Mutations at the GABA receptor selectivity filter: a possible role for effective charges

Abstract

An important feature of ligand-gated ion channels is their exquisite ability to discriminate between ions. Still, little is known about the mechanisms underlying, or structural determinates of, this ability. We examined the structural elements underlying the ionic selectivity of rho1 GABA receptors expressed in Xenopus oocytes and human embryonic kidney cells using site-directed mutagenesis and two-electrode voltage-clamp or patch-clamp techniques. The wild-type GABA receptor was chloride selective, with a small but significant permeability to potassium (PNa+ : PK+ : PCl- = 0 : 0.03 :1). Mutation of an alanine to glutamate at position 291 (thought to be located at the intracellular end of the second transmembrane domain), formed a channel that exhibited little discrimination among ions (0.70:0.87:1), while deletion of a neighbouring proline (290) was chloride selective, but had elevated cation permeabilities compared to the wild-type channel (0.12 : 0.14 : 1). Together, the two mutations (DeltaP290/A291E) caused a reversal of selectivity (2.72 : 3.59 : 1). We also examined the effects of neutralizing and reversing the charge of the adjacent, and highly conserved, arginine. Mutation of the neighbouring arginine to glutamate (R292E) increased the cation permeability similar to the DeltaP290/A291E double mutant (2.4 : 3.0 : 1), whereas neutral mutations at this position (R292M or R292C) retained chloride selectivity (0 : 0.11 : 1.0 and 0 : 0.14 : 1.0, respectively). Our experiments suggest that the effective charge near the presumed intracellular mouth of the pore is critical for ionic selectivity.

Ligand-Gated Channels

“…Also, in this channel it was possible to directly change the charge at position 0 . Neutralizing this charge made it slightly less anion-selective than the WT GABA Rs, but it still remained anion-selective, whereas replacing the positive charge with a negative one R E did make the channel weakly cation-selective P P [35]; see also discussion in [7]. Recently it has also been shown that placing a negative charge in the 2 position in the GABA R (a single mutation of the 2 proline to a glutamate P E) did invert its charge selectivity from being anion-selective P P to cation-selective (with P P ) [36].…”

Mixed antagonistic effects of the ginkgolides at recombinant human ρ1 GABAC receptors

“…The residue at position 2′ has been shown to influence response kinetics, ion permeability, selectivity and conductance of the GABA C receptors (Qian et al., 1999; Wotring et al., 2003, 2008; Carland et al., 2004a,b; Filippova et al., 2004; Zhu et al., 2007). Ginkgolide B has been shown by molecular modelling to be structurally similar to PTX (Ivic et al., 2003) and the ginkgolides only differ from one another in the hydroxyl substituents.…”

Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates

“…The size of the pore tapered from 25 A on the extracellular side to 3 A on the intracellular side. The location of residues known to be involved in ion selectivity [59][60][61] are appropriately located in the TM1-2 loop (Fig. 2A).…”