Crary J I, Dean D M, Maroof F, Zimmerman A L
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.
J Gen Physiol. 2000 Dec;116(6):769-80. doi: 10.1085/jgp.116.6.769.
We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.
我们之前发现，来自两栖动物视杆细胞的天然环核苷酸门控（CNG）阳离子通道会被二酰基甘油（DAG）类似物直接且可逆地抑制，但对这种抑制机制了解甚少。我们最近确定，在饱和cGMP浓度下，DAG完全抑制克隆的牛视杆（Brod）CNG通道，而仅部分抑制克隆的大鼠嗅觉（Rolf）通道（Crary，J.I.，D.M. Dean，W. Nguitragool，P.T. Kurshan和A.L. Zimmerman。2000。《普通生理学杂志》116：755 - 768；本期）。在此，我们报告，在嗅觉上皮和心脏中发现的Rolf和小鼠CNG通道（Molf）的S2 - S3环中第204位的点突变，增加了DAG敏感性，使其达到Brod通道的水平。将该残基从野生型甘氨酸突变为谷氨酸（Molf G204E）或天冬氨酸（Molf G204D），显著增加了DAG敏感性，而不改变明显的cGMP或cAMP亲和力及效能。然而，与野生型嗅觉通道不同，这些突变体表现出电压依赖性门控，具有明显的激活和失活动力学。有趣的是，这些突变体对局部麻醉剂丁卡因的抑制也更敏感。用色氨酸残基取代第204位甘氨酸（Rolf G204W）不仅产生了电压依赖性门控以及对DAG和丁卡因的敏感性增加，还表现出明显的激动剂亲和力和cAMP效能降低。序列比较表明，S2 - S3环中第204位的甘氨酸高度保守，我们的研究结果表明其改变可能对通道门控和抑制产生关键影响。
