Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas LT-50162, Lithuania.
Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80015, USA.
Biomed Pharmacother. 2024 May;174:116550. doi: 10.1016/j.biopha.2024.116550. Epub 2024 Apr 8.
Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca, Mg, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca-regulated kinases, such as Ca/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.
生理和病理过程，如内稳态、胚胎发生、发育、肿瘤发生和细胞运动，依赖于通过间隙连接（GJIC）的细胞间通讯。连接蛋白（Cx）为基础的 GJ 通道由相邻细胞中的两个相对的半通道组成，为细胞间的电和代谢通讯提供直接途径。GJ 电导的主要调节剂是跨连接电压、细胞内 pH 值、Ca、Mg 和磷酸化。GJIC 的化学调节剂正在用于各种依赖细胞间通讯的疾病的治疗。在这项研究中，我们使用分子对接、双全细胞膜片钳和 Western blot 技术，研究了在表达外源性 Cx43（全长和截断在氨基酸 258 处）和其他连接蛋白（Cx36、Cx40、Cx45 和 Cx47）的 HeLa 细胞以及表达内源性 Cx43 的 Novikoff 和 U-87 细胞中，连接蛋白磷酸化对 GJ 化学门控的影响，通过α-蒎烯和其他 GJ 抑制剂（辛醇、卡波酮、甲氟喹、细胞内 pH 值、甘草次酸和七氟醚）的作用。我们发现，Ca 调节激酶，如 Ca/钙调蛋白依赖性激酶 II、非典型蛋白激酶 C、细胞周期蛋白依赖性激酶和 Pyk2 激酶，可能通过 Cx43 C 端的磷酸化，变构调节α-蒎烯的效力。所鉴定的新现象是 Cx 同工型、抑制剂和细胞类型依赖性的。总的来说，这些结果表明，对于那些受体磷酸化依赖性的化合物，可能会特别关注开发针对伴有高激酶活性的疾病的靶向治疗，如心律失常、癫痫、中风、原发性震颤、炎症和癌症。
