Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels.
Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels.
复制标题
保守脯氨酸残基在跨膜段 4 电压传感器功能和 L 型钙通道门控中发挥关键作用。
DOI：
10.1073/pnas.96.4.1357
复制
发表时间：
1999
影响因子：
11.1
通讯作者：
Varadi,G
中科院分区：
文献类型：
--
作者：
Yamaguchi,H;Muth,JN;Varadi,M;Schwartz,A;Varadi,G
第四跨膜段（S4）已被证明在电压门控通道中起电压传感器的作用。在膜去极化时，一段S4向外移动并引发许多构象变化，最终导致通道开放。保守的脯氨酸残基位于电压依赖性Ca 2+通道中基序I和III的S4中间。由于脯氨酸经常在蛋白质的螺旋结构中引入“扭结”，这些残基可能在电压传感器中具有内在功能。在这里，我们报告说，S4脯氨酸的去除导致通道开放时间的显着缩短，而额外的脯氨酸在基序IIS4和IVS4中的相应位置的引入延长通道开放时间。具有脯氨酸残基的S4s的数量与通道的平均开放时间呈明显的正相关。与在S4区域中没有脯氨酸的通道相比，经诱变以在所有四个S4中具有脯氨酸的通道的平均开放时间长>11倍。此外，脯氨酸在S4s减慢激活动力学和转移的电压依赖性的激活和失活的超极化方向。我们的研究结果强烈表明，脯氨酸残基的S4是至关重要的稳定开放状态的通道。此外，这表明基序IS 4和IIIS 4比基序IS 4和IVS 4更有效地促进通道开放。
The fourth transmembrane segment (S4) has been shown to function as a voltage sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that ultimately lead to channel opening. Conserved proline residues are in the middle of the S4 of motifs I and III in voltage-dependent Ca2+channels. Because proline often introduces a “kink” into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Here, we report that the removal of S4 prolines results in a dramatic shortening of channel open time whereas the introduction of extra prolines to the corresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlation with the mean open time of the channel. The mean open time was >11-fold longer for a channel mutagenized to have prolines in all four S4s compared with a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence of activation and inactivation in a hyperpolarized direction. Our results strongly suggest that proline residues in the S4s are critical for stabilizing the open state of the channel. Moreover, it is suggested that motif IS4 and IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.