Clark A G, Murray D, Ashley R H
Department of Biochemistry, University of Edinburgh, Scotland, United Kingdom.
Biophys J. 1997 Jul;73(1):168-78. doi: 10.1016/S0006-3495(97)78057-3.
Many intracellular membranes contain ion channels, although their physiological roles are often poorly understood. In this study we incorporated single anion channels colocalized with rat brain endoplasmic reticulum (ER) ryanodine-sensitive Ca(2+)-release channels into planar lipid bilayers. The channels opened in bursts, with more activity at negative (cytoplasm-ER lumen) membrane potentials, and they occupied four open conductance levels with frequencies well described by the binomial equation. The probability of a protomer being open decreased from approximately 0.7 at -40 mV to approximately 0.2 at +40 mV, and the channels selected between different anions in the order PSCN > PNO3 > PBr > PCl > PF. They were also permeant to cations, including the large cation Tris+ (PTris/PCl = 0.16). Their conductance saturated at 170 pS in choline Cl. The channels were inactivated by 15 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and blocked with low affinity (KD of 1-100 microM) by anthracene-9-carboxylic acid, ethacrynic acid, frusemide (furosemide), HEPES, the indanyloxyacetic acid derivative IAA-94, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and Zn2+. Unlike protein translocation pores, the channels were unaffected by high salt concentrations or puromycin. They may regulate ER Ca2+ release, or be channel components en route to their final cellular destinations. Alternatively, they may contribute to the fusion machinery involved in intracellular membrane trafficking.
许多细胞内膜都含有离子通道，尽管它们的生理作用通常还了解甚少。在本研究中，我们将与大鼠脑内质网（ER）兰尼碱敏感的Ca(2+)释放通道共定位的单阴离子通道整合到平面脂质双分子层中。这些通道呈爆发式开放，在负（细胞质-内质网腔）膜电位时活性更高，它们占据四个开放电导水平，其频率可用二项式方程很好地描述。原聚体开放的概率从-40 mV时的约0.7降至+40 mV时的约0.2，并且这些通道在不同阴离子之间的选择顺序为PSCN > PNO3 > PBr > PCl > PF。它们对阳离子也具有通透性，包括大阳离子Tris+（PTris/PCl = 0.16）。在氯化胆碱中它们的电导在170 pS时达到饱和。这些通道被15 microM的4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）灭活，并被蒽-9-羧酸、依他尼酸、速尿（呋塞米）、HEPES、茚满氧基乙酸衍生物IAA-94、5-硝基-2-(3-苯丙基氨基)-苯甲酸盐（NPPB）和Zn2+以低亲和力（KD为1 - 100 microM）阻断。与蛋白质转运孔不同，这些通道不受高盐浓度或嘌呤霉素的影响。它们可能调节内质网Ca2+释放，或者是在其最终细胞目的地途中的通道成分。或者，它们可能有助于参与细胞内膜运输的融合机制。
