Ion Channel Properties of a Cation Channelrhodopsin, <i>Gt</i>_CCR4

• Main Authors: Shunta Shigemura, Shoko Hososhima, Hideki Kandori, Satoshi P. Tsunoda
• Format: Article
• Language: English
• Published: MDPI AG 2019-08-01
• Series: Applied Sciences
• Subjects: microbial rhodopsin; channelrhodopsin; electrophysiology; optogenetics
• Online Access: https://www.mdpi.com/2076-3417/9/17/3440

We previously reported a cation channelrhodopsin, <i>Gt</i>_CCR4, which is one of the 44 types of microbial rhodopsins from a cryptophyte flagellate, <i>Guillardia theta</i>. Due to the modest homology of amino acid sequences with a chlorophyte channelrhodopsin such as <i>Cr</i>_ChR2 from <i>Chlamydomonas reinhardtii</i>, it has been proposed that a family of cryptophyte channelrhodopsin, including <i>Gt</i>_CCR4, has a distinct molecular mechanism for channel gating and ion permeation. In this study, we compared the photocurrent properties, cation selectivity and kinetics between well-known <i>Cr</i>_ChR2 and <i>Gt</i>_CCR4 by a conventional path clamp method. Large and stable light-induced cation conduction by <i>Gt</i>_CCR4 at the maximum absorbing wavelength (530 nm) was observed with only small inactivation (15%), whereas the photocurrent of <i>Cr</i>_ChR2 exhibited significant inactivation (50%) and desensitization. The light sensitivity of <i>Gt</i>_CCR4 was higher (EC₅₀ = 0.13 mW/mm²) than that of <i>Cr</i>_ChR2 (EC₅₀ = 0.80 mW/mm²) while the channel open life time (photocycle speed) was in the same range as that of <i>Cr</i>_ChR2 (25~30 ms for <i>Gt</i>_CCR4 and 10~15 ms for <i>Cr</i>_ChR2). This observation implies that <i>Gt</i>_CCR4 enables optical neuronal spiking with weak light in high temporal resolution when applied in neuroscience. Furthermore, we demonstrated high Na⁺ selectivity of <i>Gt</i>_CCR4 in which the selectivity ratio for Na⁺ was 37-fold larger than that for <i>Cr</i>_ChR2, which primarily conducts H⁺. On the other hand, <i>Gt</i>_CCR4 conducted almost no H⁺ and no Ca²⁺ under physiological conditions. These results suggest that ion selectivity in <i>Gt</i>_CCR4 is distinct from that in <i>Cr</i>_ChR2. In addition, a unique red-absorbing and stable intermediate in the photocycle was observed, indicating a photochromic property of <i>Gt</i>_CCR4.