Analysis of light-gated ion channels in green algae

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Figure …. Amino acid residue alignment of 7 TM domains of known channelrhodopsins with EeChR1. Genbank accession numbers are given in brackets. PsChR, Platymonas (Tetraselmis) subcordiformis channelrhodopsin (JX983143); CrChR1, Chlamydomonas reinhardtii channelrhodopsin 1, aka Chlamydomonas sensory rhodopsin A (AF508965); VcChR1, Volvox carteri channelrhodopsin 1 (EU622855); CaChR1, Chlamydomonas (Chloromonas) augustae channelrhodopsin 1 (JN596951); CyChR1, Chlamydomonas yellowstonensis channelrhodopsin 1 (JN596948); HpChR1, Haematococcus pluvialis channelrhodopsin 1 (JN596950); DsChR1, Dunaliella salina channelrhodopsin 1 (JQ241364); PstChR1, Pleodorina starrii channelrhodopsin 1 (JQ249903); CrChR2, Chlamydomonas reinhardtii channelrhodopsin 2, aka Chlamydomonas sensory rhodopsin B (AF508966); VcChR2, Volvox carteri channelrhodopsin 2 (ABZ90903); CraChR2, Chlamydomonas raudensis channelrhodopsin 2 (JN596949); PstChR2, Pleodorina starrii channelrhodopsin 2 (JQ249904); MvChR1, Mesostigma viride channelrhodopsin 1 (JF922293); PgChR1, Pyramimonas gelidicola channelrhodopsin 1 (JQ241366); EeChR1, Eudorina elegans channelrhodopsin 1.

Conserved Glu residues (E) in helix B are highlighted light green, Glutamate, the possible proton donor, is highlited in red box; a conserved Lys residue (K) in helix B is highlighted yellow; homologs of the protonated Schiff base counterions Asp85 and Asp212 in bacteriorhodopsin are highlighted red; a His residue (H) in the position of the Schiff base proton donor Asp96 in BR is highlighted blue; a homolog of Glu87 of CrChR1, responsible for its pH-dependent color tuning and fast photocurrent inactivation, is highlighted orange; Tyr and Asn in the position of Tyr226/Asn187 (in CrChR1/CrChR2, respectively), identified as one of the molecular determinants of differences in spectra, desensitization, and current kinetics, are highlighted as light blue and pink, respectively; homologs of Ser63, Glu90 and Asn258 implicated in control of ion selectivity in CrChR2 are highlighted in olive; a homolog of Ser136 implicated in regulation of the size of the channel pore in CrChR2 is highlighted dark green; a homolog of Lys154 that contributes to a vestibule on the extracellular side of the channel pore in C1C2 is highlighted purple. The residues that form the conserved hydrophobic retinal-binding pocket and the conserved cluster at the extracellular vestibule of the cation-conducting pathway, indicated by red and green asterisks under the alignment respectively. Blue asterisks indicate the possible proton acceptors from the Schiff base. Cysteine residues involved in dimerization are indicated in green boxes. Tyrosine, which is buried in the putative pore exit, is enclosed in Blue boxes. Identical and conservatively substituted residues are shown on gray background.

Phylogenetic Analysis of EeChR1

14 channelrhodopsin proteins form algae were reported and are accessible in databases of National Center for Biotechnology Information (Supplementary data…..). Previous study revealed that homology of channelrhodopsins sequences is restricted to 7 TM helix domain {Kianianmomeni, 2009 #283129} {Sineshchekov, 2013 #288045}. A phylogenetic classification of channelrhodopsin proteins was performed based on trimmed alignments of 15 channelrhodopsin proteins including EeChR1 (Fig….) (see Supplemental data 4). Fig… shows alignment of N-terminal part of proteins including seven-transmembrane helices. Helix 3 contain some consensus motfis. Additionally, several other residues are highly conserved within the channelrhodopsin proteins and also present in EeChR1. Consensus motifs of 15 channelrhodopsin proteins at the 7 TM helices visualized usins online sequence logo maker ( are shown as sequences logos in Supplemental Data 5.

An unrooted phylogenetic tree of channelrhodopsin proteins in membrane spanning domains was inferred using the NJ (neighbor-joining) method (Saitou and Nei, 1987) with Poisson model and 2000 bootstrap reiterations (Fig…..). The topology of the tree indicated that EeChR1 was closer to a branch of channelrhodopsin 1 proteins, as expected. The closest relative of EeChR1 is VcChR1 and a branch including CrChR1 and PstChR1. Likwise, other channelrhodopsins branch according to their evolutionary distances, therefore, MvChR1 and PgChR1 were located far from other. This results confirm the homology analysis of 7TM domain of EeChR1 to other channelrhodopsins wich shows highest identity to VcChR1 and CrChR1 with 77% and 72% identity score respectively, and the lowest homology with PgChR1 with 43% identity score. A cluster including 4 ChR2 proteins (CrChR2, VcChR2, CraChR2, PstChR2) was located far from a cluster with 8 ChR1 proteins (CrChR1, VcChR1, CaChR1, CyChR1, DChR1, HpChR1, PstChR1, EeChR1) wich is somehow more closely related together.

Fig. ….. An unrooted tree of phylogenetic relationship between sequences of membrane spanning domains from channelrhodopsin proteins calculated using the neighbor-joining method (Saitou and Nei, 1987) of MEGA software package version 6 (Tamura K, Stecher G et al. 2013). The numbers associated with each node indicates the bootstrap probability of the cluster at the node obtained using 20000 resampled data set. The analysis is based on the trimmed alignment given in Fig… and Supplemental data…. Accession Nos. are represented in Fig…. except for EeChR1.

3.2 Collaboration for Spectroscopic analyses

3.2.1 Expression plasmid construction

In order to functional expression andin vitrotranslation, the full length cDNA of EChR1 gene encoding 743-residues was assembled in the ampicillin resistance Xenopus expression vector pGEMHE (Liman, Tytgat, & Hess, 1992). The final construct contains 2229 nucleotides from ATG to the stop codon. The EChR1 cDNA target sequence was synthesized using two overlapping fragments. Fragment A (nucleotide1- nucleotide1768) was amplified using forward and reverse primers that recognition sites for restriction enzymes BamHI and AocI-salI were introduced to them, respectively. Fragment B contained 1461 bp (nucleotide1769– nucleotide12229) of downstream sequence with subsequent AocI and HindIII restriction sites. During PCR, using reverse primer, restriction sites NotI and HindIII were added at the 3' end of the this fragment. The PCR fragments were cloned into the pGEM-T easy vector (Promega, Madison, WI) and cut out with respective restriction enzymes. The 768 bp BamHI /SalI synthesized fragment was ligated with a BamHI / SalI linearized pGEMHE vector. The downstream 1461 bp fragment was cloned into this plasmid using AocI/ HindIII digestion. Final constructed vector with 5249 bp contained EChR1 cDNA in 2229 bp. Subsequently NotI site was used to insertion of 741 bp YFP coding sequence. Final constructed vector contained 5982 bp. The coding region of the resulting plasmid was confirmed by DNA sequencing. Vector map and sequence are available at Fig…. and Supplemental data….., respectively.

Figure …. …….. Left: Expression construct map of EChR1 cDNA in ampicillin resistance Xenopus expression vector pGEMHE, 5249 bp. Right: Expression construct map of EChR1 cDNA+ EYFP in ampicillin resistance Xenopus expression vector pGEMHE, 5982 bp. Amplified fragment ligation sites with restriction enzymes are shown.

3.2.2 Results of Spectroscopic analyses

Spectroscopic analyses of EeChR1 was done at work group of Prof. Georg Nagel, at the department of Botany I, University of Würzburg, Germany. The above mentioned vector encoding EeChR1 was expressed in oocytes of Xenopus laevis. Illumination with blue (476nm) and green (532nm) light induced current in EeChR1. This measurement was done with Ori BaCl2 buffer pH 4.6 under -60mV. Wild-type EeChR1 exhibited photocurrent upon heterologous expression and obvious currents were elicited upon illumination with blue and green light. Similar as other ChR1, EeChR1 current is also smaller than ChR2.

Measurement of action spectra or wavelength dependence of EeChR1 was performed with Ori BaCl2 buffer pH 4.6 under -60mV. Different filters were used to generate light of certain wavelength from white light. The action spectra main peak for EeChR1 is at around 510nm.

To investigate the ionic selectivity of the light-induced permeability, EeChR1 current was measured in buffer with different pH. Ori BaCl2 buffers with different pH (4.6, 7.6, 9) were used for this test. Lowering thr pH increased the photocurrent, suggested a selectivity bias for proton of EeChR1, which is similar like other channelrhodopsin 1 proteins.

Voltage dependence of photocurrents of EeChR1 was measurement under differen potential with Ori BaCl2 buffer, pH7.6. Higher current under higher potential showed a channel-like character of EeChR1 protein.

Fig. …….. Photocurrent of EeChR1 recorded during illumination of oocytes with Blue (left, 476nm) and Green (right, 532nm) light. Obvious currents were detected upon illumination with blue and green light.

Fig. ...... Action spectra of EeChR1 at pH 4.6 under -60mV. Y axis stands for a ratio of current per photon at certain wavelength to the current per photon at 517nm. The main peak is at around 510nm.

Fig. ….. Dependence of EeChR1 photocurrent on pH. Higher current at lower pH suggested a selectivity bias for proton of EeChR1, which is similar like other ChR1.

Fig…Voltage dependence of photocurrents of EeChR1. Higher current under higher potential showed a channel-like character of EeChR1.