Chloride Concentration Dependency of the Electrogenic Activity of Halorhodopsin

Abstract

The capacitive photoelectric current responses of the halorhodopsins from Halobacterium salinarum (shR) and from Natronobacterium pharaonis (phR) were studied using membrane fragments adsorbed onto a thin polyester film. The electric current of shR was not much affected by ionic strength or cations present in the medium (Na+, K+, Li+, Mg2+, or Ca2+), but was greatly influenced by the Cl- concentration. It increased biphasically as the Cl- concentration increased from 0 to 5 M, then decreased and almost vanished at around 10 or 12 M. Apparent Kd's of about 0.1 and 6 M were deduced for the Kd of Cl- uptake sites. We had to assume a sigmoidal increase of Cl- binding with a Hill coefficient of about 8 at the cytoplasmic, Cl- release site(s). The half-maximum Cl- concentration for the sigmoidal binding was about 7.5 M. The electric current of phR had a maximum around 30 mM Cl- and biphasically decreased at higher Cl- concentrations. The apparent Kd for the Cl- uptake site was 5 mM. The biphasic decrease in the transport activity was explained by assuming a sum of simple hyperbolic type binding (Kd = 0.2 M) and sigmoidally increasing binding with a Hill coefficient of 10 on the cytoplasmic side. The half-maximum concentration of the latter cooperative binding was 5.6 M. This great difference between the apparent affinity of the release site of shR and that of phR can explain the previously reported difference between the Cl- dependency of their photocycles. These results also suggest that there may be multiple Cl- binding sites in the Cl- transport pathway. A simple sequence of Cl- transport steps based on a multiion channel model is proposed.

Genetically encoded molecular tools for light-driven silencing of targeted neurons

“…1A), has a reversal potential of approximately –400 mV (85), and although it comes from an archaeon that lives in very high salinity environments, the optimal chloride concentration for its operation is actually very close to that found in neurons (in contrast to that of other halorhodopsins such as the H. salinarum halorhodopsin, which demands far higher chloride concentrations) (86). This may be one of the reasons why it works in some neurons in some species (Fig.…”

Light-Activated Ion Pumps and Channels for Temporally Precise Optical Control of Activity in Genetically Targeted Neurons

“…We discuss in the following sections "Conductance, permeability, and context" and "Kinetics and wavelength selectivity" of halorhodopsins and bacteriorhodopsins for these two classes separately, followed by a joint discussion of the "Mutants and variants" and genomic "Diversity" in a unified section. (83,84). In the absence of any signal peptide sequences to improve trafficking and membrane localization, Halo has been reported to generate 40-100 pA of hyperpolarizing current (12,18,66), with the differences in measured photocurrents between studies largely attributable to the power and wavelength of excitation used.…”

Engineering synthetic optogenetic networks for biomedical applications

“…The history of optogenetic networks [18][19][20][21][22] is summarized in Figure 1. The first described microbial opsin, bacteriorhodopsin, dates back to the 1970s [23,24].…”