Microbial Rhodopsins: Structural–Functional Mechanisms and the Design of Optogenetic Silencing Tools—With Emphasis on Halorubrum Archaeorhodopsins

Microbial rhodopsins are light-sensitive membrane proteins that use retinal as a covalent chromophore and adopt a seven-transmembrane α-helical fold. They are widely distributed across archaea, bacteria, algae, and fungi and mediate diverse functions, including light-driven ion transport, light-gated ion conduction, and photosensory signaling. Since the discovery of bacteriorhodopsin in 1971, the microbial rhodopsin family has expanded continuously, and channelrhodopsin-based optogenetics has revolutionized neuroscience by enabling millisecond-scale optical control of neuronal activity. However, compared with excitatory tools, optogenetic silencing tools have developed more slowly and remain limited by insufficient photosensitivity, poor membrane localization, ion-homeostasis perturbation, and phototoxicity. Because these properties are largely constrained by the structural and functional features of the parent microbial proteins, the discovery and in-depth characterization of natural rhodopsins remain essential for tool innovation. This narrative review focuses on ion-pumping archaeorhodopsins, with emphasis on the genus Halorubrum and on HeAR, and describes how the literature was selected. We first summarize the functional classification, structural basis, photocycle, and ion-pumping mechanisms of microbial rhodopsins. We then discuss the diversity of Halorubrum archaeorhodopsins and the structural–functional features of HeAR, a recently characterized archaeorhodopsin from a halophilic archaeon isolated from Ejinoor Salt Lake in Inner Mongolia, China. HeAR absorbs at 550 nm (dark-adapted) and 560 nm (light-adapted), essentially the same maximum as AR3 (559 nm). Its Schiff-base counterion Asp-95 has a pKa of 3.5 ± 0.1, about one unit higher than BR Asp-85 (≈2.6) and AR3 Asp-95 (2.5 ± 0.2), but matched by AR4 Asp-85 (3.6); thus, this is a property that HeAR shares with its closest relative rather than one that sets it apart. Its photocycle (≈100 ms) is prolonged—roughly ten times longer than the overall cycle of BR (11 ms), of AR4 (≈10 ms) and of the O-decay of AR3 (τ ≈ 14 ms)—while its M intermediate forms and decays faster than in BR; relative to AR3 and AR4, however, the M decay is slower (3.3 ms against ≈390 µs and 0.55/3 ms) and the overall cycle is longer. These are established in vitro properties of the heterologously expressed protein, and because a pump moves a fixed, small number of charges per photocycle, low-light performance remains a hypothesis to be tested rather than a demonstrated advantage. In this review “red-shifted” is used in one sense only: HeAR’s 550–560 nm window lies ≈80–90 nm to the red of excitatory channelrhodopsins (≈470 nm); relative to BR it is blue-shifted by ≈18 nm, and relative to AR3 the two absorption maxima coincide. We further review the evolution of optogenetic silencing tools, from inward chloride pumps and outward proton pumps to light-gated anion channels and potassium channels, and discuss structure-guided rational design strategies, including structure prediction, molecular dynamics simulation, directed evolution, and machine learning-assisted engineering. Finally, we outline current challenges and future opportunities in phototoxicity, deep-tissue light delivery, clinical translation and the exploitation of halophilic archaeal resources, and we specify which experiments are required before HeAR can be judged as a silencing tool. Clinically validated optogenetics is kept separate throughout from speculative applications of newly characterized archaeorhodopsins. This review aims to provide a comparative framework linking the structural–functional properties of natural rhodopsins to the rational design of next-generation optogenetic silencing tools.