Potassium-selective channelrhodopsins

Abstract

Since their discovery 21 years ago, channelrhodopsins have come of age and have become indispensable tools for optogenetic control of excitable cells such as neurons and myocytes. Potential therapeutic utility of channelrhodopsins has been proven by partial vision restoration in a human patient. Previously known channelrhodopsins are either proton channels, non-selective cation channels almost equally permeable to Na⁺ and K⁺ besides protons, or anion channels. Two years ago, we discovered a group of channelrhodopsins that exhibit over an order of magnitude higher selectivity for K⁺ than for Na⁺. These proteins, known as "kalium channelrhodopsins" or KCRs, lack the canonical tetrameric selectivity filter found in voltage- and ligand-gated K⁺ channels, and use a unique selectivity mechanism intrinsic to their individual protomers. Mutant analysis has revealed that the key residues responsible for K⁺ selectivity in KCRs are located at both ends of the putative cation conduction pathway, and their role has been confirmed by high-resolution KCR structures. Expression of KCRs in mouse neurons and human cardiomyocytes enabled optical inhibition of these cells' electrical activity. In this minireview we briefly discuss major results of KCR research obtained during the last two years and suggest some directions of future research.

Keywords: ion channels; ion selectivity; optogenetics; photocurrent.

Figure 1

TM3 residue motifs conserved in different families of microbial rhodopsins. Carboxylated residues are shown in red; polar residues in green; aromatic residues in violet; and small non-polar residues in orange. The red rectangle highlight that the DTD motif is only found in KCRs, BCCRs, and haloarchaeal H⁺ pumps. Abbreviations: HcKCR1 and HcKCR2, Hyphochytrium catenoides kalium channelrhodopsins 1 and 2, respectively; CovKCR1, Colponema vietmamica kalium channelrhodopsin 1; WiChR, Wobblia inhibitory channelrhodopsin; GtCCR1 and GtCCR4, Gulliardia theta cation channelrhodopsins 1 and 4, respectively; RsCCR1, Rhodomonas salina cation channelrhodopsin 1; RlCCR1, Rhodomonas lens cation channelrhodopsin 1; HsBR and HwBR, Halobacterium salinarum and Haloquadratum walsbyi bacteriorhodopsins, respectively; aR3, archaeorhodopsin 3; cR3, cruxrhodopsin 3; CrChR1, Chlamydomonas reinhardtii channelrhodopsin 1; MvChR1, Mesostigma viride channelrhodopsin 1; GtACR1, Gulliardia theta anion channelrhodopsin 1; HfACR1, Hondaea fermentalgiana anion channelrhodopsin 1; NsXeR, Nanosalina sp. xenorhodopsin; PoXeR, Parvularcula oceani xenorhodopsin; SzR1-3, schizorhodopsins 1-3, respectively; AntR, Antarctic rhodopsin; HsHR, Halobacterium salinarum halorhodopsin; NpHR, Natronomonas pharaonis halorhodopsin; HmHR, Haloarcula marismortui halorhodopsin; NmClR, Nonlabens marinus chloride-pumping rhodopsin; PoClR, Parvularcula oceani chloride-pumping rhodopsin; FR, Fulvimarina rhodopsin; LmClR, Lewinella maritima chloride-pumping rhodopsin; DeNaR, Dokdonia eikasta natrium rhodopsin; GlNaR, Gillisia limnaea natrium rhodopsin; IaNaR, Indibacter alkaliphilus natrium rhodopsin; PoNaR, Parvularcula oceani natrium rhodopsin.

Figure 2

Photocurrents generated by the indicated KCRs at –40 mV under physiological ionic conditions in response to a 1-s light pulse, the duration of which is shown by the colored bars. The current traces were normalized at the peak value. The numbers show desensitization (reduction of photocurrent at the end of the light pulse in % of the peak value) and the time of half-amplitude reduction of photocurrent after the light is turned off. The grey area shows the s.e.m. (n=5–7 cells).

Figure 3

The residues in TM2, TM3 and TM7 that determine K⁺ selectivity of KCRs, as demonstrated by mutation analysis. The lower rows show the substitutions of the residues conserved in HcKCR1 and HcCCR that nevertheless led to a decrease in K⁺ selectivity in the former. The color code is as in Fig. 1. For more detailed explanation see the text.