Abstract
The GABA type A receptor (GABA(A)R) is a member of the pentameric ligand gated ion channel (pLGIC) family that mediates ionotropic neurotransmission. Residues in the intracellular loop domain (ILD) have recently been shown to define part of the ion permeation pathway in several closely related members of the pentameric ligand gated ion channel family. In this study, we investigated the role the ILD of the GABA(A)R α1 subunit plays in channel function. Deletion of the α1 ILD resulted in a significant increase in GABA EC(50) and maximal current amplitude, suggesting that the ILD must be intact for proper receptor function. To test this hypothesis, we conducted a mutagenic screen of all amino acids harboring ionizable side chains within this domain to investigate the contribution of individual charged residues to ion permeation. Using macroscopic and single channel voltage-clamp recording techniques, we found that mutations within a subdomain of the α1 ILD near M3 altered GABA apparent affinity; interestingly, α1(K312E) exhibited reduced partial agonist efficacy. We introduced point mutations near M4, including α1(K383E) and α1(K384E), that enhanced receptor desensitization. Mutation of 5 charged residues within a 39-residue span contiguous with M4 reduced relative anion permeability of the channel and may represent a weak intracellular selectivity filter. Within this subdomain, the α1(K378E) mutation induced a significant reduction in single channel conductance, consistent with our hypothesis that the GABA(A)R α1 ILD contributes directly to the permeation pathway.
Highlights
Homologs from Gloeobacter violaceus and Erwinia chrysanthemi [1]; anionic channels include the glycine receptor (GlyR), heteromeric GABAA receptor, and homomeric GABAC receptor
The lack of homology in the intracellular loop domain (ILD) makes it difficult to extrapolate these findings across the entire pentameric ligand gated ion channel (pLGIC) family, and the ILD role in permeation has yet to be established for the GABA type A receptor (GABAAR)
We replaced the ILD with seven lysine residues to investigate the effect of charge on ion permeation and channel gating (IL-7K)
Summary
Conductance to identify the MA residues, which control this phenomenon [32, 35] (Fig. 1). The lack of homology in the ILD makes it difficult to extrapolate these findings across the entire pLGIC family, and the ILD role in permeation has yet to be established for the GABAAR. These studies have focused solely on the MA stretch and have largely ignored the portion between M3 and MA. In this study we chose to modify only the ␣1 subunit, leaving the 2 and ␥2s subunits intact to establish a role for the ␣1 ILD in controlling GABAAR activity, not its involvement in trafficking and signaling pathways. Our results indicate that subdomains of the M3-M4 intracellular loop are responsible for controlling separate components of channel function
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