Characterizing the amiloride potentiation site in the GABAA ρ1 receptor




Snell, Heather D.
Gonzales, Eric B.


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γ- amino butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and targets the ionotropic GABAA receptors. GABAC, or GABAA rho, is a subclass of GABAA receptors composed entirely of rho (ρ) subunits and are located on the axonal terminal of retinal bipolar cells, where it not only exhibits a tonic inhibitory current, but also regulates the GABAA and other GABAA rho synaptic currents (Jones et al 2011). GABAA-rho exhibits unique properties, such as insensitivity to select antagonists of the heteromeric GABAA receptors (Korpi et al., 2002). A group of ligands, which possess a guanidine group, have been shown to influence GABAA receptors. This includes the acid sensing ion channel (ASIC) ligand, amiloride. Our previous work elucidated the intrinsic activity of the guanidine compound amiloride as having an allosteric modulatory effect on the human GABAA rho1 receptor, but the exact mechanism, or site of interaction, remains unknown. Homology modeling of amiloride interacting with ethanol sensitive GLIC, a bacterial ligand gated ion channel of known structure, has yielded possible residues that might form the amiloride site in the GABAA rho1 subunits of the receptor. We hypothesize mutating these residues in the GABAA rho1 receptor will eliminate the allosteric modulatory effect of amiloride, and thus reveal the site of interaction with the receptor. Point mutations will be introduced through polymerase chain reaction (PCR), and whole cell electrophysiology will be utilized to assess the intrinsic activity of amilorde following introduction of the mutated residue. Our findings suggest that there are functional, as well as therapeutic, implications for the use of guanidino compounds in targeting the GABA-A rho1 receptor mediated activity. This site of action could be a unique allosteric binding site in the GABAA rho1 receptor, and thus could be utilized as a target for therapeutics not only for the GABAA rho receptor family, but also other subunits in the GABAA receptor family.