The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch)1 from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue–green light-drivable proton pump from the fungus Leptosphaeria maculans4 (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of ‘optogenetic’ voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Sequences are available to download from GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers: GU045593 (mammalian codon-optimized Arch), GU045594 (mammalian codon-optimized Arch fused to GFP), GU045595 (mammalian codon-optimized Mac), GU045596 (mammalian codon-optimized Mac fused to GFP), GU045597 (ss-Prl-Arch), GU045598 (ss-Arch-GFP-ER2) and GU045599 (ss-Prl-Arch-GFP).
E.S.B. acknowledges funding by the NIH Director’s New Innovator Award (DP2 OD002002-01), as well as the NSF (0835878 and 0848804), the McGovern Institute Neurotechnology Award Program, the Department of Defense, NARSAD, the Alfred P. Sloan Foundation, Jerry and Marge Burnett, the SFN Research Award for Innovation in Neuroscience, the MIT Media Lab, the Benesse Foundation, and the Wallace H. Coulter Foundation. X.H. acknowledges the Helen Hay Whitney Foundation and NIH 1K99MH085944. The authors thank E. Klinman for help with transfections, R. Desimone for advice, J. Lin for technical aid on intracellular pH measurements, K. Ihara for discussions about archaerhodopsins, and M. Hemann and N. Gershenfeld and the Center for Bits and Atoms for use of their respective laboratory facilities.
Author Contributions B.Y.C., X.H. and E.S.B. designed experiments, analysed data and wrote the paper. B.Y.C. and X.H. carried out experiments. A.S.D. assisted with electrophysiological recording. X.Q., M.L. and A.S.C. assisted with molecular biology, virus making, and transfections. M.A.H. performed Monte Carlo modelling. P.E.M., G.M.B. and Y.L. created hippocampal and cortical neural cultures.
This file contains Supplementary Figures 1-3 with Legends, Supplementary Notes, Supplementary Tables 1-3, Supplementary Methods and Supplementary References.
About this article
Multidimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents
Journal of Biological Chemistry (2019)