Molecular mechanism of calcium inhibition in viral channelrhodopsins

Viral channelrhodopsins (VCR1s) are giant-virus-encoded light-gated channels permeable to monovalent and divalent cations, including Na⁺ and Ca²⁺ ions, and inhibited by millimolar Ca²⁺ concentrations. Here, we combine X-ray crystallography, time-resolved UV-vis spectroscopy, and ATR-FTIR spectroscopy to investigate molecular mechanisms of ion permeation and Ca²⁺-dependent inhibition in OLPVR1. An atomic resolution structure of OLPVR1 obtained in the presence of 10 mM CaCl₂ and 900 mM NaCl reveals a transient intracellular Ca²⁺ binding site near T87 and T88, close to the retinal cofactor. Upon photoactivation, this Ca²⁺ ion prevents a key rearrangement of the intracellular gate required for ion translocation, namely the flip of E44, thereby disrupting ion conduction. Instead, illumination leads to the accumulation of Na⁺ ions between E44, S208 and the carbonyl oxygen of retinal-binding residue K204. Our findings reveal the molecular basis of Ca²⁺-dependent inhibition in VCR1s and provide a foundation for engineering enhanced tools for calcium optogenetics.