RESUMO
The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina , Humanos , Transporte Biológico , Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/química , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/ultraestrutura , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Transmissão Sináptica , Imidazóis/química , Sarcosina/análogos & derivados , Piperidinas/químicaRESUMO
The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.
Assuntos
Bupropiona , Modelos Moleculares , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Norepinefrina , Piperazinas , Humanos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/química , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Norepinefrina/metabolismo , Bupropiona/metabolismo , Bupropiona/química , Bupropiona/farmacologia , Piperazinas/farmacologia , Piperazinas/química , Piperazinas/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Transporte Biológico , Sódio/metabolismo , Conformação Proteica , Ligação Proteica , TiazóisRESUMO
The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants1-3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.
Assuntos
Benzotropina , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina , Dopamina , Humanos , Apoproteínas/metabolismo , Apoproteínas/química , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Benzotropina/metabolismo , Benzotropina/farmacologia , Sítios de Ligação , Cocaína/farmacologia , Cocaína/metabolismo , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Metilfenidato/metabolismo , Metilfenidato/farmacologia , Modelos Moleculares , Piperazinas/metabolismo , Piperazinas/farmacologia , Ligação Proteica , Conformação ProteicaRESUMO
Sodium-calcium exchanger proteins influence calcium homeostasis in many cell types and participate in a wide range of physiological and pathological processes. Here, we elucidate the cryo-EM structure of the human Na+/Ca2+ exchanger NCX1.3 in the presence of a specific inhibitor, SEA0400. Conserved ion-coordinating residues are exposed on the cytoplasmic face of NCX1.3, indicating that the observed structure is stabilized in an inward-facing conformation. We show how regulatory calcium-binding domains (CBDs) assemble with the ion-translocation transmembrane domain (TMD). The exchanger-inhibitory peptide (XIP) is trapped within a groove between the TMD and CBD2 and predicted to clash with gating helices TMs1/6 at the outward-facing state, thus hindering conformational transition and promoting inactivation of the transporter. A bound SEA0400 molecule stiffens helix TM2ab and affects conformational rearrangements of TM2ab that are associated with the ion-exchange reaction, thus allosterically attenuating Ca2+-uptake activity of NCX1.3.
Assuntos
Cálcio , Trocador de Sódio e Cálcio , Humanos , Compostos de Anilina/farmacologia , Cálcio/metabolismo , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/químicaRESUMO
The R-type voltage-gated calcium channel CaV2.3 is predominantly located in the presynapse and is implicated in distinct types of epileptic seizures. It has consequently emerged as a molecular target in seizure treatment. Here, we determined the cryo-EM structure of the CaV2.3-α2δ1-ß1 complex in the topiramate-bound state at a 3.0 Å resolution. We provide a snapshot of the binding site of topiramate, a widely prescribed antiepileptic drug, on a voltage-gated ion channel. The binding site is located at an intracellular juxtamembrane hydrophilic cavity. Further structural analysis revealed that topiramate may allosterically facilitate channel inactivation. These findings provide fundamental insights into the mechanism underlying the inhibitory effect of topiramate on CaV and NaV channels, elucidating a previously unseen modulator binding site and thus pointing toward a route for the development of new drugs.
Assuntos
Anticonvulsivantes , Canais de Cálcio Tipo R , Microscopia Crioeletrônica , Topiramato , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Topiramato/química , Topiramato/farmacologia , Humanos , Regulação Alostérica/efeitos dos fármacos , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/metabolismo , Sítios de Ligação , Modelos Moleculares , Células HEK293 , Conformação Proteica , Frutose/química , Frutose/análogos & derivados , Frutose/metabolismo , Animais , Proteínas de Transporte de CátionsRESUMO
Choline is a vital nutrient and a precursor for the biosynthesis of essential metabolites, including acetylcholine (ACh), that play a central role in fetal development, especially in the brain. In cholinergic neurons, the high-affinity choline transporter (CHT1) provides an extraordinarily efficient reuptake mechanism to reutilize choline derived from intrasynaptical ACh hydrolysis and maintain ACh synthesis in the presynapse. Here, we determined structures of human CHT1 in three discrete states: the outward-facing state bound with the competitive inhibitor hemicholinium-3 (HC-3); the inward-facing occluded state bound with the substrate choline; and the inward-facing apo open state. Our structures and functional characterizations elucidate how the inhibitor and substrate are recognized. Moreover, our findings shed light on conformational changes when transitioning from an outward-facing to an inward-facing state and establish a framework for understanding the transport cycle, which relies on the stabilization of the outward-facing state by a short intracellular helix, IH1.
Assuntos
Colina , Proteínas de Membrana Transportadoras , Humanos , Colina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Acetilcolina/metabolismoRESUMO
The voltage-gated calcium channel CaV1.2 is essential for cardiac and vessel smooth muscle contractility and brain function. Accumulating evidence demonstrates that malfunctions of CaV1.2 are involved in brain and heart diseases. Pharmacological inhibition of CaV1.2 is therefore of therapeutic value. Here, we report cryo-EM structures of CaV1.2 in the absence or presence of the antirheumatic drug tetrandrine or antihypertensive drug benidipine. Tetrandrine acts as a pore blocker in a pocket composed of S6II, S6III, and S6IV helices and forms extensive hydrophobic interactions with CaV1.2. Our structure elucidates that benidipine is located in the DIII-DIV fenestration site. Its hydrophobic sidechain, phenylpiperidine, is positioned at the exterior of the pore domain and cradled within a hydrophobic pocket formed by S5DIII, S6DIII, and S6DIV helices, providing additional interactions to exert inhibitory effects on both L-type and T-type voltage gated calcium channels. These findings provide the structural foundation for the rational design and optimization of therapeutic inhibitors of voltage-gated calcium channels.
Assuntos
Canais de Cálcio Tipo L , Canais de Cálcio Tipo L/metabolismo , Estrutura Secundária de ProteínaRESUMO
Efforts on developing transient receptor potential vanilloid 1 (TRPV1) drugs for pain management have been hampered by deleterious hypo- or hyperthermia caused by TRPV1 agonists/antagonists. Here, we compared the effects of four antagonists on TRPV1 polymodal gating and core body temperature (CBT) in Trpv1+/+, Trpv1-/-, and Trpv1T634A/T634A. Neither the effect on proton gating nor drug administration route, hair coverage, CBT rhythmic fluctuations, or inflammation had any influence on the differential actions of TRPV1 drugs on CBT. We identified the S4-S5 linker region exposed to the vanilloid pocket of TRPV1 to be critical for hyperthermia associated with certain TRPV1 antagonists. PSFL2874, a TRPV1 antagonist we discovered, is effective against inflammatory pain but devoid of binding to the S4-S5 linker and inducing CBT changes. These findings implicate that biased allosteric mechanisms exist for TRPV1 coupling to nociception and CBT regulation, opening avenues for the development of non-opioid analgesics without affecting CBT.