對鈉離子通道不活化閘門D3-4 Linker與不活化電壓感受器S4/D4間交互作用之探討

Abstract

電位開關性鈉離子通道廣泛存在於生物體中可興奮性細胞之細胞膜上，是產生動作電位最主要的離子通道之一。當細胞膜去極化時，鈉離子通道中的電壓感受器第四穿膜區域(S4)因感應到電場變化而使鈉離子通道活化，遂打開孔道使鈉離子以正向回饋的方式大量進入細胞，造成電位急遽上升，使細胞達到閾值而產生動作電位。隨後鈉離子通道的不活化閘門(inactivation gate)則由於蛋白結構發生形變而塞住通道洞口，造成通道進入不活化態而無法通透離子。本實驗將大鼠腦IIA型鈉離子通道(Nav1.2)表現於南非爪蟾的卵上，並藉由膜內翻轉片膜箝制(inside-out patch clamp)的電生理技術來記錄其野生型及突變型的電流變化。本研究選定鈉離子通道第三四結構區域連接段(D3-4 linker)中的 IFMT不活化閘門中的I1488、F1489與第四結構區域第四穿膜區段(S4/D4)上的R+1軸線上的疏水性胺基酸I1633、I1636、L1639皆突變成帶有巰基(-SH)的胺基酸cysteine(C)，並從胞內給予鎘(cadmium)離子、還原劑Beta-mercaptoethanol等試劑，觀察這些試劑對於cysteine上巰基的修飾作用，藉以窺知兩者間可能的交互作用關係。實驗結果顯示：單突變I1488C、F1489C在不活化曲線中大幅向右移動約23mV，位於S4/D4 R+1軸的單突變I1633C、L1639C同時影響活化與不活化作用。鎘離子分別對於單突變I1488C、F1489C、I1633C、I1636C有明顯的修飾作用，但對於雙突變的影響則相對微小，根據雙循環突變分析(double mutant cycle analysis)，可以判斷I1488C、F1489C與I1633C、I1636C之間具有交互作用。總結以上觀察，我們認為在鈉離子通道活化、S4/D4向外移動的過程中可能與不活化閘門之間有著密切的交互作用，而I1488、F1489與I1633、I1636之間的直接交互作用可能就是造成鈉離子通道活化-不活化的偶合關鍵。

Voltage-gated sodium channels constitute the major basis of action potential generation and are present in most excitable cells. The 4th transmembrane segment (S4) in each domain of the Na+ channel moves in response to membrane potential alterations and thus cause gating conformational changes of the channel. We recorded the current changes of rat brain type IIA Na+ channels which were expressed in Xenopus laevis’ oocytes with voltage clamp and inside-out patch techniques. To investigate the possible interactions between the IFMT inactivation gate and the “R+1” axis in S4/D4, we made cysteine mutations at I1488 and F1489 in the inactivation gate D3-4 linker (the linker between domain 3 and domain 4) and I1633, I1636, L1639 in the “R+1” axis in S4/D4 (the fourth transmembrane segment of domain 4). We applied Cd2+ ions and reducing agent Beta-mercaptoethanol from cytoplasmic side to modify the sulfhydryl group(-SH) on cysteine. As a result, I1488C and F1489C mutations caused rightward shift of the inactivation curve by~23mV. I1633C mutant channel has a similar effect on the activation and inactivation curve, and so does L1639C. Cd2+ ions has a strong effect on the I1488C, F1489C, I1633C or I1636C mutant channels. However, there is only a much less action on the corresponding double mutant channels. With double mutant cycle analysis, we propose that there may be direct interactions between I1488C, F1489C and I1633C, I1636C which could constitute the biophysical basis of the coupling between the D3-4 linker inactivation gate and S4/D4 movement in the Na+ channel.