研究造成原發性肢端紅痛症之突變鈉離子通道Nav1.7其功能性影響

Abstract

原發性肢端紅痛症是一種自體顯性遺傳的神經疾病，其病症為肢端的灼燒疼痛感，此症狀會經由外在環境溫熱或運動刺激所引起。目前已發現電壓門控鈉離子通道Nav1.7上之α-subunit的突變(基因為SCN9A)，是導致肢端紅痛症的主要原因，此病症同時也是第一個由基因突變所致的遺傳性神經疼痛症。在台灣我們最近發現三個SCN9A錯義突變，其中一個為家族遺傳性(I136V)，兩個為偶發性突變(I848T, V1316A)，這當中V1316A是尚未被報導過的突變。I136V突變位置在第一區之第一穿膜區段(DIS1)，I848T及V1316A分別位在第二及第三區之第四、五穿膜連接區段(DIIS4/S5, DIIIS4/S5)。為了瞭解這些突變鈉離子通道的分子病理，我們使用中國倉鼠卵巢細胞進行全細胞電生理記錄。這些突變之鈉離子通道皆在電壓依賴性活化產生過極化偏移，在穩定快速去活化表現出去極化偏移，且比起原生型，突變通道能更快從去活化恢復。由於熱會引發並加劇病症，藉由降低患部溫度病人能得到紓緩，我們於是進而比較溫度(25°C及35°C)對原生型與突變鈉離子通道的影響。在35°C，I136V及V1316A突變通道比起原生型，在電壓依賴性活化仍產生過極化偏移，即使原生型通道本身比起在25°C時已有顯著之過極化偏移。升溫則在三個突變通道之穩定去活化皆產生顯著去極化偏移，但原生型通道並不受影響。這些特性改變都可能造成所表達的神經細胞過度興奮，特別是在溫度提昇的狀態下更是如此。由於目前對於肢端紅痛症尚無絕對有效的治療方法，我們試圖利用電生理的方式替帶有這三種突變的病人篩選治療藥物，藉由分析突變型與原生型Nav1.7通道對lidocaine及mexiletine抑制效果的IC50，我們發現lidocaine很可能不適合用來治療這些病人，而如同臨床觀察所示，mexiletine用於治療帶有I848T突變的病人有減緩症狀的功效，如同I848T突變型通道展現對mexiletine抑制較低的IC50。本研究中我們報導一個嶄新的突變，V1316A，同時也提供這些突變鈉離子通道受到升溫影響的分子機制，這些將有助於了解肢端紅痛症的症狀表現，且對於發展有效治療方法也將有所助益。

Primary erythromelalgia (PE) is an autosomal dominant neurological disorder characterized by severe burning pain and erythema in the extremities upon heat stimuli or moderate exercise. Mutations in human SCN9A gene, which encodes the α–subunit of the voltage-gated sodium channel, Nav1.7, were found to be responsible for PE. Three missense mutations of SCN9A gene have recently been identified in Taiwanese patients including a familial (I136V) and two sporadic mutations (I848T, V1316A). V1316A is a novel mutation that has not been characterized. Topologically, I136V is located in DI/S1 segment of the sodium channle, and both I848T and V1316A are located in S4-S5 linker region of DII and DIII domains, respectively. To characterize the electrophysiological properties of these mutant channels, we over-expressed the mutant and wild type channels in Chinese Hamster ovary (CHO-K1) cells and employed the whole-cell patch clamp recordings. The mutant channels showed hyperpolarizing shift in voltage dependent activation, depolarizing shift in steady-state fast inactivation, and faster recovery from inactivation as compared with wild type channel. Since warmth can trigger and exacerbates symptoms and patients can relieve pain by cooling the affected areas, the electrophysiological properties of these mutant and wild type channels at room temperature, 25°C and at a higher one, 35°C were evaluated. At 35°C, I136V and V1316A mutant channels still exhibit a further hyperpolarizing shift in activation as compared with wild type channel, even though wild type channel also produced a significant hyperpolarizing shift compared to that of 25°C. Increased temperature resulted in significant depolarizing shift in steady-state fast inactivation in all three mutant channels except for the wild type channel. These property changes may contribute to hyperexcitability of sensory neurons that express these mutant channels, especially at high temperature. To search for an effective treatment for these patients, we tested the IC50 values of selective sodium channel blockers such as lidocaine and mexiletine. The mutant channels exhibited higher IC50 values for lidocaine compared to wild type channel, which suggests that lidocaine would not be a suitable treatment choice. IC50 for mexiletine is lower for I848T mutant channel. The result is in part comparable to clinical observations that mexiletine alleviates symptoms in the patients with I136V and I848T mutations but not the patient with V1316A mutation.