Division of Soft Matter, Graduate School of Life Science, Hokkaido University, Sapporo, 060-0810, Japan.
Division of Macromolecular Functions, Department of Biological Science, School of Science, Hokkaido University, Sapporo, 060-0810, Japan.
Sci Rep. 2021 Apr 12;11(1):7908. doi: 10.1038/s41598-021-86812-z.
Ion channel proteins are physiologically important molecules in living organisms. Their molecular functions have been investigated using electrophysiological methods, which enable quantitative, precise and advanced measurements and thus require complex instruments and experienced operators. For simpler and easier measurements, we measured the anion transport activity of light-gated anion channelrhodopsins (ACRs) using a pH electrode method, which has already been established for ion pump rhodopsins. Using that method, we successfully measured the anion transport activity and its dependence on the wavelength of light, i.e. its action spectra, and on the anion species, i.e. its selectivity or preference, of several ACRs expressed in yeast cells. In addition, we identified the strong anion transport activity and the preference for NO of an ACR from a marine cryptophyte algae Proteomonas sulcata, named PsuACR_353. Such a preference was discovered for the first time in microbial pump- or channel-type rhodopsins. Nitrate is one of the most stable forms of nitrogen and is used as a nitrogen source by most organisms including plants. Therefore, PsuACR_353 may play a role in NO transport and might take part in NO-related cellular functions in nature. Measurements of a mutant protein revealed that a Thr residue in the 3 transmembrane helix, which corresponds to Cys102 in GtACR1, contributed to the preference for NO. These findings will be helpful to understand the mechanisms of anion transport, selectivity and preference of PsuACR_353.
离子通道蛋白是生物体内具有重要生理功能的分子。它们的分子功能已通过电生理学方法进行了研究，该方法能够进行定量、精确和先进的测量，因此需要复杂的仪器和经验丰富的操作人员。为了进行更简单和更容易的测量，我们使用 pH 电极法测量了光门控阴离子通道蛋白（ACR）的阴离子转运活性，该方法已经针对离子泵视蛋白建立。使用该方法，我们成功测量了几种在酵母细胞中表达的 ACR 的阴离子转运活性及其对光波长的依赖性，即其作用光谱，以及对阴离子种类的依赖性，即其选择性或偏好性。此外，我们从海洋隐藻 Proteomonas sulcata 中鉴定出一种 ACR 的强阴离子转运活性和对 NO 的偏好性，命名为 PsuACR_353。在微生物泵或通道型视蛋白中首次发现了这种偏好性。硝酸盐是氮最稳定的形式之一，被包括植物在内的大多数生物用作氮源。因此，PsuACR_353 可能在 NO 转运中发挥作用，并可能参与自然界中与 NO 相关的细胞功能。对突变蛋白的测量表明，3 个跨膜螺旋中的一个 Thr 残基（对应于 GtACR1 中的 Cys102）有助于对 NO 的偏好性。这些发现将有助于理解 PsuACR_353 的阴离子转运、选择性和偏好性的机制。
