Light-Driven Pumps

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Microbial rhodopsins behaving as light-driven ion pumps were long known to exist in halophilic archaebacteria [1] and were discovered recently in bacteria (Beja, Aravind et al. 2000) and some eukaryotes [2]. Proteins like bacteriorhodopsins, proteorhodopsins and archaerhodopsins extrude protons from the cytoplasm, building up a proton gradient used for the production of ATP. Others like halorhodopsins are used by certain halobacteria to maintain their osmotic balance by transporting chloride into their cytoplasm (Muller and Oren 2003). Both types thus generate a hyperpolarizing photocurrent which can be used to silence neuronal activity. Interestingly these pumps operate at different peak sensitivity wavelengths compared to ChR2, opening the possibility of coexpressing them with ChR2 to achieve bidirectional control of the same cell. Zhang et al. [3] provided the proof of principle that this is indeed possible by coexpressing ChR2 and the halorhodopsin from Natronomonas pharaonis (NpHR) in acute brain slices and in C. elegans. But translating these light-driven pumps into usable tools was not as straightforward as for ChR2. A common problem with these proteins was impaired subcellular localization which decreased their tolerability when expressed at high levels. In particular, the proteins would accumulate at successive steps along the secretary pathway to the cell surface. A number of candidates isolated from various species were tested and modified by adding a series of trafficking signal peptides to improve their membrane localization [4]. Two families of pumps emerged as promising light-activated silencers: the series of proteins derived from NpHR (the latest being eNpHR3.0 described in [4]) and the archaerhodopsins Arch from Halorubrum sodomense [5] and ArchT from Halorubrum genus [6]. The diversity and biophysical properties of microbial opsins and their use in neuroscience were reviewed extensively [7, 8, 9] (Yizhar, Fenno et al. 2011). State-of-art methodologies to deploy these tools in mammalian cells were reviewed in detailed by Chow et al. [10].

Summary of available tools

Acronym Full Name Description Accession # Refs
Proton Pumps
BR Bacteriorhodopsin Outward light-driven proton pump from Halobacterium salinarum (a.k.a. Halobacterium halobium). CAA23744
Mac Outward light-gated proton pump from Leptosphaeria maculans Mac the first retinal-based proton pump isolated from a eukaryote (the fungus Leptosphaeria maculans). Mac-expressing neurons can be hyperpolarized using blue-green light (542 nm). AAG01180 [2]
Arch archaerhodopsin-3 Light-driven proton pump from the archaebacteria Halorubrum sodomense". BAA09452 [5]
ArchT Light-driven proton pump from the archaebacteria Halorubrum genus. Han et al., 2011
Chloride Pumps
NpHR Halorhodopsin from Natromonas pharaonis. EF474018 [3] [11]
eNpHR2.0 Second-generation NpHR. This second-generation NpHR displays reduced globular accumulation in the endoplasmic reticulum (ER). eNpHR2.0 is the result of the fusion of the FCYENEV ER export motif from a vertebrate inward rectifier potassium channel to the NpHR C

terminus.

[12]
eNpHR3.0 Third-generation NpHR. This third-generation NpHR displays improved membrane targeting (and therefore larger photocurrents). eNpHR3.0 was obtained by adding the trafficking signal from Kir2.1 at the C terminus of the NpHR-YFP fusion protein. [4]

Properties of available variants

Acronym Species* λ max (nm) Desensitization*** (1 - Is-s/Ipeak) Sensitivity (EC50) (mW/mm2) Refs
Mac Leptosphaeria maculans [2]
NpHR Natronomonas pharaonis [3] [11]
eNpHR2.0 Natronomonas pharaonis [12]
eNpHR3.0 Natronomonas pharaonis [4]
Arch (archaerhodopsin-3) Halorubrum sodomense [5]
ArchT Halorubrum sodomense

List of available constructs

pcDNAs

AAV Plasmids

DiO (FLEX) plasmids

The DiO (Double-floxed Inverted ORF) strategy is also known as the flip-excision (FLEX) switch and was first described in [13].

Name Description Map Lab Addgene
Halorhodopsin (NpHR)
pAAV-Ef1a-DIO-eNpHR 3.0-EYFP everyvector.com Deisseroth lab Plasmid 26966
pAAV-double floxed-eNpHR-EYFP-WPRE-pA Deisseroth lab Plasmid 20949
Arch and ArchT
AAV-FLEX-Arch-GFP Plasmid for adeno-associated virus (AAV) Cre-dependent cell-type selective expression of Arch-GFP. Boyden lab Plasmid 22222
AAV-FLEX-ArchT-GFP Plasmid for adeno-associated virus (AAV) Cre-dependent cell-type selective expression of ArchT-GFP. From the Boyden lab. Available through the UNC Gene therapy center
pAAVs with YFP only (for control experiments)
pAAV-Ef1a-DIO EYFP Deisseroth lab Plasmid 27056
pAAV-EF1a-double floxed-EYFP-WPRE-HGHpA Deisseroth lab Plasmid 20296
pAAV-EF1a-double floxed-mCherry-WPRE-HGHpA Deisseroth lab Plasmid 20299

Plasmids with specific promoters

Name Description Map Lab Addgene
Halorhodopsin (NpHR)
pAAV-CaMKIIa-eNpHR3.0-EYFP eNpHR 3.0 (with trafficking signal and ER Export Signal) everyvector.com Deisseroth lab Plasmid 26971
pAAV-hSyn-eNpHR 3.0-EYFP everyvector.com Deisseroth lab Plasmid 26972

Plasmids with non-specific promoters

Name Description Map Lab Addgene
Arch and ArchT
AAV-CAG-ArchT-GFP Plasmid for adeno-associated virus (AAV) expression of ArchT-GFP under the CAG promoter. From the Boyden lab. Available through the UNC Gene therapy center

Lentivirus Plasmids

Plasmids with specific promoters

Name Description Map Lab Addgene
Halorhodopsin (NpHR)
pLenti-CaMKIIa-eNpHR 3.0-EYFP Deisseroth lab Plasmid 26970
pLenti-CaMKIIa-eNpHR-EYFP-WPRE Deisseroth lab Plasmid 20946
pLenti-hSyn-eNpHR 3.0-EYFP Deisseroth lab Plasmid 26775
Arch and ArchT
FCK-Arch-GFP Plasmid for lentiviral excitatory neuron expression of Arch-GFP. Contains 2nd-generation lentiviral backbone, CaMKII promoter, yellow/green-light driven proton pump Arch (Archaerhodopsin from H. sodomense, aka ar-3), GFP. Link Boyden lab Plasmid 22217
FCK-ss-Prl-Arch-GFP Plasmid for lentiviral excitatory neuron expression of ss-Prl-Arch-GFP. Contains CaMKII promoter, ss-Prl enhanced trafficking sequence, yellow/green-light driven proton pump Arch (Archaerhodopsin from H. sodomense, aka ar-3), GFP. Slightly higher currents than Arch. Link Boyden lab Plasmid 22224
FCK-ss-Arch-GFP-ER2 Plasmid for lentiviral excitatory neuron expression of ss-Arch-GFP-ER2. Contains CaMKII promoter, ss enhanced trafficking sequence, yellow/green-light driven proton pump Arch (Archaerhodopsin from H. sodomense, aka ar-3), GFP, ER2 enhanced trafficking sequence. Currents are of the same magnitude as Arch. Link Boyden lab
FCK-Arch-CFP Plasmid for lentiviral excitatory neuron expression of Arch-CFP. Contains 2nd-generation lentiviral backbone, CaMKII promoter, yellow/green-light driven proton pump Arch (Archaerhodopsin from H. sodomense, aka ar-3), CFP. Link Boyden lab Plasmid 22221

List of available transgenic lines

Strain Name Type Description Lab Jackson Labs stock # Ref
NpHR
Orexin/Halo [14]
eNpHR
B6;SJL-Tg(Thy1-HOP/EYFP)2Gfng/J This line (founder line 2) has the expression of eNpHR::YFP directed to neural cells by the the murine thymus cell antigen 1 (Thy1) promoter region. This line exhibits high expression in layer 5 pyramidal neurons at cortex, dentate gyrus, thalamus, superior colliculus, inferior culliculus, brainstem, amydagala and cerebellum. Guoping Feng 012332
B6;SJL-Tg(Thy1-HOP/EYFP)4Gfng/J This line (founder line 4) has the expression of eNpHR::YFP directed to neural cells by the the murine thymus cell antigen 1 (Thy1) promoter region. This line exhibits high expression in layer 2/3 and layer 5 pyramidal neurons at cortex, dentate gyrus, thalamus, superior colliculus, inferior culliculus, brainstem, amydagala and cerebellum. Guoping Feng 012334

References

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  1. Error fetching PMID 10101275: [Mukohata1999]
  2. Error fetching PMID 15860584: [Waschuk2005]
  3. Error fetching PMID 17410168: [Zhang2007]
  4. Error fetching PMID 20303157: [Gradinaru2010]
  5. Error fetching PMID 20054397: [Chow2010]
  6. Error fetching PMID 21876722: [Boyden2001]
  7. Error fetching PMID 21692661: [Fenno2011]
  8. Error fetching PMID 21601097: [Chow2011]
  9. Error fetching PMID 17375185: [Han2007]
  10. Error fetching PMID 18677566: [Gradinaru2008]
  11. Error fetching PMID 21775598: [Tsunematsu2011]
  12. Error fetching PMID 21811444: [Han2001]
  13. Error fetching PMID 21363959: [Yizhar]
All Medline abstracts: PubMed HubMed