Glutamate residue 90 in the predicted transmembrane domain 2 is crucial for cation flux through channelrhodopsin 2

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Abstract

Channelrhodopsin 2 (ChR2) is a microbial-type rhodopsin with a putative heptahelical structure that binds all-trans-retinal. Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions of these residues significantly altered ChR2 function as revealed by two-electrode voltage-clamp recordings of light-induced currents from Xenopus laevis oocytes expressing the respective mutant proteins. Specifically, replacement of E90 by lysine or alanine resulted in differential effects on H+- and Na+-mediated currents. Our results are consistent with this glutamate side chain within the proposed TM2 contributing to ion flux through and the cation selectivity of ChR2.

Highlights

Channelrhodopsin 2 is a light-activated cation channel widely used in neurobiology. ► We show its transmembrane segment 2 to be crucial for ion conductance. ► Transmembrane segment 2 forms a hydrophilic amphipathic helix. ► Glutamate 90 located within this helix determines cation flux and selectivity.

Introduction

Channelrhodopsin 2 (ChR2) is a retinylidene protein of 737 amino acids that is expressed in the eye spot of the green alga Chlamydomonas reinhardtii and supposed to evoke photoresponses [1], [2]. Illumination of Xenopus laevis oocytes expressing ChR2 with blue light (λmax = 480 nm) induces a transient peak current followed by a persisting stationary one, carried mainly by protons and other monovalent cations. Additionally, Ca2+, but not Mg2+, also permeates ChR2 [1], [3]. The presumptive ion pore of ChR2 is located within the first 315 amino acids, as a correspondingly truncated ChR2 retains the electrophysiological properties of the full-length protein [1]. ChR2 shows 15–20% sequence homology to the microbial-type rhodopsins bacteriorhodopsin (BR), halorhodopsin (HR) and sensory rhodopsins (SRs). Their seven transmembrane domain structure [4], [5], [6], [7], [8] is consistent with hydropathy plots of ChR2 [2], and spectroscopic data as well as measurements on lipid bilayers suggest that ChR2 is a leaky proton pump [9]. The precise topology, the mechanism of its proton pump activity, as well as the location of its ion channel pore, however, remain elusive.

Here, we investigated whether a cluster of glutamates localized at positions 90, 97 and 101 of the predicted TM2 of ChR2 is implicated in ion permeation and/or cation selectivity. Neutral substitution of E97 has been previously shown to result in modest changes in current amplitude [10]. We report that charge inversions at these positions impair ChR2 currents, and that residue E90 appears to be particularly important for both H+ and Na+ conductance. Our results suggest that TM2 contributes to the lining and selectivity filter of ChR2’s intrinsic ion channel.

Section snippets

Plasmids and cRNA synthesis

cDNAs encoding a truncated version of ChR2 (AA1–315) or a C-terminal fusion of the truncated ChR2 sequence to YFP (ChR2-YFP; see [1]) were cloned into pGemHE [11] using its BamHI and XhoI sites. Mutations were introduced by site-directed mutagenesis using the QuickChange Mutagenesis Kit (Agilent Technologies, Santa Clara, USA) and confirmed by sequencing. After linearization at a unique NheI site, these ChR2-encoding plasmids were used as templates for cRNA synthesis with the mMessage T7 Ultra

Charged residues in TM2 might form an amphipathic helix

To identify residues of ChR2 that could be involved in the formation of the ion channel, we aligned ChR2 to ChR1, another light-gated channel protein from Chlamydomonas reinhardtii originally thought to be H+-selective [15], [16]. Assuming that membrane permeating cations should be stabilized by hydrophilic or negatively charged side chains, we identified three glutamate residues, E90, E97 and E101, within the predicted α-helical TM2 (Fig. 1A; see also Refs. [2], [10]). These residues and a

Discussion

In this study, we investigated whether three glutamate residues located within the predicted TM2 of ChR2 contribute to the channel-like cation conductance of this light-activated proton pump. Based on hydropathy plots and sequence comparison with ChR1 and BR, we identified four charged amino acids (E90, K93, E97 and E101) within the putative TM2 that might form an amphipathic helix suitable to provide for hydrophilic lining of the leak channel of ChR2. Assuming an α-helical arrangement of TM2,

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    1

    Present address: Faculté des Sciences Pharmaceutiques et Biologiques, University Paris Descartes, 75006 Paris, France.

    2

    Present address: Merz Pharmaceuticals GmbH, 60318 Frankfurt, Germany.

    3

    Present address: Institute for Biochemistry and Molecular Medicine, University Erlangen-Nürnberg, 91054 Erlangen, Germany.

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