The α1-GABAA photoswitch-ready mutant (PhoRM) mice harbor the α1T125C knock-in allele. When used in conjunction with a photo-switchable tethered ligand (PTL), the resulting light-regulated GABAA receptor (α1-LiGABAR) allows optogenetic control at the synapse of a modified inhibitory neurotransmitter receptor that also retains its normal expression, trafficking and activity.
Richard H Kramer, University of California, Berkeley
The Gabra1 gene encodes the gamma-aminobutyric acid type A receptor (GABAA) α1 subunit. GABA is the major inhibitory neurotransmitter in the mammalian brain where it exerts its effects largely through GABAA receptors; ligand-gated chloride channel hetero-pentamers containing two α, two β and one tertiary subunit. The α subunit contributes to GABA binding and determines gating kinetics and subcellular localization of the receptor. Chloride conductance of these channels can also be modulated by agents such as benzodiazepines that bind to different regions of the GABAA receptor.
The α1-GABAA photoswitch-ready mutant (PhoRM) mice harbor the α1T125C knock-in allele that encodes a modified GABAA α1 subunit with a cysteine residue in the α subunit loop E (near the GABA-binding site located at the β-α subunit interface). The α1T125C-containing GABAA hetero-pentamer functions normally, with the added functionality that it may bind ligands specific to the T125C moiety.
Specifically, when conjugated with a complimentary photo-switchable tethered ligand (PTL; see description below), the resulting light-regulated GABAA receptor (α1-LiGABAR) allows photo-control of the endogenous neurotransmitter receptor. Before photo-switched to the antagonizing configuration, the α1-LiGABAR functions as its wildtype receptor counterpart: neither the cysteine substitution nor PTL conjugation alone affects the characteristic properties of the parent receptor (including allosteric modulation at the benzodiazepine site or anion permeability of the channel). α1-LiGABAR functions like the wildtype receptor in 380 nm light (cis conformation), and is strongly inhibited in 500 nm light (trans conformation).
Mice homozygous for the α1T125C allele are viable and fertile, with no reported spontaneous synaptic or behavioral abnormalities.
The photo-switchable tethered ligand (PTL) is a modular-based compound consisting of three parts:
i. a cysteine-reactive maleimide group that covalently binds to the Cys125 residue in the α subunit loop E (near the GABA-binding site located at the β-α subunit interface) and creates a highly stable S-C bond on the GABAA receptor.
ii. a photosensitive azobenzene group core that facilitates the photoswitch by changing from cis to trans conformation in 380 nm light or 500 nm light, respectively.
iii. a GABAA receptor ligand that, when bound to the orthosteric (neurotransmitter binding) site of the receptor, displaces GABA and causes GABAAR inhibition.
Therefore, the complete PTL allows light maniputation of receptor activity by anchoring itself to the Cys125 residue and, depending upon the light-dependent conformation of the azobenzene core, the GABA binding site is either absent of the GABAAR ligand (380 nm light cis; normal GABAAR function) or occupied by the GABAAR ligand (500 nm light trans; inhibits function).
The specific PTL structure suitable for use with these α1-GABAA PhoRM mice is described in Lin et al. 2015 Neuron 88:879. Researchers may acquire the PTL by contacting the donating investigator directly - Dr. Richard H. Kramer (University of California, Berkeley).
The α1T125C knock-in allele was designed by Dr. Richard H. Kramer (University of California, Berkeley) to introduce a threonine-to-cysteine mutation at codon 152 (T152C counting from the start codon; or T125C in the mature peptide sequence) into exon 5 of the gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1 gene (Gabra1) on chromosome 11. The targeting vector also had a loxP-flanked neomycin selection cassette upstream of exon 5, as well as a C-to-T silent mutation in the exon 5 DNA sequence (to create a HindIII site upstream of T152C for genotyping).
The targeting vector was electroporated into 129/SVJ-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred with C57BL/6J for germline transmission and to establish the colony.
Offspring were bred with germline Cre recombinase-expressing mice (on a C57BL/6N genetic background) and the neo cassette was deleted.
The donating investigator reports that the resulting α1T125C mice were bred with C57BL/6 for one generation (and the cre-expressing allele was removed), and then bred to Crl:CD1(ICR) for one generation. In 2016, α1T125C males on the mixed genetic background (C57BL/6;CD1;129) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
|Allele Name||targeted mutation 1.1, Richard Kramer|
|Allele Type||Targeted (Not Applicable)|
|Allele Synonym(s)||alpha1-GABAsubA/subPhoRM; alpha1T125C; PhoRM|
|Gene Symbol and Name||Gabra1, gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1|
|Strain of Origin||129|
|Molecular Note||The allele was designed to introduce a threonine-to-cysteine mutation at codon 152 (T152C counting from the start codon; or T125C in the mature peptide sequence) into exon 5 of the gene. The targeting vector also includes a loxP-flanked neomycin selection cassette upstream of exon 5, as well as a C-to-T silent mutation in the exon 5 DNA sequence (to create a HindIII site upstream of T152C for genotyping). Cre-mediated recombination removed the floxed neo cassette.|
Homozygous mice are viable and fertile, with no reported spontaneous synaptic or behavioral abnormalities. When maintaining a live colony, heterozygous mice may be bred together, to wildtype littermates, or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
When using the α1-GABAA PhoRM (α1T125C knock-in) mouse strain in a publication, please cite the originating article(s) and include JAX stock #028965 in your Materials and Methods section.