PASMCs from wild-type mice were immunolabeled for ANO1 and Ca_V1.2 (C) or ANO1 and IP₃R (D). All three proteins were preferentially localized to the periphery of the cells. (D and F) Line profiles of the areas indicated by the white dashed lines in C and E. The fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the location of the PM. ANO1 and Ca_V1.2 show strong immunolabeling at the PM (D). (E) IP₃R shows some intracellular immunolabeling, with moderate peaks present at the periphery showing an enhancement of protein localization to peripheral coupling sites. Source data are available for this figure: SourceData F8.

Figure 9.

Superresolution imaging of ANO1, Ca_V1.2, and IP₃R at the PM of PASMCs from wild-type mice. (A and B) Superresolution images of PASMCs labeled for ANO1 and Ca_V1.2 (A) or ANO1 and IP₃R (B) were imaged using GSDIM in epifluorescence mode. Epifluorescence images are shown in the inset for reference, with the yellow box demonstrating the region for GSD imaging. (C and D) Coordinate-based colocalization (CBC) of ANO1-Ca_V1.2 (C) or ANO1 and IP₃R (D) where −1 shows mutual exclusion and +1 shows high correlation. (E and F) Superresolution images of PASMCs labeled with ANO1 and Ca_V1.2 (E) or ANO1 and IP₃R (F) were imaged using GSDIM in TIRF mode. Enlargements of the white boxes are shown to the right, highlighting the adjacent location of the protein pairs. (G) Nearest neighbor analysis of the protein pairs demonstrates that the single-molecule localizations of Ca_V1.2 (blue) and IP₃R (red) are both located closer to ANO1 than would be expected for a randomly distributed protein (black and grey). (H) An example of Voronoï-based segmentation of Ca_V1.2 single-molecule localizations (top) and the corresponding identified clusters are shown in yellow with the outline of the identified clusters denoted by the red line (bottom). (I) Cluster sizes of ANO1 (top), Ca_V1.2 (middle), and IP₃R (bottom) as determined using SR-Tesseler analysis as shown in H.

Figure 10.

Membrane cholesterol depletion with MβCD causes the internalization of ANO1 and Ca_V1.2 proteins. (A and C) Freshly isolated PASMCs from wild-type mice were immunolabeled for ANO1 and Ca_V1.2 before (A) or after (C) a 30-min exposure to MβCD (3 mg/ml; MβCD) to deplete membrane cholesterol and disrupt lipid rafts. The two ion channel proteins were preferentially localized to the periphery of the cells in control conditions as similarly shown in Fig. 8. (B–D) Line profiles of the areas indicated by the white dashed lines in A and C are respectively displayed in B and D. For these plots, the fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the location of the PM. ANO1 and Ca_V1.2 show strong immunolabeling at the PM in control condition (C) and translocation toward the center core of the cell after exposure to MβCD (D). The cells from A and C were isolated from the same mouse. (E and F) Graphs summarizing the effects of exposing PASMCs to MβCD on the distribution of ANO1 (magenta bars) and Ca_V1.2 (green bars), respectively. Measurements were performed as described in the text and consisted in normalizing membrane fluorescence to total cell fluorescence. For each dataset, the mean is indicated by a large, filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells; for the control group (E): ANO1 and Ca_V1.2: N = 3, n = 43; for the MβCD group (F): ANO1 and Ca_V1.2: N = 3, n = 35. *** indicates a significant difference between means with P < 0.001.

Figure 11.

Disruption of lipid rafts with MβCD inhibits EC coupling mediated by 5-HT. (A) Mean cumulative dose–response curves to 5-HT in mouse pulmonary arteries from wild-type C57/BL6 mice in control condition (black circles, Control; N = 14), a 10-min exposure to 10 μM CPA (blue squares; N = 4), a 10-min exposure to 10 μM CaCC_Inh-A01 (blue circles; N = 6), or after a 30-min incubation with MβCD (3 mg/ml) to deplete membrane cholesterol and disrupt lipid rafts (upper triangles; N = 5). The data for the control, CPA, and CaCC_Inh-A01 groups were reproduced from Fig. 1 B to facilitate comparisons. Each data point is a mean ± SEM of net contractile force normalized to the second high K⁺–Krebs solution-induced contraction (see description in Materials and methods). (B–E) The summarized data presented in B–E show the effects of exposure of pulmonary arteries from conditional and smooth muscle-specific GCaMP6f mice (SMC-GCaMP6f) to a 30-min exposure with 3 mg/ml of MβCD on the frequency of GCaMP6f Ca²⁺ oscillations (B; Control, n = 58; MβCD, n = 78), peak GCaMP6f Ca²⁺ transients (C; Control, n = 58; MβCD, n = 70), area under the curve of GCaMP6f Ca²⁺ transients (D; Control, n = 58; MβCD, n = 70) and FWHM GCaMP6f Ca²⁺ transients (E; Control, n = 58; MβCD, n = 70). The Control (light blue boxes) and MβCD (light gray boxes) datasets in B–F are from the same animals (N = 4). For each dataset in the latter panels, the mean is indicated by a filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. For all panels, *** indicates a significant difference between means with P < 0.001.

Figure 12.

Hypothetical models of EC coupling involving ANO1, Ca_V1.2, and IP₃R during agonist-mediated contraction of mouse pulmonary arterial smooth muscle cells. (A) General uniform model depicting the activation of ANO1 by both Ca²⁺ release from IP₃-sensitive SR Ca²⁺ stores and Ca²⁺ entry through Ca_V1.2. In this model, the three ion transporters are evenly distributed in the membrane and are not physically coupled. The depolarization is maintained by the positive feedback loop established by Ca_V1.2-mediated activation of Cl⁻ efflux through ANO1 and its impact on the state of activation of Ca_V1.2 through regulation of membrane potential. (B) Schematic diagram illustrating the local interaction of ANO1, Ca_V1.2 with IP₃R and their impact on membrane potential, Ca²⁺ entry, and contraction. In this model, the three ion channels are physically coupled in a restricted number of sites (Super Cluster) distributed across the long axis of the cell (shown as red boxes in the bottom diagram) and are organized for compartmentalized Ca²⁺ signaling as highlighted by the yellow gradient area between the PM and segments of the SR in the close vicinity of the PM (top diagram). These compartmentalized areas serve the role of trigger sites for initiating Ca²⁺ waves that can then propagate through Ca²⁺-induced Ca²⁺ release (CICR). Ca²⁺ entry through Ca_V1.2, which is maintained by the depolarization caused by ANO1, supports the microenvironment that is necessary to promote the propagation of the Ca²⁺ waves by CICR and to reload SR Ca²⁺ stores. PASMC: pulmonary artery smooth muscle cell; IP₃: inositol-triphosphate; G_qPCR: G_q-protein coupled receptor; PLC: phospholipase C; SERCA2: type 2 Ca²⁺-ATPase of the SR.