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Published August 2008 | Published
Journal Article Open

eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications


Temporally precise inhibition of distinct cell types in the intact nervous system has been enabled by the microbial halorhodopsin NpHR, a fast light-activated electrogenic Cl^− pump. While neurons can be optically hyperpolarized and inhibited from firing action potentials at moderate NpHR expression levels, we have encountered challenges with pushing expression to extremely high levels, including apparent intracellular accumulations. We therefore sought to molecularly engineer NpHR to achieve strong expression without these cellular side effects. We found that high expression correlated with endoplasmic reticulum (ER) accumulation, and that under these conditions NpHR colocalized with ER proteins containing the KDEL ER retention sequence. We screened a number of different putative modulators of membrane trafficking and identified a combination of two motifs, an N-terminal signal peptide and a C-terminal ER export sequence, that markedly promoted membrane localization and ER export defined by confocal microscopy and whole-cell patch clamp. The modified NpHR displayed increased peak photocurrent in the absence of aggregations or toxicity, and potent optical inhibition was observed not only in vitro but also in vivo with thalamic single-unit recording. The new enhanced NpHR (eNpHR) allows safe, high-level expression in mammalian neurons, without toxicity and with augmented inhibitory function, in vitro and in vivo.

Additional Information

© 2008 The Author(s). This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Received 22 May 2008; Revised 13 June 2008; Accepted 17 June 2008. Published online 2 August 2008. K.D. is supported by CIRM, McKnight, Coulter, Klingenstein, NSF, NIMH, NIDA, the NIH Pioneer Award, and the Kinetics Foundation. V.G. is supported by a Stanford Graduate Fellowship. K.R.T. is supported by NARSAD. We thank Feng Zhang and Joanna Mattis for providing us with the nhNpHR construct, and Andrew Hsu for assistance with cloning. We also thank the entire Deisseroth lab for useful discussions. The materials and methods described herein are freely distributed and supported by the authors (www.stanford.edu/group/dlab).

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