Dunn S M
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
J Biol Chem. 1989 Jul 5;264(19):11053-60.
Transverse tubule membranes isolated from rabbit skeletal muscle consist mainly of sealed vesicles that are oriented primarily inside out. These membranes contain a high density of binding sites for 1,4-dihydropyridine calcium channel antagonists. The presence of functional voltage-dependent calcium channels in these membranes has been demonstrated by their ability to mediate 45Ca2+ efflux in response to changes in membrane potential. Fluorescence changes of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine, have shown that transverse tubule vesicles may generate and maintain membrane potentials in response to establishing potassium gradients across the membrane in the presence of valinomycin. A two-step procedure has been developed to measure voltage-dependent calcium fluxes. Vesicles loaded with 45Ca2+ are first diluted into a buffer designed to generate a membrane potential mimicking the resting state of the cell and to reduce the extravesicular Ca2+ to sub-micromolar levels. 45Ca2+ efflux is then measured upon subsequent depolarization. Flux responses are modulated with appropriate pharmacological specificity by 1,4-dihydropyridines and are inhibited by other calcium channel antagonists such as lanthanum and verapamil.
从兔骨骼肌分离出的横管膜主要由主要呈内翻取向的封闭囊泡组成。这些膜含有高密度的1,4-二氢吡啶钙通道拮抗剂结合位点。这些膜中功能性电压依赖性钙通道的存在已通过它们响应膜电位变化介导45Ca2+外流的能力得到证实。电压敏感染料3,3'-二丙基-2,2'-硫代二碳菁的荧光变化表明，在缬氨霉素存在下，横管囊泡可能会响应跨膜建立钾梯度而产生并维持膜电位。已开发出一种两步法来测量电压依赖性钙通量。首先将加载有45Ca2+的囊泡稀释到一种缓冲液中，该缓冲液旨在产生模拟细胞静息状态的膜电位，并将囊泡外的Ca2+降低到亚微摩尔水平。然后在随后的去极化时测量45Ca2+外流。通量反应可被1,4-二氢吡啶以适当的药理学特异性调节，并被镧和维拉帕米等其他钙通道拮抗剂抑制。
