ANO1、CaV1.2和IP3R在小鼠肺动脉平滑肌中形成EC偶联的局部单元。

IF 3.3 2区 医学 Q1 PHYSIOLOGY Journal of General Physiology Pub Date : 2023-11-06 Epub Date: 2023-09-13 DOI:10.1085/jgp.202213217
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
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引用次数: 0

摘要

肺动脉(PA)平滑肌细胞(PASMC)对与Gq蛋白受体信号传导偶联的激动剂产生血管张力。这种激动剂刺激振荡的钙波,钙波的频率驱动收缩的强度。这些Ca2+事件由多种离子通道调节,包括电压门控钙通道(CaV1.2)、Tmem16a或Anoctamin-1(ANO1)编码的钙激活氯化物(CaCC)通道,以及通过肌醇三磷酸受体(IP3R)从肌浆网释放Ca2+。尽管这些钙事件已经被表征,但尚不清楚这些钙振荡是如何导致这些肌肉细胞持续收缩的。我们使用ANO1的平滑肌特异性消融和药理学工具来确定ANO1、CaV1.2和IP3R在表达Ca2+生物传感器GCaMP3或GCaMP6的小鼠PA平滑肌的收缩和细胞内Ca2+信号传导特性中的作用。ANO1的药理学阻断或遗传消融、CaV1.2或IP3R的抑制、或Ca2+储存耗竭同样抑制5-HT诱导的张力和细胞内Ca2+波。共免疫沉淀实验表明,抗ANO1抗体能够同时下调CaV1.2和IP3R。共聚焦和超分辨率纳米显微镜显示,ANO1在野生型小鼠PASMC质膜处或附近与CaV1.2和IP3R共组装。我们得出结论,稳定的5-HT诱导的PA收缩是由包括ANO1、CaV1.2和IP3R的微环境支持的随机和局部Ca2+事件的整合引起的。在该模型中,ANO1和CaV1.2将间接支持IP3R的周期性Ca2+释放事件和细胞内Ca2+波的传播。
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ANO1, CaV1.2, 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 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.

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来源期刊
CiteScore
6.00
自引率
10.50%
发文量
88
审稿时长
6-12 weeks
期刊介绍: General physiology is the study of biological mechanisms through analytical investigations, which decipher the molecular and cellular mechanisms underlying biological function at all levels of organization. The mission of Journal of General Physiology (JGP) is to publish mechanistic and quantitative molecular and cellular physiology of the highest quality, to provide a best-in-class author experience, and to nurture future generations of independent researchers. The major emphasis is on physiological problems at the cellular and molecular level.
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