Bechem M, Pott L
Pflugers Arch. 1985 May;404(1):10-20. doi: 10.1007/BF00581485.
Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.
利用单个低电阻膜片钳微电极，研究了培养的豚鼠心房心肌球在电压钳去极化过程中Ca电流的情况。微电极内充以含Cs⁺作为主要阳离子的溶液，以阻断K⁺电流，并加入高浓度的各种Ca螯合剂（乙二醇双四乙酸、次氮基三乙酸、柠檬酸盐、二吡啶羧酸），以防止Ca内流引起细胞内Ca活性升高。加载20 mM乙二醇双四乙酸（[(乙二氧基)-二乙二胺四乙酸]）或次氮基三乙酸的心肌细胞的Ca电流，在膜电位-25至+45 mV之间呈现双相失活时间进程。快速相随着膜电位越来越正向而减小。在加载柠檬酸盐或二吡啶羧酸的细胞中，小去极化时失活可忽略不计或不存在。在最大电流流动的膜电位范围内（0至+10 mV），观察到单相缓慢失活时间进程。在更正向的膜电位下，失活减慢。在此条件下失活的量与细胞的电流密度有关。对于给定膜电位，降低ICa幅度的条件，如细胞外施加阻断离子（Co²⁺、Cd²⁺）、预处理去极化或Ca通道的“衰减”，分别导致加载柠檬酸盐或二吡啶羧酸的细胞中失活减慢或完全消除。加载这些Ca螯合剂的细胞未显示ICa电压依赖性失活的任何症状。在所述条件下，以电流钳模式记录动作电位。用乙二醇双四乙酸透析后，典型的“三角形”心房动作电位会出现持续500至800 ms的平台期。使用含柠檬酸盐的微电极溶液时，动作电位持续时间通常为几秒。结果首次表明，心脏ICa的失活可显著减慢甚至消除。它们为该电流的失活取决于Ca内流而非膜电位这一假说提供了进一步有力支持。用乙二醇双四乙酸（次氮基三乙酸）观察到的快速失活相可能反映了这类Ca螯合剂结合反应的缓慢动力学。
