WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

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 identification of 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 a so far unmatched preference for K + over Na + , stable photocurrents under continuous illumination, and a prolonged open-state lifetime. Showing high expression levels in cardiac myocytes and neurons, WiChR allows single- and two-photon inhibition at low irradiance and reduced tissue heating. Therefore, we recommend WiChR as the long-awaited efficient and versatile optogenetic inhibitor.

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

“…Several natural and engineered ChRs have been shown to exhibit a higher selectivity for H +3 , Na +4 , or Cl −5-7 compared to the originally discovered ChR1 and ChR2 of Chlamydomonas reinhardtii. More recently, ChRs with a high K + -selectivity were also discovered [8][9][10] . Accordingly, when expressed in neuronal cells or tissues, ChRs enable multidirectional manipulation of electrical cellular activity.…”

“…Accordingly, when expressed in neuronal cells or tissues, ChRs enable multidirectional manipulation of electrical cellular activity. Whereas non-selective cation-conducting ChRs (CCRs) generally depolarize cells and promote action potential firing, anionconducting ChRs (ACRs) and K + -selective ChRs (KCRs) suppress spiking upon illumination 8,9,11 . However, to date, no Ca 2+ -selective ChRs have been identified or engineered.…”

Potassium-selective channelrhodopsins

“…Out of all known ChRs, protein sequences of HcKCRs show the highest homology to those of BCCRs (including conservation of the DTD motif in TM3, Figure 1), although their source organism is phylogenetically very distant from cryptophytes. Close homologs of HcKCRs have been found in other stramenopile and colponemid protists, and in metagenomic samples [41,42], but not in cryptophytes. Close homologs of KCRs form a distinct branch of the phylogenetic tree together with KCRs, but most of these channels are not K + selective [41,43], which provides a unique possibility to identify the residues involved in K + selection by comparative analysis of their sequences.…”

“…Close homologs of KCRs form a distinct branch of the phylogenetic tree together with KCRs, but most of these channels are not K + selective [41,43], which provides a unique possibility to identify the residues involved in K + selection by comparative analysis of their sequences. Only six K + selective homologs are currently known: in addition to HcKCRs, these are WiChR from Wobblia lunata [42], B1ChR2 from Bilabrum sp. [42], and CovKCR1 and CovKCR2 from Colponema vietnamica [41].…”

A self-inactivating invertebrate opsin with resistance to retinal depletion optically drives biased signaling toward Gβγ-dependent ion channel modulation

“…Activated GPCR promotes dissociation of G protein a and βγ subunits, each of which drives downstream pathways. Thus, animal opsin-induced cellular responses tend to be complicated 17 , but Ga- and Gβγ-dependent cellular responses do not seem to be separable due to the 1:1 stoichiometry of Ga and Gβγ subunits.…”