測定微生物視紫質之膜片箝制系統的建立

Abstract

微生物視紫質 (microbial rhodopsin) 為一類由七個穿膜螺旋 (transmembrane helix) 及全反式視黃醛發色團 (all-trans-retinal chromophore) 所組成的感光膜蛋白質。在自然界中，微生物視紫質參與多項生理功能，如離子運輸及趨光性 (phototaxis)。此類蛋白質在特定波長的光刺激下行使其功能，並產生一系列稱為光週期 (photocycle) 的構形改變 (conformational change)。因此，對離子幫浦型微生物視紫質而言，光週期的反應動態與離子選擇性便成為其運輸活性的決定因子。在嗜鹽古菌 (halophilic archaea) 中，有兩種廣受關注的離子幫浦型微生物視紫質，即菌視紫質 (bacteriorhodopsin, BR) 與氯視紫質 (halorhodopsin, HR)。菌視紫質與氯視紫質分別為氫離子及氯離子幫浦，其功能皆會導致細胞過極化 (hyperpolarization)，該特性也促成兩者於光控基因生物學上的應用。為探究微生物視紫質的離子運輸對細胞內電位之影響，我們將菌視紫質與氯視紫質表現於人類胚胎腎細胞株 HEK293 及大腸桿菌 (Escherichia coli) 中，以建立適用於兩表現系統的膜片箝制記錄 (patch-clamp recording) 裝置。相較於 HEK293 細胞株的廣泛應用，大腸桿菌須透過巨型原膜體 (giant spheroplast) 的製備使其見用於電生理研究。巨型原膜體乃透過抑制細胞分裂及移除細胞壁來產生。本研究選用 Haloarcula marismortui BRI 的 D94N 突變型（亦稱 highly expressible BR, HEBR）與 Natronomonas pharaonis 的氯視紫質 (NpHR) 作為研究光週期與離子運輸相關性之蛋白質標的。我們發現 HEBR 的光週期時間常數約為其野生型的 50 倍長，而 NpHR 者則與前人實驗相符。在 HEK293 細胞實驗中，HEBR、NpHR 兩者皆成功進行轉染。後續的全細胞電壓箝制實驗 (whole-cell voltage-clamp recording) 則能在負細胞電位下記錄到 HEBR 轉染株的光電流 (photocurrent)。大腸桿菌的膜片箝制實驗方面，本研究提出以下有助於製備表現菌視紫質與氯視紫質之巨型原膜體的步驟：1）誘導蛋白質表現後，不進行過夜 2°C 培養、2）以置換緩衝溶液的方式終止細胞壁裂解反應、3）以 2°C 進行保存。在確認 NpHR 於大腸桿菌巨型原膜體中的功能性後，我們正致力於完成細胞膜封接 (membrane seal) 以在大腸桿菌中測量光電流。本研究旨在以膜片箝制實驗研究微生物視紫質光週期對離子運輸的影響，並以此分析方法建立一適用真核及原核表現系統之光控基因生物學工具篩選平台。

Microbial rhodopsins (MRhos) are a family of light-sensitive membrane proteins composed of seven transmembrane helices and an embedded all-trans-retinal (ATR) chromophore. These proteins exert diverse functions, participating in ion transport and phototaxis. Upon light stimulation of specific wavelengths, mRhos function while undergoing a series of conformational changes termed photocycle. Photocycle kinetics and ion selectivity thus determine the transport activity of ion-pumping mRhos. Bacteriorhodopsin (BR) and halorhodopsin (HR) are two well-studied mRhos from halophilic archaea. BR and HR are a proton and a chloride pump, respectively. Both mRhos’ functionality leads to cell hyperpolarization, inspiring their optogenetic applications in neuroscience. To investigate how ion transport of mRhos affects the whole-cell potential, we expressed the proteins in HEK293 and Escherichia coli cells. We aimed to construct a patch-clamp device applicable to both expression systems. HEK293 is a cell line readily amenable to patch-clamp measurements. On the other hand, previous studies applied E. coli cells in patch-clamp recordings by preparing giant spheroplasts via inhibition of cell division and removal of the cell wall. We selected the D94N mutant of Haloarcula marismortui BRI (a highly expressible BR, HEBR) and the HR from Natronomonas pharaonis (NpHR) to investigate the effects of photocycle on ion transport. We found that the photocycle time constant of HEBR is about 50-fold longer than its wild type. The photocycle kinetics of our NpHR construct is comparable to previous studies. BR and HR constructs were successfully transfected into HEK293 cells. Subsequent whole-cell voltage-clamp recordings displayed the photocurrents of HEBR transfectants under membrane voltages of -60 to 20 mV. For patch-clamp experiments on E. coli giant spheroplasts, we identified favorable preparation procedures for BR- and HR-expressing cells: 1) skipping overnight refrigeration after induction, 2) terminating the cell wall digestion by buffer replacement, and 3) storing at 2°C. The functionality of NpHR was confirmed in giant spheroplasts, and we aimed to attain photocurrents after forming the membrane seal. Through further analyzing the correlation between the photocycle kinetics and ion transport of mRhos, our patch-clamp system has the potential to be a handy screening platform for optogenetic tools using HEK293 and E. coli cells.