Endophilin A2 Influences Volume-Regulated Chloride Current by Mediating ClC-3 Trafficking in Vascular Smooth Muscle Cells

Abstract

Background: Previous research has demonstrated that ClC-3 is responsible for volume-regulated Cl -current (ICl.vol) in vascular smooth muscle cells (VSMCs). However, it is still not clear whether and how ClC-3 is transported to cell membranes, resulting in alteration of ICl.vol. Methods and Results:Volume-regulated chloride current (ICl.vol) was recorded by whole-cell patch clamp recording, and Western blotting and co-immunoprecipitation were performed to examine protein expression and protein-protein interaction. Live cell imaging was used to observe ClC-3 transporting. The results showed that an overexpression of endophilin A2 could increase ICl.vol, while endophilin A2 knockdown decreased ICl.vol. In addition, the SH3 domain of endophilin A2 mediated its interaction with ClC-3 and promotes ClC-3 transportation from the cytoplasm to cell membranes. The regulation of ClC-3 channel activity was also verified in basilar arterial smooth muscle cells (BASMCs) isolated from endophilin A2 transgenic mice. Moreover, endophilin A2 increase VSMCs proliferation induced by endothelin-1 or hypo-osmolarity. Conclusions:The present study identified endophilin A2 as a ClC-3 channel partner, which serves as a new ClC-3 trafficking insight in regulating ICl.vol in VSMCs. This study provides a new mechanism by which endophilin A2 regulates ClC-3 channel activity, and sheds light on how ClC-3 is transported to cell membranes to play its critical role as a chloride channel in VSMCs function, which may be involved in cardiovascular diseases. mechanism, coupling intracellular ClC-3 to cell membrane ICl. vol for VSMC functions.

Endophilin A2 regulates calcium-activated chloride channel activity via selective autophagy-mediated TMEM16A degradation

“…2b ). Similar results were found in BASMCs freshly isolated from endophilin A2 transgenic mice [ 22 ] and their littermate controls (Fig. 3c ).…”

“…For primary BASMC isolation, 4-week-old Sprague–Dawley rats were used. Endophilin A2 transgenic mice were generated as described previously [ 22 ]. All experiments were approved by and conducted in accordance with the ethical guidelines of the Sun Yat-Sen University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.…”

EndophilinA2 protects against angiotensin II‐induced cardiac hypertrophy by inhibiting angiotensin II type 1 receptor trafficking in neonatal rat cardiomyocytes

“…Once AT1‐R is activated, G protein‐dependent and G protein‐independent downstream signaling pathways (PI3 K/Akt, calcineurin, MAPKs, and Jak/STAT) are initiated. Among them, ERS is known to contribute to the development of many cardiovascular diseases, including atherosclerosis, ischemic cardiomyopathy, hypertension, cardiac hypertrophy, and heart failure . ERS indicates a lack of cellular homeostasis and induces protein synthesis in cardiomyocytes, this action results in the mass accumulation of unfolded proteins.…”

“…In vascular smooth muscle cells, the interaction of EndoA2 with ClC-3 enhances ClC-3 trafficking from the cytoplasm to the membrane. 13 In addition, our previous studies suggested that EndoA2 significantly inhibited apoptosis in rat basilar artery smooth muscle cells via direct binding with Bax and preventing Bax translocation to the mitochondria. 14 Thus, these studies indicate that EndoA2 is involved in vascular diseases.…”

Molecular mechanism of Fast Endophilin-Mediated Endocytosis

“…They were reported to do so at resting Ca 2+ concentration (100-300 nM) through their linker regions between the BAR and the SH3 domain [84]. Endophilin was also reported to interact with the Chloride ClC-3 channel [85], the small-conductance calcium activated K ca 2.3 channel [86] and the gamma-aminobutiric acid GABA receptor B [87], which is a ligand-gated Chloride channel (Table 1), but these interactions have not been followed up to date. The binding of Endophilin to several ion channels is intriguing because it is not believed that channels are actively endocytosed, and even less so rapidly in a ligand-or stimuli-dependent manner.…”