Serre V, Ildefonse M, Bennett N
C.E.A., URA CNRS n. 520, Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
J Membr Biol. 1995 Jul;146(2):145-62. doi: 10.1007/BF00238005.
The effect of sulfhydryl reagents on the activity of the cGMP-gated channel from bovine retinal rods was studied by measurements of 8-Br-cGMP-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol blockers) as well as diamide (dithiol-disulfide conversion agent) have a dual effect on the channels activity: at low concentration, they increase the apparent affinity for cyclic nucleotide ("activation") at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturating nucleotide concentration, while mersalyl is shown to reduce the activity of the channels in bilayer experiments ("inhibition"). Nitric oxide precursors have no effect. The results suggest that blocking at least 1 of the 3 cytoplasmic cysteine residues situated close to the cGMP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinity for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersalyl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridge), and may concern a process of channel inactivation. The 3 cytoplasmic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.
通过测量8-溴-cGMP-（cGMP）诱导的视杆细胞膜囊泡中的钙外流以及记录通过整合到平面脂质双分子层中的通道的8-溴-cGMP依赖性钠电流，研究了巯基试剂对牛视网膜视杆细胞cGMP门控通道活性的影响。N-乙基马来酰亚胺和汞撒利（巯基阻断剂）以及二酰胺（二硫醇-二硫化物转化剂）对通道活性有双重影响：在低浓度时，它们增加了对环核苷酸的表观亲和力（“激活”），同时导致核苷酸结合的协同性丧失；在较高浓度时，N-乙基马来酰亚胺和二酰胺在饱和核苷酸浓度下会使钙释放的幅度和初始速率降低，而汞撒利在双层实验中显示会降低通道活性（“抑制”）。一氧化氮前体没有影响。结果表明，通过N-乙基马来酰亚胺、汞撒利或二酰胺（在两个亚基之间形成二聚体）阻断每个通道亚基中靠近cGMP结合位点的3个胞质半胱氨酸残基中的至少1个，会增加对核苷酸的亲和力。抑制作用是通过阻断另外2个胞质巯基中的至少1个（N-乙基马来酰亚胺、汞撒利、氧化型谷胱甘肽）或另外2个（二酰胺、亚基内桥）产生的，并且可能涉及通道失活过程。当通道处于开放状态时，这3个胞质巯基是可及的，但当通道处于关闭状态时则不可及（或不可及得多）。
