RESUMO
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a genetic form of epilepsy that is caused by mutations in several genes, including genes encoding for the α4 and ß2 subunits of the nicotinic acetylcholine (nACh) receptor. Pentameric α4ß2 nACh receptors are the most abundant nicotinic receptor in the mammalian brain and form two stoichiometries, the (α4)3(ß2)2 and (α4)2(ß2)3 receptors that differ in their physiological and pharmacological properties. The purpose of this study was to investigate how ADNFLE mutations ß2V287M, ß2V287L or α4T293I manifest themselves in different receptor stoichiometries. We expressed wild-type and mutant receptors in Xenopus oocytes and measured the response to ACh and other agonists at both receptor stoichiometries. For all three mutations, the efficacy of ACh at (α4)2(ß2)3 receptors was increased. At (α4)3(ß2)2 receptors, the efficacy of activation was increased both when two molecules of agonist, either ACh or the site-selective agonist sazetidine-A, were bound at the α4-ß2 interfaces, and when a third ACh molecule was bound at the α4-α4 site. Regardless of stoichiometry, the mutations increased the current elicited by low concentrations of ACh. Further, the smoking cessation agents, nicotine, varenicline and cytisine increased activation of mutant (α4)3(ß2)2 receptors, while only nicotine increased activation of mutant (α4)2(ß2)3 receptors. Chronic exposure of all agonists reduced ACh-activation levels at low and high ACh concentrations. From this, we concluded that mutations that cause ADNFLE manifest themselves in a change in efficacy regardless of the stoichiometry of the receptor.
Assuntos
Epilepsia do Lobo Frontal/genética , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Alcaloides/farmacologia , Animais , Azocinas/farmacologia , Epilepsia do Lobo Frontal/fisiopatologia , Feminino , Mutação , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Oócitos/fisiologia , Quinolizinas/farmacologia , Vareniclina/farmacologia , Xenopus laevisRESUMO
The α4ß2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4ß2 nAChR, (α4)2(ß2)3 and (α4)3(ß2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4ß2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4ß2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(ß2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(ß2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(ß2)2 nAChRs.
Assuntos
Aconitina/análogos & derivados , Antagonistas Nicotínicos/química , Receptores Nicotínicos/química , Aconitina/química , Animais , Sítios de Ligação , Cisteína/química , Escherichia coli/metabolismo , Feminino , Ligantes , Maleimidas/química , Mutagênese Sítio-Dirigida , Oócitos/citologia , Ligação Proteica , Conformação Proteica , Ratos , Receptores Nicotínicos/fisiologia , Proteínas Recombinantes/química , Xenopus laevisRESUMO
We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.
Assuntos
Teste de Tolerância a Glucose , Resistência à Insulina/genética , Proteína de Leucina Linfoide-Mieloide/genética , Sequência de Aminoácidos , Animais , Genes Letais , Histona-Lisina N-Metiltransferase , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína de Leucina Linfoide-Mieloide/química , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Ligand-gated ion channels efficiently couple neurotransmitter binding to the opening of an intrinsic ion channel to generate the post-synaptic potentials that are characteristic of fast synaptic transmission. In the Cys-loop family of ligand-gated ion channels, the ligand-binding site is approximately 60 Å above the channel gate. Structural modelling of related proteins and mutagenesis studies led to the hypothesis that loops 2 and 7 of the extracellular domain may couple ligand binding to receptor activation. Mutating loop 2 residues of the glycine receptor to cysteine reveals an alternating pattern of effect upon receptor function. Mutations A52C, T54C and M56C produced a threefold right-shift in EC(50) . In contrast, a 30-fold right-shift was seen for mutations E53C, T55C and D57C. Loop 2 conformational changes associated with ligand binding were assessed by measuring the rate of covalent modification of substituted cysteines by charged methane thiosulfonate reagents. We show for the first time state-dependent differences in the rate of reaction. A52C and T54C are more accessible in the resting state and M56C is more accessible in the activated state. These results demonstrate that loop 2 does undergo a conformational change as part of the mechanism that couples ligand binding to channel opening.
Assuntos
Receptores de Glicina/química , Receptores de Glicina/metabolismo , Transdução de Sinais/fisiologia , Sítios de Ligação/genética , Linhagem Celular Transformada , Cisteína/genética , Relação Dose-Resposta a Droga , Glicina/farmacologia , Humanos , Ativação do Canal Iônico/genética , Ligantes , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/genética , Mutagênese/fisiologia , Mutagênese Sítio-Dirigida/métodos , Técnicas de Patch-Clamp/métodos , Conformação Proteica , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Receptores de Glicina/genética , Transdução de Sinais/genética , Reagentes de Sulfidrila/farmacologia , Transfecção/métodosRESUMO
The Cys-loop receptor family of ligand-gated ion channels (LGICs) play a key role in synaptic transmission in the central nervous system of animals. Recent advances have led to the elucidation of two crystal structures of related prokaryotic LGICs and the electron micrograph derived structure of the acetylcholine receptor from Torpedo marmorata. Here, we review the structural and biochemical data that form our understanding of the structure of the channel pore. We introduce original data from the glycine receptor using the substituted-cysteine accessibility technique and show that while the helical structure of the segment that surrounds the channel pore is generally agreed, the location of the channel gate, the pore diameter and the structure that forms the entry to the channel pore are likely to differ between receptors. The fundamental structural differences between anion and cation selective receptors and how these differences are related to the pore structure are also considered.
Assuntos
Cisteína/química , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Canais Iônicos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cisteína/genética , Cisteína/metabolismo , Humanos , Ativação do Canal Iônico/genética , Canais Iônicos/genética , Ligantes , Dados de Sequência Molecular , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Torpedo/genética , Torpedo/metabolismoRESUMO
The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.