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 Ca ²⁺ 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.