Pierre Jean Corringer

Nicotine upregulates its own receptors through enhanced intracellular maturation.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smokers brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N‐terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7‐V201–5HT3 chimera. Mutations E18Q or E44Q abolish calcium‐enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161–172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161–172 domain in the calcium insensitive 5‐hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160–174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161–172) domain may simultaneously contribute to calcium and agonist binding.

Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the “linking tunnel.” These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.

Paradoxical allosteric effects of competitive inhibitors on neuronal α7 nicotinic receptor mutants

MUTATION of the conserved leucine residue, in the second transmembrane domain of the neuronal α7 acetylcholine receptor to a threonine (L247T) causes pleiotropic alterations of receptor properties. In this study we examined the effects of competitive inhibitors on the α7-L247T physiological responses. While the α7 competitive inhibitor dihydro-β-erythroidine evoked a current comparable to that induced by ACh, other inhibitors such as methyllycaconitine (MLA) and α-bungarotoxin (α-Bgt) caused a blockade of α7-L247T to ACh activation. When applied in the absence of ACh, MLA or α-Bgt reduced the cell leakage current, showing that α7-L247T displays a significant fraction (10%) of spontaneously open channels. These data can be interpreted in terms of an allosteric model, assuming that the L247T mutant possesses a low isomerization constant L and that MLA and α-Bgt stabilize the closed, resting state.

An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H‐bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced α4 residues into the α7/5HT3 chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non‐additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole‐cell patch–clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5‐fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high‐affinity α‐bungarotoxin binding (100‐fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric–kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter‐backbone H‐bond between positions 152 and 193, revealed in α4 and in α7 double mutant but not in α7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors

BACKGROUNDnMutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC.nnnMETHODSnWe directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes.nnnRESULTSnEtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50xa0μM picrotoxin to the media, a technique that may have applications in expression of other ion channels.nnnCONCLUSIONSnOur results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.