鈉離子通道S4/D1與S4/D4區段與 通道活化及不活化反應之關係

Abstract

中文摘要 大鼠腦 IIA型鈉離子通道是一種電位開關性的離子通道，由四個相似的結構區域所組成。每個區域包含六條穿膜蛋白(S1~S6)，在S4上因為被發現有規則分布的鹼性胺基酸以α螺旋(α-helix)的方式排列，因此被認為其和感測電場的變化有關，且可隨膜電位之變化而運動，是目前公認的電位感測器。近年對於S4的運動方式主要有兩種說法：第一種是S4利用旋轉並往細胞外位移的方式運動，且S4所處的環境主要是由蛋白質所構成；第二種是S4位於通道蛋白質的外圍與細胞膜接觸，利用擺動的方式移動。在四個S4當中，位在第四結構區域上的S4 (S4/D4)被認為和鈉離子通道的不活化反應最相關。本文即藉由S3與S4間之連接段(S3-4 linker)和S4本身之非鹼性胺基酸的突變，來探討S4在去極化時移動的方式與距離，以及包圍在S4外側之主要電阻區的環境與位置。而我們主要把S3-4 linker與S4分成三條軸線來探討︰S4中帶正電荷的鹼性胺基酸序列為”R”軸線，此序列延伸到不帶有鹼性胺基酸的S3-4 linker上亦稱”R”軸線；”R”軸線的上一個厭水性胺基酸序列稱為”R-1”軸線；下一個厭水性胺基酸序列則稱”R+1”軸線。在將第一結構區域與第四結構區域S3-4 linker上的胺基酸和S4上非鹼性胺基酸的位置一一突變成鹼性胺基酸arginine之後，我們發現鈉離子通道的第一結構區域或是第四結構區域S3-4 linker上，有許多胺基酸位置的突變會造成活化曲線或是不活化曲線向左偏移的現象，此現象尤以”R-1”軸線最明顯，”R+1”軸線和”R”軸線次之，我們認為”R-1”軸線可能最具有能將S4固定於休息態的能力。並且在S4的外圍所包覆的主要電阻區和S4之間相接觸的情形，令人意外地，卻並不緊密，我們只能看出某些胺基酸在去極化的過程中有經過主要電阻區的可能性。不過在去極化的過程中，S4可能移動9個胺基酸的距離，並轉動180度的行動模式，則似乎仍可能與Shaker鉀離子通道是共通的。另外我們在每個結構區域S3-4 linker R1之前一個胺基酸位置的突變結果中看出，不同結構區域各自在通道開關機制當中可能各扮演不同的角色。其中第四結構區域和不活化反應的關係非常密切，也呼應了第四結構區域在構造上與不活化反應的密切相關性，而第一結構區域在活化反應當中可能扮演相當重要的角色。

Abstract Rat brain type IIA Na+ channel is a member of the voltage-gated Na+ channel which is composed of four structurally similar domains (I-IV). Each of the domain has six transmembrane segments (S1-S6). Because S4 contains regularly spaced basic amino acids, it has been considered as the voltage sensor of the channel. S4 presumably moves in response to membrane electric field change, leading into different gating conformations of the channel. There are chiefly two models explaining the S4 movement: the helical screw (S4 move translationally and rotationally in a gating canal which is composed of part of the channel protein), and the paddle model (S4 lying at the channel periphery and moving as one unit with S3b). Among the four S4s, the one in domain 4 (S4/D4) is especially implicated in Na+ channel inactivation and has been shown to move externally upon membrane depolarization. The amino acids in S3-4 linker and S4 are devided into three axes: the positive basic residues in S4 are called the “R” axis, extending to the hydrophobic residues in S3-4 linker ; the hydrophobic residues ahead of the “R” axis are called the “R-1” axis, and the next ones are the “R+1” axis. We made point mutations of the uncharged residues into arginine in the S3-4 linker and S4, and used two-electrode voltage clamp technique to study the effect of the mutation on channel gating. We found that many arginine mutations on S3-4 linker of domain one and domain four produced negative (hyperpolarized) shifts of the activation curve or inactivation curve, especially in the “R-1” axis, suggesting the essential role of this axis in stabilizing the channel in the resting state. The “impedance zone” surrounding S4 is surprisingly much wider in the Na+ channel than in the Shaker K+ channel. However, it seems plausible that like the S4 in the Shaker channel, the S4 in the Na+ channel are likely to move ~9 residues with a rotation of ~180° during channel activation/inactivation. According to the results of mutations of S3-4 linker at the position just preceding R1, we considered that different domains play different roles during channel activation/inactivation: domain four plays an important role only in channel inactivation, and domain one very likely is involved primarily in channel activation.