eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications
Key findings
Screening of signal peptides and ER export sequences identified a halorhodopsin variant (eNpHR) combining an N-terminal signal peptide from the β subunit of nicotinic acetylcholine receptor with a C-terminal ER export sequence (FCYENEV) from Kir2.1. The modified protein prevented endoplasmic reticulum accumulation observed with wild-type NpHR, with only 1 possible aggregate detected in >400 cultured hippocampal neurons versus widespread cytoplasmic accumulations in NpHR-expressing cells.
Whole-cell patch clamp recordings in cultured hippocampal neurons infected with titer-matched virus showed eNpHR generated 75% higher peak photocurrents compared to NpHR (68.1 ± 7.2 pA versus 38.9 ± 6.8 pA, P=0.008) after 10 days expression. Confocal microscopy confirmed increased plasma membrane localization without colocalization with KDEL-labeled ER proteins, while maintaining identical membrane resistance (116.8 versus 113.5 mΩ, P=0.87).
Lentiviral transduction in adult mouse hippocampus CA1 and thalamus at high titer (10^10 i.u./ml) showed eNpHR eliminated aggregates observed with NpHR after 10 days expression with enhanced membrane localization extending into distal dendritic processes. Single-unit optrode recordings in thalamus demonstrated reversible yellow light-induced inhibition of neuronal activity in vivo, while non-transduced control tissue showed no light response.