Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes
Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes
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DOI：
10.1113/jphysiol.1996.sp021392
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发表时间：
1996-06-01
影响因子：
5.5
通讯作者：
Torre, V
中科院分区：
文献类型：
--
作者：
Bucossi, G;Eismann, E;Torre, V
1.将牛视杆cGMP门控通道α亚基的赖氨酸346和谷氨酸372之间的推定孔区域中具有带电或极性残基的氨基酸突变为不同的氨基酸。将编码野生型（即α-亚基）或突变体通道的mRNA注射到非洲爪蟾卵母细胞中。当谷氨酸363突变为天冬酰胺、丝氨酸或丙氨酸时，在突变通道中由稳定cGMP浓度激活的电流下降。当谷氨酸363突变为天冬氨酸、谷氨酰胺或甘氨酸时，当苏氨酸359、360和364突变为丙氨酸时，或当孔区域中的其他带电残基被中和时，没有观察到电流下降。电流下降量及其时间过程具有明显的电压依赖性。在突变体E363 A中，在-100 mV下电流下降在约1.5 s内，但在+100 mV下约6 s内。在同一突变体中，电流在+100 mV时下降到其初始水平的约55%，在-100 mV时下降到约10%。突变体E363 A、E363 S和E363 N的电流下降仅中度依赖于cGMP浓度（从10 μ M至1000 μ M），并且不是由突变体通道对cGMP的亲和力降低引起的。对单通道电流波动的分析表明，电流下降主要是由于开路概率的降低.野生型通道对二甲基铵是不可渗透的。当谷氨酸363被一个更小的残基如丝氨酸取代时，突变通道变得对二甲基铵具有渗透性。在突变体通道中观察到的电流下降使人联想到配体门控通道的脱敏和电压门控通道的失活。这些结果还表明，门控和渗透通过cGMP门控通道从牛棒是内在耦合的，谷氨酸363是控制门控和最西部地区的孔的分子结构的一部分。
1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha-subunit of the cGMP-gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild-type, i.e. the alpha-subunit, or mutant channels was injected in Xenopus laevis oocytes.2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when threonine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized.3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at -100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at -100 mV.4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 mu M) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single-channel level indicated that current decline was primarily caused by a decrease of the open probability.5. The wild-type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium.6. The current decline observed in mutant channels is reminiscent of desensitization of ligand-gated channels and of inactivation of voltage-gated channels. These results suggest also that gating and permeation through the cGMP-gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore.