Zimmerman A L, Karpen J W, Baylor D A
Department of Neurobiology, Stanford University School of Medicine, California 94305.
Biophys J. 1988 Aug;54(2):351-5. doi: 10.1016/S0006-3495(88)82966-7.
Excised inside-out membrane patches are useful for studying the cGMP-activated ion channels that generate the electrical response to light in retinal rod cells. We show that strong ionic current across a patch changes the driving force on the current by altering the ionic concentration near the surface membrane, an effect somewhat like that first described by Frankenhaeuser and Hodgkin (1956) in squid axons. The dominant concentration change occurs in the solution adjacent to the cytoplasmic (inner) surface of the membrane, where diffusion is impaired by intracellular material that adheres to the patch during excision. The magnitude and time course of the ionic changes are consistent with the expected volume of this material and with an effective diffusion coefficient about an order of magnitude less than that in free solution. Methods are described for correcting current transients observed in voltage clamp experiments, so that channel gating kinetics can be obtained without contamination by changes in driving force. We suggest that restricted diffusion may occur in patches excised from other types of cells and influence rapid kinetic measurements.
外翻式膜片对于研究视网膜视杆细胞中产生光电反应的cGMP激活离子通道很有用。我们发现，通过改变表面膜附近的离子浓度，穿过膜片的强离子电流会改变电流的驱动力，这种效应有点类似于Frankenhaeuser和Hodgkin（1956年）在鱿鱼轴突中首次描述的效应。主要的浓度变化发生在与膜的细胞质（内）表面相邻的溶液中，在切除过程中，细胞内物质附着在膜片上，阻碍了扩散。离子变化的幅度和时间进程与这种物质的预期体积一致，并且有效扩散系数比自由溶液中的有效扩散系数小一个数量级左右。本文描述了校正电压钳实验中观察到的电流瞬变的方法，以便在不受驱动力变化影响的情况下获得通道门控动力学。我们认为，在从其他类型细胞中切除的膜片中可能会发生受限扩散，并影响快速动力学测量。
