Characterization of the Effects of Charged Residues in the Intracellular Loop on Ion Permeation in α1 Glycine Receptor Channels

Abstract

The Cys loop receptor channels mediate fast synaptic transmission in the nervous system. The M2-demarcated transmembrane pore is an important determinant of their ion permeation properties. Portals within the intracellular domain are also part of the permeation pathway in cationic Cys loop receptors, with charged residues in a helical MA stretch partially lining these openings profoundly affecting channel conductance. It is unknown whether analogous portals contribute to the permeation pathway in anionic Cys loop receptors. We therefore investigated the influence of charged residues within the proposed MA stretch on functional properties of the homomeric glycine ␣1 receptor. Up to eight basic residues in the MA stretch were concurrently mutated to a negatively charged glutamate, and wild-type and mutant subunits were expressed in HEK-293 cells. Mutation of all eight residues produced a non-functional receptor. The greatest reduction in conductance at negative membrane potentials (from 92.2 ؎ 2.8 to 60.0 ؎ 2.2 picosiemens) was observed with glutamate present at the 377, 378, 385, and 386 positions (the 4E subunit). Inclusion of additional glutamate residues within this subunit did not decrease conductance further. Neutralizing these residues (the 4A subunit) caused a modest decrease in conductance (80.5 ؎ 2.3 picosiemens). Outward conductance at positive potentials was not markedly affected. Anion to cation selectivity and concentration-response relationships were unaffected by the 4A or 4E mutations. Our results identify basic residues affecting conductance in the glycine receptor, suggesting that portals are part of the extended permeation pathway but that the M2-demarcated channel pore is the dominant determinant of permeation properties in glycine receptors.Rapid and coordinated communication between neurons at synapses in the central nervous system is critical for the maintenance of normal brain function. Key to this process is the Cys loop superfamily of ligand-gated ion channels (LGICs), 4 which includes the cation-selective nicotinic acetylcholine (ACh) and 5-hydroxytryptamine type 3 (5-HT 3 ) and the anion-selective glycine and ␥-aminobutyric acid type A (GABA A ) receptors.LGICs convert the chemical messages conveyed by neurotransmitters into excitatory or inhibitory electrical signals via the selective conduction of ions. The polarity of the most permeant ion (ion charge selectivity) and the rate at which the ions permeate (the single channel conductance, ␥) are major determinants of how this synaptic signal affects neuronal excitability.Cys loop LGICs are composed of five protein subunits arranged around a central ion-conducting channel pore. Each of the five subunits consists of a large extracellular domain harboring the neurotransmitter binding site, four membranespanning domains (M1-M4) and, in mammalian LGICs at least, a large intracellular loop connecting M3-M4. The electron microscopic images of the Torpedo marmorata nicotinic ACh receptor (1-3) provide our most complete picture of the structure...

Discrete M3-M4 Intracellular Loop Subdomains Control Specific Aspects of γ-Aminobutyric Acid Type A Receptor Function

“…The mutation of charged residues near M4 has been shown to alter single channel conductance in homomeric 5-HT 3 R and GlyRs; in both cases the mutations were made within a putative homolog of the MA stretch (29,32). In our study ␣1(K378E) induced a significant decrease in single channel chord conductance ( Figs.…”

“…At present, the theory of ion permeation in pLGICs relies on the concept that canonical rings of charged residues make integral electrostatic interactions with permeant ions to control permeation and selectivity (10,12). Recent functional data in 5-HT 3 R, nAChR, and GlyR demonstrated that the ILD also plays a role in this process (29,31,32,35). Therefore, we hypothesized that deletion of the ␣1 ILD of the GABA A R would change the effective geometry of the permeation pathway.…”

“…Previous studies of pLGIC ILDs have focused on receptor surface stability, mobility (16,17,22), phosphorylation (26,27), binding interactions with accessory proteins (23), and trafficking machinery (19). Recent studies point to an intracellular component of desensitization (42)(43)(44), and intracellular control of conductance has been linked to the MA stretch (29,34), but this has yet to be confirmed for the GABA A R.…”

The Basic Property of Lys385 Is Important for Potentiation of the Human α1 Glycine Receptor by Ethanol

“…Regarding GlyRs, it was shown that replacement of all basic amino acids present in the IL to negatively charged residues produced a strong impact on the GlyR, rendering it nonfunctional. In addition, simultaneous mutations of arginine and lysine at positions 377, 378, 385, and 386 to glutamate reduced the conductance of the channel, showing their importance in channel function (Carland et al, 2009). Furthermore, the correct assembly of GlyR seems to depend on the presence of the RFRRKRR cluster in the intracellular loop (Sadtler et al, 2003).…”

“…For example, it was reported that the IL regulates nAChR and GlyR channel gating via G␤␥ binding (Fischer et al, 2005;Yevenes et al, 2006). In addition, a single residue (Arg436) in the IL of the human 5-HT 3A receptor was found to regulate channel conductance (Deeb et al, 2007;Carland et al, 2009). Furthermore, another study showed that the most important residue controlling the actions of G␤␥ and ethanol on GlyRs was Lys385 (Yevenes et al, 2008).…”