Qu Y, Rogers J, Tanada T, Scheuer T, Catterall W A
Department of Pharmacology, University of Washington, Seattle 98195.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3289-93. doi: 10.1073/pnas.91.8.3289.
Cardiac rH1 Na+ channel alpha subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line expressing cardiac Na+ channels (SNa-rH1) was isolated. Mean Na+ currents of 2.2 +/- 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per micron2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 microM) and had voltage-dependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 microM) decreased this current approximately 33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8- to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 microM) or Ca(2+)-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microinjected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effect of OAG. Single-channel recordings from SNa-rH1 cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation of neuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 microM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.
通过转染，将心脏rH1 Na⁺通道α亚基在中国仓鼠肺1610细胞系的细胞中表达，并分离出表达心脏Na⁺通道的稳定细胞系（SNa-rH1）。记录到的平均Na⁺电流为2.2±1.0 nA，这相当于在Na⁺电流峰值时每平方微米约有1 - 2个活跃的细胞表面通道密度。所表达的心脏Na⁺电流对河豚毒素具有抗性（Kd = 1.8 μM），并且具有与新生心室肌细胞中Na⁺电流相似的电压依赖性特性。用1-油酰基-2-乙酰基-sn-甘油（OAG）（10 μM）激活蛋白激酶C，在-114 mV的钳制电位下使该电流降低约33%，在-94 mV时降低56%。峰值电流的这种降低部分是由于稳态失活在超极化方向上有8至14 mV的偏移。Na⁺通道激活未改变。OAG对SNa-rH1细胞和新生大鼠心肌细胞的作用相似，只是当在低钙（1 μM）或无钙溶液中孵育的心肌细胞中激活蛋白激酶C时，失活的时间进程会短暂或持续减慢，但在SNa-rH1细胞中不受影响。OAG对心脏Na⁺电流的作用在先前已显微注射蛋白激酶C肽抑制剂的细胞中被阻断，但用cAMP依赖性蛋白激酶的肽抑制剂则未被阻断，这表明蛋白激酶C负责OAG的作用。来自SNa-rH1细胞的单通道记录显示，OAG降低了通道开放的概率，但电导率未受影响。OAG并未诱导在神经元和骨骼肌Na⁺通道的PKC调节中观察到的晚期Na⁺通道开放。因此，在正常舒张期去极化时用10 μM OAG导致的Na⁺电流大幅降低是由于通道在去极化时未能开放。这种Na⁺电流降低可能对心脏细胞兴奋性产生深远影响。
