RESUMEN
BACKGROUND AND PURPOSE: P2X receptors are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains. EXPERIMENTAL APPROACH: Here, we examine the impact of P2X2 receptor inter-subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. KEY RESULTS: We demonstrate that predicted extracellular domain inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. CONCLUSION AND IMPLICATIONS: We provide the first structural mapping of the mutational distribution across the human population in a ligand-gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand-gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.
Asunto(s)
Activación del Canal Iónico , Mutación Missense , Receptores Purinérgicos P2X2 , Adenosina Trifosfato/metabolismo , Humanos , Mutación , Receptores Purinérgicos P2X2/genéticaRESUMEN
The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/fisiología , Iones/metabolismo , Proteínas Musculares/metabolismo , Canales de Potasio/metabolismo , Línea Celular , Microscopía por Crioelectrón/métodos , AMP Cíclico/metabolismo , Células HEK293 , HumanosRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.
Asunto(s)
AMP Cíclico/química , AMP Cíclico/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Dominios Proteicos , Sitios de Unión , Electrofisiología , Células HEK293/fisiología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Activación del Canal Iónico , Cinética , Simulación de Dinámica Molecular , Conformación Proteica , Isoformas de Proteínas , TermodinámicaRESUMEN
P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.
Asunto(s)
Agonistas del Receptor Purinérgico P2X/metabolismo , Receptores Purinérgicos P2X2/química , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células HEK293 , Humanos , Unión Proteica , Agonistas del Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X2/metabolismo , Xenopus laevisRESUMEN
The α4ß2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4ß2)2α4 and (α4ß2)2ß2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4ß2)2α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4ß2)2α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4ß2)2α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4ß2)2α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4ß2)2α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.
Asunto(s)
Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Animales , Ligandos , Receptores Nicotínicos/genética , Xenopus laevisRESUMEN
Nicotinic acetylcholine receptor (nAChR) α4 and ß2 subunits assemble in two alternate stoichiometries to produce (α4ß2)(2)α4 and (α4ß2)(2)ß2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/ß2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or ß2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or ß2(+)/ß2(-) interfaces. Using fully concatenated (α4ß2)(2)α4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4ß2)(2)α4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the α4(+)/(α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4ß2)(2)α4 nAChRs.