WiChR, a highly potassium selective channelrhodopsin for low-light two-photon neuronal inhibition

Abstract

The electric excitability of muscle, heart and brain tissue relies on the precise interplay of Na+- and K+-selective ion channels. The involved ion fluxes are controlled in optogenetic studies using light-gated channelrhodopsins (ChRs). While non-selective cation-conducting ChRs are well-established for excitation, K+-selective ChRs (KCRs) for efficient inhibition have only recently come into reach. Here, we report the molecular analysis of recently discovered KCRs from the stramenopile Hyphochytrium catenoides and identify a novel type of hydrophobic K+-selectivity filter. Next, we demonstrate that the KCR signature motif is conserved in related stramenopile ChRs. Among them, WiChR from Wobblia lunata features an unmatched 80-fold preference for K+ over Na+, stable photocurrents under continuous illumination and a prolonged open state lifetime. Well expressed in neurons, WiChR allows two-photon inhibition at low irradiance and reduced tissue heating, recommending WiChR as the long-awaited efficient and versatile optogenetic inhibitor.

Structural foundations of potassium selectivity in channelrhodopsins

“…In both models, Asp116 forms sidechain hydrogen bonds with Ser70 in the middle of TM2 and Arg 244 at the cytoplasmic end of TM7. In Hc KCR1, the S70A mutation decreased P K /P Na (23), but Ser70 is conserved in Hc CCR and does not render this channel K + -selective. Considering the results of our mutant analysis (Fig.…”

“…In WiChR1 and a KCR from the stramenopile Bilabrum sp. (B1ChR2; (23)), the residue corresponding to Tyr222 of HcKCRs is Phe.…”

“…In W iChR1 and a KCR from the stramenopile Bilabrum sp. ( B1 ChR2; (23)), the residue corresponding to Tyr222 of Hc KCRs is Phe. Both these KCRs showed higher P K /P Na values than Hc KCR1, but the Hc KCR1_Y222F mutant exhibited a decrease rather than increase in the K + selectivity (23).…”

“…( B1 ChR2; (23)), the residue corresponding to Tyr222 of Hc KCRs is Phe. Both these KCRs showed higher P K /P Na values than Hc KCR1, but the Hc KCR1_Y222F mutant exhibited a decrease rather than increase in the K + selectivity (23). Therefore, the Phe for Tyr substitution is unlikely responsible for the larger P K /P Na of W iChR1 and B1 ChR2, as compared to that of Hc KCR1.…”

“…In Gt CCR2 (one of BCCRs), deprotonation of the Asp96 homolog (Asp98) occurs >10-fold faster than reprotonation of the Schiff base and is required for cation channel opening (13). When we were preparing our manuscript for submission, a preprint was published reporting patch clamp analysis of Hc KCR1 mutants (23). Its results provide an independent validation of the conclusions drawn in our study.…”