RESUMEN
Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/metabolismo , Lípidos/química , Animales , Ligandos , Mutagénesis , XenopusRESUMEN
With the long-term goal of using a chimeric approach to dissect the distinct lipid sensitivities and thermal stabilities of the pentameric ligand-gated ion channels (pLGIC), GLIC and ELIC, we constructed chimeras by cross-combining their extracellular (ECD) and transmembrane (TMD) domains. As expected, the chimera formed between GLIC-ECD and ELIC-TMD (GE) responded to protons, the agonist for GLIC, but not cysteamine, the agonist for ELIC, although GE exhibited a 25-fold decrease in proton-sensitivity relative to wild type. The chimera formed between ELIC-ECD and the GLIC-TMD (EG) was usually toxic, unless it contained a pore-lining Ile9'Ala gain-of-function mutation. No significant improvements in expression/toxicity were observed with extensive loop substitutions at the ECD/TMD interface. Surprisingly, oocytes expressing EG-I9'A responded to both the ELIC agonist, cysteamine and the GLIC agonist, protons - the latter at pH values ≤4.0. The cysteamine- and proton-induced currents in EG-I9'A were inhibited by the GLIC TMD pore blocker, amantadine. The cysteamine-induced response of EG-I9'A was also inhibited by protons at pH values down to 4.5, but potentiated at lower pH values. Proton-induced gating at low pH was not abolished by mutation of an intramembrane histidine residue previously implicated in GLIC TMD function. We show that the TMD plays a major role governing the thermal stability of a pLGIC, and identify three distinct mechanisms by which agonists and protons influence the gating of the EG chimera. A structural basis for the impaired function of GE is suggested.
Asunto(s)
Canales Iónicos Activados por Ligandos/metabolismo , Células Procariotas/metabolismo , Animales , Quimera/metabolismo , Cristalografía por Rayos X/métodos , Cisteamina/metabolismo , Histidina/metabolismo , Activación del Canal Iónico/fisiología , Ligandos , Modelos Moleculares , Mutación/genética , Oocitos/metabolismo , Dominios Proteicos/fisiología , Protones , Xenopus laevis/metabolismoRESUMEN
The role of the outermost transmembrane α-helix in both the maturation and function of the prokaryotic pentameric ligand-gated ion channels, GLIC and ELIC, was examined by Ala scanning mutagenesis, deletion mutations, and mutant cycle analyses. Ala mutations at the M4-M1/M3 interface lead to loss-of-function phenotypes in GLIC, with the largest negative effects occurring near the M4 C terminus. In particular, two aromatic residues at the M4 C terminus form a network of π-π and/or cation-π interactions with residues on M3 and the ß6-ß7 loop that is essential for both maturation and function. M4-M1/M3 interactions appear to be optimized in GLIC with even subtle structural changes at this interface leading to detrimental effects. In contrast, mutations along the M4-M1/M3 interface of ELIC typically lead to gain-of-function phenotypes, suggesting that these interactions in ELIC are not optimized for channel function. In addition, no cluster of interacting residues involving the M4 C terminus, M3, and the ß6-ß7 loop was found, suggesting that the M4 C terminus plays little role in ELIC maturation or function. This study shows that M4 makes distinct contributions to the maturation and gating of these two closely related homologs, suggesting that GLIC and ELIC exhibit divergent features of channel function.
Asunto(s)
Membrana Celular/metabolismo , Multimerización de Proteína , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Receptores de Glicina/genética , Eliminación de SecuenciaRESUMEN
The gating of pentameric ligand-gated ion channels is sensitive to a variety of allosteric modulators that act on structures peripheral to those involved in the allosteric pathway leading from the agonist site to the channel gate. One such structure, the lipid-exposed transmembrane α helix, M4, is the target of lipids, neurosteroids, and disease-causing mutations. Here we show that M4 interactions with the adjacent transmembrane α helices, M1 and M3, modulate pLGIC function. Enhanced M4 interactions promote channel function while ineffective interactions reduce channel function. The interface chemistry governs the intrinsic strength of M4-M1/M3 inter-helical interactions, both influencing channel gating and imparting distinct susceptibilities to the potentiating effects of a lipid-facing M4 congenital myasthenic syndrome mutation. Through aromatic substitutions, functional studies, and molecular dynamics simulations, we elucidate a mechanism by which M4 modulates channel function.
Asunto(s)
Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/metabolismo , Regulación Alostérica , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
Membrane lipids are potent modulators of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable versus non-activatable conformations, as well as influencing the transitions between these conformational states. Of note, some membranes stabilize an electrically silent uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions. The uncoupled nAChR, however, does transition to activatable conformations in relatively thick lipid bilayers, such as those found in lipid rafts. In this review, we discuss current understanding of lipid-nAChR interactions in the context of increasingly available high resolution structural and functional data. These data highlight different sites of lipid action, including the lipid-exposed M4 transmembrane α-helix. Current evidence suggests that lipids alter nAChR function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These interactions have also been implicated in both the folding and trafficking of nAChRs to the cell surface. We review current mechanistic understanding of lipid-nAChR interactions, and highlight potential biological roles for lipid-nAChR interactions in modulating the synaptic response. This article is part of a Special Issue entitled: Lipid-protein interactions.
Asunto(s)
Lípidos de la Membrana/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Nicotínicos/química , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Receptores Nicotínicos/metabolismoRESUMEN
With the availability of high resolution structural data, increasing attention has focused on the mechanisms by which drugs and endogenous compounds allosterically modulate nicotinic acetylcholine receptor (nAChR) function. Lipids are potent modulators of the nAChR from Torpedo. Membrane lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations, as well as influencing the transitions between these conformational states. Structural and functional data highlight a role for the lipid-exposed M4 transmembrane α-helix of each subunit in lipid sensing, and suggest that lipids influence gating by altering the binding of M4 to the adjacent transmembrane α-helices, M1 and M3. M4 has also been implicated in both the folding and trafficking of nAChRs to the cell surface, as well as in the potentiation of nAChR gating by neurosteroids. Here, we discuss the roles of M4 in the folding, trafficking, and allosteric modulation of nAChRs. We also consider the hypothesis that variable chemistry at the M4-M1/M3 transmembrane α-helical interface in different nAChR subunits governs the capacity for potentiation by activating lipids. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.