Identification of a β Subunit TM2 Residue Mediating Proton Modulation of GABA Type A Receptors

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (␣1 and ␣1␤) and GABA A (␣1␤2␥2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine ␣1␤ receptor, replacing Lys with Glu in ␣1 reduces single-channel conductance by 41%, but has no effect in the ␤ subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the ␣1␤2␥2 GABA A receptor, where only mutations in ␣1 or ␤2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.Channels in the nicotinic superfamily are formed by five subunits arranged quasi-symmetrically around the central pore. Structural information from the Torpedo acetylcholine nicotinic receptor and related prokaryotic channels, such as GLIC and ELIC (all cation-permeable), shows that the conduction path for ions starts with a wide vestibule formed by the extracellular domains (1-3). This tapers to a narrower transmembrane region, which is lined mainly by the second transmembrane domains (M2) of each of the five subunits. In eukaryotic channels, this is connected to the cytoplasm by openings in the subunits intracellular domains, which are formed by the M3-M4 loop. The rate of ion flow along this pathway (and therefore the single-channel conductance) is strongly affected by the nature of the residues that line it, especially those that line the narrowest parts. In particular, there are rings of charged amino acids at the extracellular and intracellular ends of the transmembrane part of the pore (positions Ϫ4Ј and 20Ј in the M2 domain (4) and around the fenestrations of the cytoplasmic domain (5). It was recently found that single-channel conductance can also be influenced by residues in the extracellular domain (ECD). The first clue came from the structure of a member of the family of ACh-binding proteins (AChBP), molluscan homopentameric soluble proteins, which are homologous to the ECD part...

Section: Discussionmentioning

confidence: 99%

In Glycine and GABAA Channels, Different Subunits Contribute Asymmetrically to Channel Conductance via Residues in the Extracellular Domain

Moroni

Meyer

Lahmann

et al. 2011

“…DEPC was used to specifically alkylate the imidazole group of histidines (22); this treatment resulted in the loss of the copper inhibition and a reduction in the P2X 4 ATP-evoked currents of the receptor. We are aware that there are controversial results reported in the literature with respect to the use of DEPC, because some authors have successfully modified histidine residues (23)(24)(25), whereas others have failed to detect significant changes (26,27). Considering that the rat P2X 4 receptor has only three histidine residues, it became challenging to establish which of these imidazole-bearing residues is responsible for the negative modulator role of copper, as well as the decreased channel activity.…”

Section: Discussionmentioning

confidence: 99%

Histidine 140 Plays a Key Role in the Inhibitory Modulation of the P2X4 Nucleotide Receptor by Copper but Not Zinc

Coddou

Morales

González

et al. 2003

To elucidate the role of extracellular histidines in the modulation of the rat P2X 4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wildtype receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X 4 receptor, which is distinct from the putative zinc facilitator binding site.The notion that trace metals such as zinc or copper are atypical brain messengers has attracted much attention in view of their emerging role in the modulation of brain excitability (1). The importance of trace metals in synaptic activity is highlighted by the description that both copper and zinc are stored in synaptic vesicles from where they are released by electrical depolarization, reaching a high micromolar concentration at the synaptic cleft (2-4). Trace metals are known to modulate a wide variety of brain ionotropic receptors such as glycine, N-methyl-D-aspartate, ␥-aminobutyric acid, nicotinic, and the novel nucleotide receptors (P2X) family (5-9). The P2X purinoceptors are homomeric or heteromeric membrane channels gated by extracellular ATP and related synthetic nucleotides; North (10) recently reviewed the principles of the molecular physiology of this family of receptors. Within the P2X receptor family, the P2X 4 is the most widely distributed in the central nervous system, including the cerebellum and the CA1 region of the hippocampus, where it has been proposed to play a role in glutamatergic synapses (11).The P2X 4 receptor is an interesting model of an ionic channel differentially modulated by trace metals. Acuñ a-Castillo et al. (12) and Coddou et al. (13) reported that zinc potentiates the ATP-evoked currents whereas copper has an inhibitory effect on the activity of this receptor. Based on these findings, Acuñ aCastillo et al. (12) proposed that trace metals modulate the activity of the P2X 4 receptor via two separate metal-binding sites. One of these sites has a preferential selectivity for copper and is characterized by a non-competitive inhibition of the ATP-gated channel acti...

“…Cell Culture and Transfection-Human embryonic kidney (HEK) cells (American Type Culture Collection CRL1573) were grown and transfected as previously documented (23). Plasmids of hGlyR cDNA clones were co-transfected in a ratio of 1:1 with enhanced green fluorescent protein (24).…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis for Zinc Potentiation at Strychnine-sensitive Glycine Receptors

Miller

Silva

Smart

2005

Self Cite

The divalent cation Zn 2؉ is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn 2؉ . Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn 2؉ potentiation site on GlyRs and account for the differential sensitivity of GlyR ␣ 1 and GlyR ␣ 2 to Zn 2؉ potentiation. In addition, juxtaposed to this Zn 2؉ site, which is located externally on the N-terminal domain of the ␣ subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn 2؉ potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn 2؉ binds to a site on the extracellular outer face of the GlyR ␣ subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.