Affolter H, Coronado R
Biophys J. 1986 Mar;49(3):767-71. doi: 10.1016/S0006-3495(86)83703-1.
The verapamil-type calcium antagonist, D600, and its charged quaternary derivative, D890, were used to assess the sidedness of blockade in single calcium channels reconstituted from purified transverse tubules of skeletal muscle. Spontaneous single channel openings were induced with the agonist Bay-K8644 and recordings were made in a two-chamber planar bilayer setup so that drugs could be delivered to either side of the channel. Micromolar drug addition resulted in a greater than 10-fold decrease in probability of open channel events (po) without a significant change in single channel currents. Changes in po occurred in parallel with changes in mean open time and both parameters could be titrated with a similar IC50. At pH 7.2, cis or trans D600 blocked with an IC50 of 5 microM but for D890 the IC50 was cis 3 microM and trans greater than 75 microM (cis is the intracellular-equivalent side as defined by the voltage-dependent activation). The asymmetry of D890 blockade indicates that the drug can readily gain access to the blocking site from the aqueous phase adjacent to the inner but not extracellular end of the channel.
维拉帕米型钙拮抗剂D600及其带电荷的季铵衍生物D890，被用于评估在由骨骼肌纯化横管重构的单钙通道中阻断作用的侧别。用激动剂Bay-K8644诱导自发的单通道开放，并在双室平面双层装置中进行记录，以便药物能够被递送至通道的任一侧。添加微摩尔浓度的药物导致开放通道事件的概率（po）降低超过10倍，而单通道电流无显著变化。po的变化与平均开放时间的变化平行，并且两个参数都可以用相似的半数抑制浓度（IC50）进行滴定。在pH 7.2时，顺式或反式D600的阻断作用的IC50为5微摩尔，但对于D890，顺式IC50为3微摩尔，反式大于75微摩尔（顺式是由电压依赖性激活所定义的细胞内等效侧）。D890阻断作用的不对称性表明该药物能够容易地从与通道内侧而非细胞外侧相邻的水相进入阻断位点。
