Light-Driven Chloride Transport Kinetics of Halorhodopsin.
Light-Driven Chloride Transport Kinetics of Halorhodopsin.
复制标题
DOI：
10.1016/j.bpj.2018.06.009
复制
发表时间：
2018-07
影响因子：
3.4
通讯作者：
Hasin Feroz;Bryan H Ferlez;Cécile Lefoulon;Tingwei Ren;Carol S. Baker;John P. Gajewski;D. J. Lugar;Sandeep Gaudana;P. Butler;Jonas Hühn;M. Lamping;W. Parak;J. Hibberd;C. Kerfeld;N. Smirnoff;M. Blatt;J. Golbeck;Manish Kumar
中科院分区：
文献类型：
--
作者：
Hasin Feroz;Bryan H Ferlez;Cécile Lefoulon;Tingwei Ren;Carol S. Baker;John P. Gajewski;D. J. Lugar;Sandeep Gaudana;P. Butler;Jonas Hühn;M. Lamping;W. Parak;J. Hibberd;C. Kerfeld;N. Smirnoff;M. Blatt;J. Golbeck;Manish Kumar
尽管人们对用于光遗传学的光驱动离子泵越来越感兴趣，但由于在体外测量缓慢运输过程和确定膜中的蛋白质浓度和/或取向方面存在挑战，目前对其运输速率的估计跨越了两个数量级。在这项研究中，据我们所知，我们首次直接定量测量了法老钠单胞菌（NpHR）阴离子泵盐紫质的光驱动Cl -输运速率。我们使用光界面电压钳测量表达nphrr的卵母细胞，获得了219（±98）Cl−/蛋白/s的传输速率，光子通量为630光子/蛋白/s。测量结果与文献报道的nphr约30%的量子效率一致，即每光子吸收0.3个异构化。为了将我们的测量结果与先前报道的20 ms速率限制步骤或35次/蛋白质/s相一致，据我们所知，我们进行了新的连续单次闪光实验，证明在连续照明下，NpHR绕过了光循环中的这一步骤。
Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl−transport rates of the anion pump halorohodopsin fromNatronomonas pharaonis(NpHR). We used light-interfaced voltage clamp measurements onNpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl−/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% forNpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination,NpHR bypasses this step in the photocycle.