Functional characterization of sodium-pumping rhodopsins with different pumping properties.

Satoshi P. Tsunoda,Yuko Kozaki,Hideki Kandori,Keiichi Inoue,Rei Abe-Yoshizumi,Matthias Prigge,Ofer Yizhar,Toru Ishizuka,Hiromu Yawo,Hendrik W. van Veen

doi:10.1371/journal.pone.0179232

Abstract

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2.All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3–5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.

Highlights

NaR is a new member of the microbial rhodopsin family, actively transporting Na+ and H+ depending on the ionic conditions
One of the ideal methods is electrophysiology by which ion transport function can be recorded under control of membrane voltage and ionic environment of both sides of cellular membrane with a high time resolution up to ~10 μs
The performance of the system greatly relies on the functional expression of a transporter of interest in mammalian cells

Summary

Introduction

Functional characterization of NaR the Human Frontier Science Program and the Israel Science Foundation (ISF #1351-12) to OY, and by a Miverva Foundation postdoctoral fellowship to MP. Of a lysine residue via Schiff base They are widely distributed through eukaryotic and prokaryotic organisms and exhibited diverse, specialized functions. Spectroscopic and X-ray studies revealed the cation transport pathway of KR2 [17,18], demonstrating that upon photoisomerization of all-trans retinal, a protonated Schiff-base provides H+ to D116 which breaks an electrical barrier. This change allows Na+ to pass through the chromophore region of the protein. Based on the functional expression of KR2 under these conditions we set out to identify new NaRs with regards to a high expression in plasma membranes of mammalian cells as well as large photocurrent amplitudes

Materials and methods

Results

Discussion