Brette F, Sallé L, Orchard C H
Department of Physiology, Medical Sciences Building, University of Bristol, Bristol, United Kingdom.
Biophys J. 2006 Jan 1;90(1):381-9. doi: 10.1529/biophysj.105.069013. Epub 2005 Oct 7.
The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.
心室肌细胞的动作电位具有持续时间长的特点，这主要是由于钙离子通过L型钙通道内流所致。钙离子内流还可触发肌浆网释放钙离子。本研究的目的是探讨被称为横管（T管）的细胞膜内陷在决定心室肌细胞钙离子内流和动作电位持续时间方面的作用。我们采用全细胞膜片钳技术记录完整大鼠心室肌细胞（即来自T管和表面肌膜）以及去管化肌细胞（即仅来自表面肌膜）的电生理活动。去管化细胞的动作电位明显短于对照细胞。相比之下，对照和去管化肌细胞的静息膜电位和动作电位幅度相似。利用动作电位波形在电压钳条件下进行实验，以量化通过钙电流的钙离子内流。去管化后钙离子内流减少了约60%，这一数值与动作电位持续时间的减少相似。我们计算得出，在方波电压钳脉冲期间，T管处的钙离子内流是细胞表面的1.3倍（分别为4.9和3.8微摩尔/升胞浆）。相比之下，在心脏动作电位期间，T管处的钙离子内流是细胞表面的2.2倍（分别为3.0和1.4微摩尔/升胞浆）。然而，每平方微米细胞表面连接膜处的钙离子内流比T管处更多（在心脏动作电位期间，以纳摩尔/平方微米计：1.43对1.06）。这种差异不太可能是由于每个部位钙通道/连接点数量的差异，因为我们估计细胞表面和T管连接点处存在相同数量的钙通道（约35个）。本研究首次提供证据表明，T管是调节心室肌细胞动作电位持续时间的关键部位。我们的数据还首次直接测量了T管钙离子内流，这与心脏兴奋 - 收缩偶联主要发生在T管二联体裂隙处的观点一致。
