KCNQ4之功能與藥物作用位點之探討

電位依賴型鉀離子通道KCNQ家族有五個成員 (KCNQ1-KCNQ5)，對於神經元細胞膜電位之穩定、神經骨骼肌動作電位的產生等皆佔有重要角色，對於治療癲癇、神經性疼痛也是新興治療目標，我們探討KCNQ家族其中一個成員KCNQ4。已知KCNQ4的異常會造成先天性非症候群聽障 (non-syndromic autosomal dominant deafness-2, DFNA2)，在高血壓與神經肌肉疾病中也扮演相關角色，因此我們想去探討KCNQ4電位依賴型鉀離子通道之功能性與藥物作用之結合位點。我們利用位點導向點突變技術將human KCNQ4或點突變KCNQ4建構於pcDNA3.1質體，轉染哺乳類細胞HEK293t表達，藉由全細胞電位鉗定技術紀錄其產生之電位依賴型鉀離子電流並分析KCNQ4鉀離子通道功能性。我們分別建構出KCNQ4點突變R168A、R488A、R490A、R492A、S494A、W242L質體並轉染HEK293t細胞株，藉由全細胞電位鉗定技術紀錄表達電流與野生型KCNQ4 (wild-type, WT) 比較分析之，並且處理鉀離子通道開啟劑retigabine後記錄表達電流。結果顯示，R168A平均二分之一活化膜電位 (half-activation voltage, V1/2) 為 -21.8±0.7 mV，W242L平均V1/2為 -21.5±1.2 mV，R492A平均V1/2為 -20.6±0.9 mV對照野生型 (WT)平均V1/2為 -17.7±1.1 mV，比較結果三者點突變不影響KCNQ4鉀離子通道之活化功能。R488A平均V1/2為 -29.7±0.5 mV，R490A平均V1/2為 -25.0±1.1 mV，S494A平均V1/2為 -29.0±0.8 mV，此三者點突變平均V1/2活化膜電位與WT比較往負極偏移約68%、42%、64%程度，顯示這幾個位點可能是KCNQ4鉀離子通道功能性關鍵位點。另外處理選擇性KCNQ家族鉀離子通道活化劑 retigabine 10 μM後，R168A平均V1/2加藥前 -21.8±0.7 mV而加藥後為 -44.6±1.0 mV，而WT平均V1/2加藥前 -17.7±1.1 mV而加藥後為 -47.8±0.7 mV，皆有尾電流活化曲線往負極活偏移之活化現象；另外處理retigabine 3 μM，因先前10μM濃度過高故先降低1/3，W242L平均V1/2為 -14.0±1.0 mV (加藥前-21.5±1.2 mV)，R488A平均V1/2為 -29.0±0.6 mV (加藥前 -29.0±0.5 mV) ，R490A平均V1/2為 -16.1±0.8 mV (加藥前 -25.0±1.1 mV)，R492A平均V1/2為 -28.9±1.3 mV (加藥前 -20.6±0.9 mV)，S494A平均V1/2為 -35.8±2.4 mV (加藥前 -29.0±0.8 mV)，推測W242、R488、R490、R492、S494可能為選擇性KCNQ家族鉀離子通道活化劑retigabine在KCNQ4上之相關作用結合位。

The family of KCNQ (Kv7) voltage-gated potassium channels consists of five members (Kv7.1 to Kv7.5). In this study, we investigated the function of KCNQ4 (Kv7.4) and its binding sites interact with KCNQ openers. We constructed human KCNQ4 and point mutant with pcDNA3.1 plasmid. KCNQ4/pcDNA3.1 was expressed in HEK293t cells and analyzed its conducted voltage-gated potassium channel current. The tail currents analysis of KCNQ4 channels was using the whole-cell patch clamp technique. We constructed R168A, W242L, R488A, R490A, R492A and S494A by site-directed point mutation. Our results showed that the half-activation potential of R168A, W242L, and R492A were -21.8±0.7 mV, -21.5±1.1 mV, -20.6±0.9 mV, respectively. There are no significant difference between WT (-17.7±1.0 mV), R168A, W242L and R492A in half-activation potential. However, the half-activation potential of R488A, R490A and S494A (-29.7±0.5 mV, -25.0±1.1 mV, -29.0±0.8 mV) was shifted more negative than that of WT. Treatment with the selective KCNQ family opener, retigabine, in WT and R168A can produce a V1/2 shifted to a more negative potentials. On the other hand, W242L, R488A, R490A, R492A and S494A did not produce a significant shift in the V1/2 of activation curves after the treatment of retigabine. These data suggested that W242, R488, R490, R492 and S494 are may be involved in the binding of retigabine to the KCNQ4 channels.