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

作者：Elizabeth J. Akin, Joydeep Aoun, Connor Jimenez, Katie Mayne, Julius Baeck, Michael D. Young, Brennan Sullivan, Kenton M. Sanders, Sean M. Ward, Simon Bulley, Jonathan H. Jaggar, Scott Earley, Iain A. Greenwood, Normand Leblanc · 发表于：The Journal of General Physiology · 年份：2023 · DOI：10.1085/jgp.202213217 · 被引用次数：21 · 研究领域：Ion channel regulation and function、Cardiac electrophysiology and arrhythmias、Ion Channels and Receptors

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 stochas...