Channel gating in bacteriorhodopsin-like channelrhodopsins with contrasting K⁺ and Na⁺ selectivity

Two closely homologous channelrhodopsins from Hyphochytrium catenoides, known as HcKCR1 (kalium channelrhodopsin 1) and HcCCR (cation channelrhodopsin), exhibit >100 times different K⁺/Na⁺ relative permeabilities and are emerging optogenetic tools for controlling neurons and cardiomyocytes. Key residue motifs and trimeric organization relate them structurally to bacteriorhodopsin-like cation channelrhodopsins (BCCRs). Here, we demonstrate that they utilize a specific gating mechanism previously suggested for the earlier-discovered BCCR from cryptophytes. Using a comparative analysis of transient absorption changes and photocurrents under single-turnover conditions, we identified an early, far-UV-absorbing photocycle intermediate that precedes channel opening in wild-type HcKCR1 and HcCCR. The UV-absorbing product is a spectrally distinct M1 state, characterized by a deprotonated Schiff base that facilitates cation passage through the channel. It is subsequently converted into an M2 state that absorbs at ∼400 nm. These photocycle steps appear to be a common feature of the entire BCCR family, as they were also found in Guillardia theta CCR2 (GtCCR2) and Rhodomonas abbreviata CCR1 (RaCCR1). Channel gating involves the transfer of a proton from Asp116, the conserved residue in the position of bacteriorhodopsin's proton donor to the Schiff base, to an unidentified residue. Here, we show that this residue is located on the cytoplasmic side of the molecule. The time course of this deprotonation correlated with the opening of the channel. The D116N mutation completely abolished HcCCR channel activity and converted HcKCR1 into a weak Na⁺ channel. Furthermore, HcCCR did not exhibit outwardly directed active proton transfer, as observed during the M2 state in HcKCR1. Finally, M2 rise coincided with the slow phase of M1 formation and channel opening in Na⁺-selective variants, but was delayed in HcKCR1 and its mutants. Our results contribute to a deeper mechanistic understanding of light-gated cation conductance in BCCRs, facilitating the further development of optogenetic tools.