探討嗜鹽古生菌氯視紫質光控生理學之應用潛力： 研究在不同酸鹼度與鹽度下之光驅動特性

Abstract

氯視紫質是類GPCR的膜蛋白質，目前被發現廣泛存在於嗜鹽古生菌中。氯視紫質具有七個穿膜螺旋及一個發光機團-視黃醛，在基態時視黃醛會傾向全反式結構，當受到特定波長的光激發後會進行異構化轉變為13-順式，藉由視黃醛的結構變化推動氯視紫質蛋白質的構型轉換，將氯離子主動運輸進入細胞內。因氯視紫質具有主動運輸氯離子進入細胞內的特性，氯視紫質被應用於光控生理學，用以降低細胞的膜電位，抑制神經細胞的活性。NpHR及HsHR這兩種氯視紫質在過去被研究的最透徹，由於NpHR擁有較高的表現量，因此最常被應用於光控生理學上。然而NpHR受光照後展現極快的氯離子運輸效率，導致氯離子快速在細胞內累積，這個現象造成劇烈的細胞膜電位變化以及細胞膜內外離子平衡失調最終導致細胞受損或死亡。因此本研究挑選六種嗜鹽古生菌的氯視紫質，NpHR、HwHR、HsHR、HmHR、HtHR與HrHR，分析在不同鹽度與酸鹼度下氯視紫質的結構穩定性、氯離子運輸效率、功能穩定性以及在神經細胞生理條件下氯離子運輸能力，藉由這些性質評估六種嗜鹽古生菌的氯視紫質應用在光控生理學的潛力。根據實驗結果發現NpHR對環境的變化最敏感，HsHR在神經細胞生理條件下擁有最慢的氯離子運輸效率，此外值得一提的是HwHR與HsHR在神經細胞生理條件下運輸氯離子的能力明顯降低。根據實驗結果，Haloarcula屬的三個氯視紫質HmHR、HtHR與HrHR在不同環境下擁有較好的結構穩定性、較NpHR稍慢的氯離子運輸速度、出色的功能穩定性以及在神經細胞的生理條件下可以維持氯離子運輸能力。因為這三個氯視紫質的氯離子運輸速度比NpHR慢，能減緩的氯離子在細胞內累積的速度，降低對細胞的壓力及損害，因此本研究認為Haloarcula屬的三個氯視紫質是可替代NpHR作為光控生理學工具的候選人。

Halorhodopsins (HRs) are GPCR-like proteins that are widely found in haloarchaea. HR has seven transmembrane helices and a retinal chromophore, which tends to keep in all-trans configuration in the ground state. Upon stimulation by light of the specific wavelength, all-trans-retinal undergoes isomerization to 13-cis form, leading to conformational changes of HRs and actively transporting chloride ions into the cells. Because HRs can generate the influx of chloride ions, HRs have been applied in optogenetics to decrease the membrane potential of cells and to inhibit neural activity. Previously, two HRs, NpHR and HsHR, are the most well-studied HRs, but due to the high expression level of NpHR, which is the most frequently used HR in optogenetics. However, NpHR exhibits an extremely high chloride ion transport rate upon light illumination, causing the rapid accumulation of chloride ions in the cells. This phenomenon leads to severe changes in membrane potential and ion imbalance between the inside and the outside of the cell membrane, contributing to cell damage and death. Therefore, this study selected six HRs, NpHR, HwHR, HsHR, HmHR, HtHR, and HrHR, from six haloarchaea species and analyzed their structural stability, chloride ion transport rate, functional stability, and chloride transport capabilities under different salt concentrations and pH levels to comprehensively evaluate their potential for application in optogenetics. According to the experimental results, we found that NpHR was the most sensitive to environmental changes than others, because of the lower structural stability of NpHR in various environments. Then, HsHR has the longest tau value of the G-state under neural physiological conditions, which means HsHR may have the lowest chloride ion transport rate in neurons. Besides, it is noteworthy that the chloride ion transport capabilities of HwHR and HsHR were significantly reduced under neural physiological conditions. On the basis of the experimental results, three HRs from the genus Haloarcula, HmHR, HtHR, and HrHR, had better structural stabilities, slower chloride ion transport rate, and excellent functional stabilities under neural physiological conditions. Because the transport efficiencies of these three HRs were lower than that of NpHR, they can slow down the over-accumulation of chloride ions inside the cells, which reduces stress and damage to neurons. Therefore, three HRs from the genus Haloarcula are the candidates as alternative HR-based optogenetic tools.