Arrestin‐Biased M3 Designer Receptor: A Novel Therapeutic Tool in the Upregulation of hERG Channel Activity

Background The human ether‐a‐go‐go‐related gene ( hERG ) encodes the pore‐forming subunit of the voltage‐gated K + channel (Kv11.1), which passes the rapidly activating delayed rectifier K + current (I Kr ). Unique gating kinetics of the hERG channel establishes the important role of I Kr in setting the cardiomyocyte action potential duration (APD) and maintaining cardiac repolarization. A reduction in I Kr due to loss‐of‐function mutations or drug interactions may cause long QT syndrome (LQTS), a cardiac electrical disorder characterized by a prolonged QT interval on an ECG. Individuals with inherited or acquired LQTS are predisposed to fatal ventricular arrhythmias (Torsade de pointes) and sudden cardiac death. Spontaneous loss‐of‐function hERG mutations reduce I Kr and cause inherited LQTS type 2. Furthermore, many medications or reductions in extracellular K + concentration (hypokalemia) have been shown to cause acquired LQTS. Since a reduction in hERG expression and I Kr cause LQTS, methods to enhance or restore hERG channel activity are recognized as appropriate treatment strategies. In particular, we discovered that M3 receptor activation increases hERG expression and I Kr through a Gq protein‐dependent PKC pathway. Although these findings establish a basis for M3‐mediated regulation of hERG activity, there is little therapeutic potential due to complex G protein signaling and heterologous M3 expression. Similar to other G protein‐coupled receptors (GPCRs), M3 receptors recruit β‐arrestins in response to ligand binding to mediate G protein‐independent signaling pathways. Such pathways may provide additional insight into the regulation of hERG channel activity and serve as a potential therapeutic target. The development of an M3 designer receptor (M3D) has allowed for exclusive β‐arrestin signaling in response to an otherwise pharmacologically inert ligand, clozapine‐N‐oxide (CNO). These designer receptors may prove to be of therapeutic benefit for upregulating hERG channel activity through a β‐arrestin‐dependent pathway. The present study aims to elucidate the role of β‐arrestin signaling in the regulation of mature hERG channel activity and to identify components involved in the signaling cascade. Methods Human embryonic kidney cells stably expressing hERG protein (hERG‐HEK cells) were transiently transfected with empty pcDNA3 or M3D plasmids, and cultured for 24 hours in the absence (control) or presence of CNO. Western blot analysis was performed to assess hERG expression in the groups. Whole‐cell patch clamp recordings were conducted to evaluate hERG channel current. Results Western blot analysis revealed that CNO‐mediated activation of M3D significantly increased hERG expression and current compared to controls. CNO treatment enhances the β‐arrestin‐M3D interaction at the plasma membrane. M3D activation via CNO enhances p‐Akt levels but not endogenous Akt levels. Increased hERG activity and p‐Akt levels in response to CNO‐mediated activation of M3D is abolished by overexpression of PTEN, a phosphoinositide 3‐kinase (PI3‐K) inhibitor. Conclusions These findings suggest that β‐arrestin signaling in response to CNO‐induced activation of M3D increases hERG channel activity through PI3‐K‐dependent phosphorylation of Akt. Support or Funding Information Canadian Institutes of Health Research and Heart & Stroke Foundation

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