Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City, 04510, Mexico.
Departamento de Biología Celular y del Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Mexico City, 04510, Mexico; Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, 98124, USA.
Biochem Biophys Res Commun. 2020 Apr 16;524(4):1030-1036. doi: 10.1016/j.bbrc.2020.02.033. Epub 2020 Feb 14.
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by a family of four genes (HCN1-4). All isoforms are expressed in the heart, HCN4 being the most abundant in the sinoatrial node (SAN). HCN channels are responsible for the "funny" current (I) associated with the generation and autonomic control of the diastolic depolarization phase of cardiac action potential. In this work we performed a proteomic analysis of HCN4 transfected in HEK293 cells. Most of the identified proteins in the HCN4 network belonged to mitochondria. The subcellular localization of HCN channels was predicted in plasma membrane, mitochondria and nucleus. Experimentally, HCN2 (full-length, truncated), HCN3 (full-length, truncated) and HCN4 (truncated) were detected in rat heart mitochondria by immunoblotting. I sensitive to ZD7288, was recorded by patch-clamp in mitoplasts from cardiomyocytes. Mitochondrial membrane potential (ΔΨm) assessment in H9c2 cells revealed that ZD7288 induced almost 50% higher hyperpolarization respect to control at 30 min. Furthermore, ZD7288 reduced oxygen consumption attributed to ATP synthesis in H9c2 cells. In conclusion, we identify for the first time functional HCN channels in mammalian cardiac mitochondria and demonstrate their impact on ΔΨm and respiration.
超极化激活环核苷酸门控 (HCN) 通道由四个基因 (HCN1-4) 家族编码。所有同工型都在心脏中表达，HCN4 在窦房结 (SAN) 中最为丰富。HCN 通道负责与心脏动作电位的舒张去极化相相关的“有趣”电流 (I) 的产生和自主控制。在这项工作中，我们对转染 HEK293 细胞的 HCN4 进行了蛋白质组学分析。HCN4 网络中大多数鉴定的蛋白质属于线粒体。HCN 通道的亚细胞定位预测为质膜、线粒体和核。实验中，通过免疫印迹在大鼠心脏线粒体中检测到 HCN2（全长、截断）、HCN3（全长、截断）和 HCN4（截断）。通过心肌细胞线粒体质膜小泡中的膜片钳记录到对 ZD7288 敏感的 I。在 H9c2 细胞中评估线粒体膜电位 (ΔΨm) 表明，与对照相比，ZD7288 在 30 分钟时诱导的超极化几乎高 50%。此外，ZD7288 降低了归因于 H9c2 细胞中 ATP 合成的耗氧量。总之，我们首次在哺乳动物心脏线粒体中鉴定出功能性 HCN 通道，并证明它们对 ΔΨm 和呼吸的影响。
