鈉離子通道第四次單元孔洞迴路與第六穿膜區中段之交互關係

Abstract

抗癲癇藥物及局部麻醉劑對於鈉離子通道有使用依賴性的抑制效果，一般相信這些藥物有一個共同的受體結合位置。部分的學者認為，此受體位於細胞內側，為鈉離子通道內口的一部分；另一些學者則認為受體是位於細胞外側。過去的研究發現，當第四次單元 (DIV) 之第五、六穿膜區連接段 (S5-S6 linker, P-loop) 的 W1716，以及 DIV S6 中段的 F1764 發生突變時，皆可顯著影響抗癲癇藥物或局部麻醉劑藥物的親和力。傳統上認為 W1716 是構成鈉離子通道外口的一部分，F1764 則是位於鈉離子通道的內面。我們從這樣的觀點出發，針對此二位置及其前後位置做各種不同的突變，以了解為何在鈉離子通道內側的 F1764 及外側的 W1716 皆可影響藥物親和力。實驗中，使用典型的抗癲癇藥物 (carbamazepine, CBZ)、典型的局部麻醉劑 (lidocaine) 以及結構上類似但不屬於此兩類藥物的 diclofenac (一種非固醇類抗發炎藥物)。對於同一突變來說，親和力變化程度有 lidocaine > CBZ > diclofenac 的關係。而對於同一氨基酸，突變成氨基酸 A 或 C 的效果要大於突變成 E 或 R 的效果。若從能量的觀點來看，W1716C / F1764A 雙突變的效果相較於兩單點突變並沒有加成性，而 W1716E / F1764R 則可能有超加成性。我們同時也測試 W1716C、F1764C 及 W1716C/F1764C 對於 Cd2+ 的敏感性，結果發現敏感度在 -30 mV 有下列關係 W1716C > F1764C > WT (野生型) ~ W1716C / F1764C，即 W1716C 與 F1764C 之敏感度在雙突變有相互抵消的效果。另一方面，從胞外給予 MTSET (2-(trimethylammonium)ethyl) methanethiosulfonate bromide，可於氨基酸 C 的側支上做共價鍵的修飾) 的結果發現，WT 及 F1764C 皆無法被有效修飾，W1716C 則在速率及程度上都有明顯的狀態依賴修飾效果。若 W1716C 配合 F1764C 形成 W1716C / F1764C 雙突變，W1716C 被修飾的程度會大幅降低，且於不活化態時要比處於休息態時減弱幅度更大。儘管 F1764C 無法被胞外MTSET 修飾，卻可被胞外MTSEA (2-aminoethyl methanethiosufonate, hydrobromide) 修飾，且修飾後並不影響電流的峰值，反而是減小殘餘的電流 (sustain current)，並將不活化曲線負向移動，扮演一開闔修飾者的角色。此外，當利用氨基酸 arginine – glutamate 形成的離子鍵來強化 W1716 及 F1764 間交互作用時，我們發現 W1716E 及 F1764R各別影響離子通道性質的情況甚小。但當兩者搭配，即在 W1716E / F1764R 雙突變離子通道中，我們發現不活化曲線斜率變得很緩且反轉膜電位大幅負向移動至 0 mV 附近。此一結果顯示 W1716 及 F1764的位置關係相當靠近，且兩者間互動關係相當精細，若改變此關係則離子通道的性質會隨之嚴重改變。從藥理上來說，W1716 及 F1764 交互作用的模式，造成了藥物的抑制效果的狀態依賴性。其次，從功能性數據的暗示，傳統上認為 W1716 在鈉離子通道外口、而 F1764 在內口的想法應有所修改；最後，從生理的角度來看，W1716 所處的鈉離子通道外口，可能並不是單純地負責離子的通透與選擇，而很可能是連結通道開闔機制及離子通透機制的重要樞紐。

Anticonvulsants and local anesthetic drugs block voltage gated sodium channels in a similar use-dependent manner, and are found to bind to a common receptor in the channels. Some researchers believe that this common receptor is located at the internal pore of sodium channels, whereas others believe that the receptor is located at the external pore of sodium channels. Both W1716 at DIV P-loop and F1764 at mid DIV S6 segment have been implicated as the putative receptor site for anticonvulsants and local anesthetics. However, according to the traditional view of sodium channel structure, W1716 is located at the external pore mouth and F1764 is located in the internal pore. In this present study, we tried to explore why and how external and internal pore could affect drug binding affinities simultaneously. To reach this goal, we tested the affinity of carbamazepine (a typical anticonvulsant, CBZ), lidocaine (a typical local anesthetic), or diclofenac (a non-steroid anti-inflammatary drug structurally similar to carbamazepine) to different mutant channels. The affinity changes showed the following pattern for all mutants: lidocaine > CBZ > diclofenac. The attenuation of drug affinity is more significant in mutating W1716 or F1764 to A or C than to E or R. While W1716C/F1764A showed no additive effect on drug binding as compared to corresponding single mutants, W1716E/F1764R showed supra-additive effects. We also characterized the sensitivity of different mutant channels to cadmium ion, and demonstrating an interesting order: W1716C > F1764C > WT ~ W1716C/F1764C. This order strongly implied that the sensitivity of W1716C and that of F1764C were cancelled mutually in double mutant channel W1716C/F1764C. Moreover, while W1716C showed its sensitivity to external MTSET in a state dependent manner, WT and F1764C had no significant modification by external MTSET. Interestingly, as in the case of cadmium sensitivity, we found that F1764C attenuated the sensitivity of W1716C to external MTSET, especially at the inactivated channel. That is to say, W1716C/F1764C mutant channel thus was less rather than more modified than W1716C mutant channel. Although F1764C can not be modified by external MTSET, it can be modified by external MTSEA. Modification of W1716C by MTSET attenuated peak current. In contrast, MTSEA blocked sustain current of F1764C and shifted its inactivation curve negatively. On the other hand, we use RE mutant to strengthen the interaction between W1716 and F1764. Both single mutant channels (W1716E and F1764R) showed little effects on channel gating or ion selectivity. However, W1716E/F1764R double mutation caused changes in the slope factor of the steady state inactivation curve and significant shift of reversal potential. These results supported that W1716 and F1764 may be located in a common region which allows state dependent interaction between these two residues. The traditional view of sodium channel pore structure thus may have to be modified to incorporate our data. From the pharmacological point of view, this state-dependent interaction may constitute the molecular basis of use-dependent drugs, such as anticonvulsants or local anesthetics. W1716 and the external pore mouth may not only be involved in ion selectivity but also serve as a key linkage between the gating machinery and the ion permeation pathway in sodium channels.