ANO1, Ca_V1.2, and IP₃R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to G_q-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 (Ca_V1.2), 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, Ca_V1.2, 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 Ca_V1.2 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 Ca_V1.2 and IP₃R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both Ca_V1.2 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, Ca_V1.2, and IP₃R. In this model, ANO1 and Ca_V1.2 would indirectly support cyclical Ca²⁺ release events from IP₃R and propagation of intracellular Ca²⁺ waves.