Cook N J, Hanke W, Kaupp U B
Proc Natl Acad Sci U S A. 1987 Jan;84(2):585-9. doi: 10.1073/pnas.84.2.585.
The cyclic GMP-dependent cation channel from bovine rod outer segments has been purified to greater than 90% homogeneity by a rapid two-step chromatographic procedure. The purified channel has an apparent molecular mass of 63 kDa as determined by NaDodSO4/gel electrophoresis. When incorporated into the membrane of liposomes, the purified protein mediates the cyclic GMP-dependent efflux of entrapped Ca2+. The reconstituted channel protein exhibits properties similar to the cyclic GMP-dependent channel observed in excised patches of the plasma membrane and in disk membranes. Cyclic GMP activated the channel cooperatively (Hill coefficient n = 3.1) with an apparent Michaelis constant of approximately 11 microM. After reconstitution of the purified protein into a planar lipid bilayer, we recorded cyclic GMP-stimulated single-channel activity. The single-channel conductance at physiological salt concentrations and in the absence of divalent cations was 26 pS. The drug l-cis-diltiazem, shown to block the cyclic GMP-dependent channel in excised patches of the plasma membrane and in isolated disks of rod outer segments, was ineffective against the purified channel.
通过快速两步色谱法，已将来自牛视杆细胞外段的环鸟苷酸依赖性阳离子通道纯化至均一性大于90%。经十二烷基硫酸钠/凝胶电泳测定，纯化后的通道表观分子量为63 kDa。当整合到脂质体膜中时，纯化的蛋白质介导被包裹的Ca2+的环鸟苷酸依赖性外流。重构的通道蛋白表现出与在质膜切除片和盘膜中观察到的环鸟苷酸依赖性通道相似的特性。环鸟苷酸以协同方式激活该通道（希尔系数n = 3.1），表观米氏常数约为11 microM。将纯化的蛋白质重构到平面脂质双分子层后，我们记录到了环鸟苷酸刺激的单通道活性。在生理盐浓度且不存在二价阳离子的情况下，单通道电导为26 pS。药物l-顺式地尔硫䓬在质膜切除片和视杆细胞外段分离盘片中可阻断环鸟苷酸依赖性通道，但对纯化的通道无效。
