Hydroxylated residues influence desensitization behaviour of recombinant α3 glycine receptor channels
Hydroxylated residues influence desensitization behaviour of recombinant α3 glycine receptor channels
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DOI：
10.1046/j.1471-4159.2002.01109.x
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发表时间：
2002-10-01
影响因子：
4.7
通讯作者：
Becker, CM
中科院分区：
文献类型：
--
作者：
Breitinger, HG;Villmann, C;Becker, CM
人甘氨酸受体亚基α 3存在于两个剪接变体（α 3 K/L）中，其中α 3L在胞质TM 3 -4环内具有15个氨基酸的额外片段。同型α 3 K甘氨酸受体表现出比α 3L受体更快的脱敏。比较了α 3L、α 3 K和三重突变体α 3L（Δ OH）= α 3L（T358 A/Y367 F/S370 A）的离子通道性质，其中剪接插入物的羟基功能已通过定点诱变去除。在HEK 293细胞中重组表达后，膜片钳记录实验显示，羟基功能的去除主要影响受体脱敏。非脱敏电流的分数对于α 3L为68 +/- 13%，对于α 3 K为21 +/- 13%，并且对于α 3L（DeltaOH）为48 +/- 16%。对于α 3L、α 3 K和三重突变体α 3L（DeltaOH），在饱和甘氨酸浓度下的脱敏时间常数分别为8.4 +/- 2.8 s、1.9 +/- 2.3 s和2.8 +/- 0.4 s。相比之下，所有三种构建体的单通道和全细胞性质相似。因此，离子通道的激活、脱敏和电导性质是由不同的结构元件独立控制的。甘氨酸受体α 3亚基的M3-4环内的羟基功能是受体脱敏的决定因素，但不是唯一的。
The human glycine receptor subunit alpha3 exists in two splice variants (alpha3K/L), with alpha3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3-4 loop. Homomeric alpha3K glycine receptors show faster desensitization than alpha3L receptors. Ion channel properties were compared of alpha3L, alpha3K, and of the triple mutant alpha3L(DeltaOH) = alpha3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site-directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch-clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non-desensitizing current was 68 +/- 13% for alpha3L, 21 +/- 13% for alpha3K, and 48 +/- 16% for alpha3L(DeltaOH) . Desensitization time constants at saturating glycine concentration were 8.4 +/- 2.8 s, 1.9 +/- 2.3 s, and 2.8 +/- 0.4 s, for alpha3L, alpha3K, and the triple mutant alpha3L(DeltaOH) , respectively. In contrast, single-channel and whole-cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3-4 loop of the glycine receptor alpha3 subunit are crucial, but not exclusive, determinants of receptor desensitization.