Cook N J, Zeilinger C, Koch K W, Kaupp U B
J Biol Chem. 1986 Dec 25;261(36):17033-9.
The protein(s) that constitute(s) the cGMP-regulated channel in vertebrate photoreceptors has been solubilized from rod outer segment membranes and reincorporated into the membrane of calcium-containing liposomes. The properties of the reconstituted channel protein were determined by studying the cGMP-stimulated efflux of Ca2+ from these liposomes. Among several detergents tested the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) proved to be the most suitable. Solubilization of channel activity was found to be optimal at a detergent concentration of about 18 mM. The presence of Ca2+ ions and phospholipids during solubilization greatly increased the channel stability. The reconstituted channel shared most but not all properties with the channel in situ. It is cooperatively activated by cGMP with an EC50 of 19 microM. The cooperativity as determined from Hill plots was n = 2.7. Unlike the cGMP-sensitive channel in the native membrane of isolated discs and excised patches of plasma membrane it is not blocked by l-cis-diltiazem. Reconstitution of this channel protein(s) may serve as a valuable tool for identifying the polypeptide composition and to study structural and functional aspects of the purified protein(s).
构成脊椎动物光感受器中cGMP调节通道的蛋白质已从视杆外段膜中溶解出来，并重新整合到含钙脂质体的膜中。通过研究cGMP刺激的Ca2+从这些脂质体中的流出，确定了重组通道蛋白的特性。在测试的几种去污剂中，两性离子去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）被证明是最合适的。发现通道活性在约18 mM的去污剂浓度下溶解最佳。溶解过程中Ca2+离子和磷脂的存在大大提高了通道的稳定性。重组通道与原位通道具有大部分但并非全部特性。它被cGMP协同激活，EC50为19 microM。根据希尔图确定的协同性为n = 2.7。与分离盘和质膜切除片的天然膜中的cGMP敏感通道不同，它不受l-顺式地尔硫䓬的阻断。这种通道蛋白的重组可作为鉴定多肽组成以及研究纯化蛋白的结构和功能方面的有价值工具。
