Haynes L W
Department of Medical Physiology, University of Calgary, Alberta, Canada.
J Gen Physiol. 1992 Nov;100(5):783-801. doi: 10.1085/jgp.100.5.783.
Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.
从鲶鱼视杆或视锥细胞外段切下内翻式膜片。在低二价缓冲盐溶液中，记录由1 mM cGMP激活的GMP门控通道的单通道电流和宏观电流。通过向膜片的胞质侧施加微摩尔浓度的l-顺式地尔硫䓬来阻断电流。阻断的浓度依赖性表明单个分子就足以阻断一个通道，并且所有通道都易于被阻断。视杆通道的解离常数比视锥通道小一个数量级，但阻断的电压依赖性几乎相同。存在阻滞剂时的宏观电流-电压关系呈内向整流，表面上类似于由非渗透性阻滞剂阻断通道的离子传导孔所引起的电压依赖性阻断。通过在记录微管溶液中加入5 microM地尔硫䓬，研究了从通道细胞外侧作用的地尔硫䓬的阻断作用。宏观电流-电压关系再次显示内向整流，这与地尔硫䓬通过阻断外侧孔起作用的观点不一致。在仅含有单个通道的视锥膜片中，测量了施加到细胞内和细胞外侧的地尔硫䓬的阻断动力学。两种情况下的解离速率相似，表明存在单个结合位点。结合速率的差异与从胞质侧更容易接近结合位点一致。从胞质侧的阻断与pH无关，这表明地尔硫䓬的离子化状态与其以电压依赖性方式阻断通道的能力无关。这些观察结果与孔道阻断阻滞剂不一致，但如果地尔硫䓬的疏水部分分隔到通道蛋白的疏水核心中，可能改变通道的门控，就可以得到解释。
