Charged Residues at the 2′ Position of Human GABAC ρ1 Receptors Invert Ion Selectivity and Influence Open State Probability

Abstract

The ability of members of the nicotinicoid superfamily of ligand-gated ion channels to selectively conduct anions or cations is critical to their function within the central nervous system. Recent work has demonstrated that residues at the intracellular end of the second transmembrane domain, between the ؊3 and 2 positions, form the ion selectivity filter of these receptors. In this study, the proline residue at the 2 position (Pro-2) at the intracellular end of the second transmembrane domain of the ␥-aminobutyric acid type C 1 subunit was mutated to glutamate (1P2E) and arginine (1P2R). Dilution potential experiments indicated that the charge selectivity of the 1P2E receptor channels had been inverted, with the channels now becoming predominantly cation selective, indicating the ability of negatively charged residues at this 2 position to control charge selectivity. The mutation was also seen to have significantly decreased agonist potency and intrinsic efficacy. In contrast, the 1P2R receptor channels were anion-selective but were now found to be constitutively open with high holding currents (inhibited by low ␥-aminobutyric acid doses and the competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid alone) and increased agonist activity. Hill coefficients of both mutants were decreased, but competitive antagonist studies indicated that their binding sites were not significantly affected.

Mechanism of Cl- Selection by a Glutamate-gated Chloride (GluCl) Receptor Revealed through Mutations in the Selectivity Filter

“…Position Ϫ1Ј is located in a constriction that extends from position 2Ј toward the bottom of the pore (20, 26 -30). Consistently, it was previously shown that a substitution of Glu at position 2Ј of the homomeric Gly␣1R converted its selectivity (31). In addition, Glu substituted at positions Ϫ3Ј or Ϫ4Ј of the ␤ subunit of a heteromeric GABA A ␣ 2 ␤ 3 ␥ 2 receptor imparted permeability to cations along with anions (32).…”

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

“…To assess the effect of the T244 mutation on receptor gating, we examined the activity of the partial agonists, muscimol and imidazole-4-acidic acid (I4AA), at ρ 1 T244S mutant receptors. The intrinsic efficacy of muscimol at 100 μM produced 79% of the maximum response of GABA at ρ 1 wildtype receptors 31 and produced 2.4 ± 0.8% (n = 5) of the maximum response of GABA (1 mM) at ρ 1 T244S mutant receptors ( Figure 4A and E). We did not observe I4AA (1 mM, n = 4) acting as an agonist at ρ 1 T244S mutant receptors ( Figure 4B).…”

Structural model of ρ1 GABAC receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function

“…Mutational studies of the GABA C receptor, including those of the N-terminal domain, have identified residues that rendered GABA C inactive, altered the sensitivity of the receptor to the agonists, or kept the protein constitutively active. [15][16][17][18][19] These studies have relied primarily on information about known locations of protein loops that contain binding residues in other homologous well-studied LGICs, such as nAChR. 20 The present study was undertaken to develop a homology model of the q1 GABA C receptor, in which detailed evolutionary analysis is used specifically to guide structure generation and to predict protein sites under strong evolutionary selection pressure.…”

“…55,56 Briefly, mRNA, obtained for each selected sequence from in vitro transcription (mMessage mMachine Ambion, Austin, TX) from linearized cDNAs, was injected into the oocytes (Drummond Nanoject II; Drummond Scientific, Broomall, PA). Oocytes were analyzed for GABA C receptor expression after 24-72 h incubation in physiological saline (100 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, and 10 mM glucose, at pH 7.2-7.4) containing 0.1 mg/mL gentamycin at [16][17][18][19] C.…”