RESUMO
Otopetrin 1 (OTOP1) is a proton-activated channel crucial for animals' perception of sour taste. Despite its significance, the gating mechanism of OTOP1 remains poorly understood. Here, we demonstrate that carvacrol activates the mouse OTOP1 (mOTOP1) channel under neutral and acidic conditions. Functional analysis showed that carvacrol enhances pH fluorescence signals in OTOP1-expressing cells, with reduced efficacy at lower pH levels. Carvacrol selectively activates mOTOP1, while mOTOP2, mOTOP3, and Chelonia mydas OTOP1 (CmOTOP1) are insensitive to carvacrol activation under neutral pH. Through chimera and point mutation experiments, swapping S134 in transmembrane segment 3 (TM3) and T247 in the TM5-6 linker abolished carvacrol activation of mOTOP1 and conferred activation on CmOTOP1, suggesting these two residues are critical for carvacrol sensitivity. These findings highlight TM3 and TM5-6 linker as pivotal gating apparatus of OTOP1 channels and potential docking sites for drug design.
Assuntos
Cimenos , Cimenos/farmacologia , Animais , Camundongos , Ativação do Canal Iônico/efeitos dos fármacos , Humanos , Células HEK293 , Concentração de Íons de Hidrogênio , Canais Iônicos/metabolismo , Canais Iônicos/genéticaRESUMO
A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss- and gain-of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel's pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.
Assuntos
Canal de Potássio KCNQ3 , Simulação de Dinâmica Molecular , Mutação , Transtornos do Neurodesenvolvimento , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Humanos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/tratamento farmacológico , Ativação do Canal Iônico/efeitos dos fármacos , Animais , Células HEK293 , Potenciais da Membrana/efeitos dos fármacosRESUMO
Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR's activity through binding to a site also comprising TM8.
Assuntos
Microscopia Crioeletrônica , Regulador de Condutância Transmembrana em Fibrose Cística , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Regulação Alostérica , Ativação do Canal Iônico/efeitos dos fármacos , Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Animais , Ligação Proteica , Aminofenóis/farmacologia , Aminofenóis/química , Aminofenóis/metabolismo , Benzodioxóis/farmacologia , MutaçãoRESUMO
TWIK-related potassium channel 1 (TREK1), a two-pore-domain mammalian potassium (K+) channel, regulates the resting potential across cell membranes, presenting a promising therapeutic target for neuropathy treatment. The gating of this channel converges in the conformation of the narrowest part of the pore: the selectivity filter (SF). Various hypotheses explain TREK1 gating modulation, including the dynamics of loops connecting the SF with transmembrane helices and the stability of hydrogen bond (HB) networks adjacent to the SF. Recently, two small molecules (Q6F and Q5F) were reported as activators that affect TREK1 by increasing its open probability in single-channel current measurements. Here, using molecular dynamics simulations, we investigate the effect of these ligands on the previously proposed modulation mechanisms of TREK1 gating compared to the apo channel. Our findings reveal that loop dynamics at the upper region of the SF exhibit only a weak correlation with permeation events/nonpermeation periods, whereas the HB network behind the SF appears more correlated. These nonpermeation periods arise from both distinct mechanisms: a C-type inactivation (resulting from dilation at the top of the SF), which has been described previously, and a carbonyl flipping in an SF binding site. We find that, besides the prevention of C-type inactivation in the channel, the ligands increase the probability of permeation by modulating the dynamics of the carbonyl flipping, influenced by a threonine residue at the bottom of the SF. These results offer insights for rational ligand design to optimize the gating modulation of TREK1 and related K+ channels.
Assuntos
Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Ativação do Canal Iônico/efeitos dos fármacos , HumanosRESUMO
P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,ßMe-ATP (α,ßMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.
Assuntos
Trifosfato de Adenosina , Receptores Purinérgicos P2X , Humanos , Ligantes , Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2X/química , Magnésio/metabolismo , Magnésio/química , Ligação Proteica , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X3/química , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X2/química , Agonistas do Receptor Purinérgico P2X/farmacologiaRESUMO
Natural ion channels are proteins embedded in the cell membrane that control many aspects of cell and human physiology by acting as gatekeepers, regulating the flow of ions in and out of cells. Advances in nanotechnology have influenced the methods for studying ion channels in vitro, as well as ways to unlock the delivery of therapeutics by modulating them in vivo. This review provides an overview of nanotechnology-enabled approaches for ion channel research with a focus on the synthesis and applications of synthetic ion channels. Further, the uses of nanotechnology for therapeutic applications are critically analyzed. Finally, we provide an outlook on the opportunities and challenges at the intersection of nanotechnology and ion channels. This work highlights the key role of nanoscale interactions in the operation and modulation of ion channels, which may prompt insights into nanotechnology-enabled mechanisms to study and exploit these systems in the near future.
Assuntos
Canais Iônicos , Nanotecnologia , Canais Iônicos/metabolismo , Humanos , Animais , Membrana Celular/metabolismo , Membrana Celular/química , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
Lysophosphatidylcholine (LPC) is a bioactive lipid present at high concentrations in inflamed and injured tissues where it contributes to the initiation and maintenance of pain. One of its important molecular effectors is the transient receptor potential canonical 5 (TRPC5), but the explicit mechanism of the activation is unknown. Using electrophysiology, mutagenesis and molecular dynamics simulations, we show that LPC-induced activation of TRPC5 is modulated by xanthine ligands and depolarizing voltage, and involves conserved residues within the lateral fenestration of the pore domain. Replacement of W577 with alanine (W577A) rendered the channel insensitive to strong depolarizing voltage, but LPC still activated this mutant at highly depolarizing potentials. Substitution of G606 located directly opposite position 577 with tryptophan rescued the sensitivity of W577A to depolarization. Molecular simulations showed that depolarization widens the lower gate of the channel and this conformational change is prevented by the W577A mutation or removal of resident lipids. We propose a gating scheme in which depolarizing voltage and lipid-pore helix interactions act together to promote TRPC5 channel opening.
Assuntos
Lisofosfatidilcolinas , Simulação de Dinâmica Molecular , Canais de Cátion TRPC , Humanos , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/química , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Animais , Ativação do Canal Iônico/efeitos dos fármacos , Células HEK293 , Mutação , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacosRESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.
Assuntos
Epilepsia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Mutação , Canais de Potássio , Propofol , Humanos , Sítios de Ligação , Microscopia Crioeletrônica , Eletrofisiologia , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/metabolismo , Células HEK293 , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/ultraestrutura , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Movimento/efeitos dos fármacos , Fenilalanina/genética , Fenilalanina/metabolismo , Polimorfismo Genético , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio/ultraestrutura , Propofol/farmacologia , Propofol/químicaRESUMO
The opening of the Torpedo CLC-0 chloride (Cl-) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H+i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H+i inhibition similar to that of the channel inactivation, a resistance to the H+i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H+i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H+i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H+i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states.
Assuntos
Canais de Cloreto , Ativação do Canal Iônico , Prótons , Canais de Cloreto/metabolismo , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/química , Canais de Cloreto/genética , Ativação do Canal Iônico/efeitos dos fármacos , Animais , Mutação , CinéticaRESUMO
Understanding the agonist concentration-response curve (CRC) is the cornerstone in pharmacology. While CRC parameters, agonist potency (EC50) and efficacy (maximum response, Imax) are well-studied, the role of unliganded gating (minimum response, Imin) on CRC is often overlooked. This study explores the effect of unliganded gating on agonist response in muscle-type acetylcholine (ACh) receptors, focusing on the underexplored role of Imin in modulating EC50 and Imax. Three Gain-of-Function (GOF) mutations that increase, and two Loss-of-Function (LOF) mutations that decrease the unliganded gating equilibrium constant (L0) were studied using automated patch-clamp electrophysiology. GOF mutations enhanced agonist potency, whereas LOF mutations reduced it. The calculated CRC aligned well with empirical results, indicating that agonist CRC can be estimated from knowledge of L0. Reduction in agonist efficacy due to LOF mutations was calculated and subsequently validated using single-channel patch-clamp electrophysiology, a factor often obscured in normalized CRC. The study also evaluated the combined impact of mutations (L0) on CRC, confirming the predictive model. Further, no significant energetic coupling between distant residues (>15 Å) was found, indicating that the mutations' effects are localized and do not alter overall agonist affinity. These findings substantiate the role of unliganded gating in modulating agonist responses and establishes a predictive model for estimating CRC parameters from known changes in L0. The study highlights the importance of intrinsic activity in receptor theory.
Assuntos
Ativação do Canal Iônico , Mutação , Agonistas Nicotínicos , Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Ativação do Canal Iônico/efeitos dos fármacos , Humanos , Agonistas Nicotínicos/farmacologia , Ligantes , Relação Dose-Resposta a Droga , Animais , Células HEK293RESUMO
ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.
Assuntos
Ativação do Canal Iônico , Venenos de Aranha , Animais , Humanos , Venenos de Aranha/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Células HEK293 , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Xenopus laevisRESUMO
Delta receptors (GluD1 and GluD2), members of the large ionotropic glutamate receptor (iGluR) family, play a central role in numerous neurodevelopmental and psychiatric disorders. The amino-terminal domain (ATD) of GluD orchestrates synapse formation and maturation processes through its interaction with the Cbln family of synaptic organizers and neurexin (Nrxn). The transsynaptic triad of Nrxn-Cbln-GluD also serves as a potent regulator of synaptic plasticity, at both excitatory and inhibitory synapses. Despite these recognized functions, there is still debate as to whether GluD functions as a "canonical" ion channel, similar to other iGluRs. A recent report proposes that the ATD of GluD2 imposes conformational constraints on channel activity; removal of this constraint by binding to Cbln1 and Nrxn, or removal of the ATD, reveals channel activity in GluD2 upon administration of glycine (Gly) and d-serine (d-Ser), two GluD ligands. We were able to reproduce currents when Gly or d-Ser was administered to clusters of heterologous human embryonic kidney 293 (HEK293) cells expressing Cbln1, GluD2 (or GluD1), and Nrxn. However, Gly or d-Ser, but also l-glutamate (l-Glu), evoked similar currents in naive (i.e., untransfected) HEK293 cells and in GluD2-null Purkinje neurons. Furthermore, no current was detected in isolated HEK293 cells expressing GluD2 lacking the ATD upon administration of Gly. Taken together, these results cast doubt on the previously proposed hypothesis that extracellular ligands directly gate wild-type GluD channels.
Assuntos
Ativação do Canal Iônico , Receptores de Glutamato , Animais , Humanos , Camundongos , Glicina/metabolismo , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Ligantes , Receptores de Glutamato/metabolismo , Serina/metabolismoRESUMO
Voltage-gated ion channels are responsible for the electrical excitability of neurons and cardiomyocytes. Thus, they are obvious targets for pharmaceuticals aimed to modulate excitability. Compounds activating voltage-gated potassium (KV) channels are expected to reduce excitability. To search for new KV-channel activators, we performed a high-throughput screen of 10,000 compounds on a specially designed Shaker KV channel. Here, we report on a large family of channel-activating compounds with a carboxyl (COOH) group as the common motif. The most potent COOH activators are lipophilic (4 < LogP <7) and are suggested to bind at the interface between the lipid bilayer and the channel's positively charged voltage sensor. The negatively charged form of the COOH-group compounds is suggested to open the channel by electrostatically pulling the voltage sensor to an activated state. Several of the COOH-group compounds also activate the therapeutically important KV7.2/7.3 channel and can thus potentially be developed into antiseizure drugs. The COOH-group compounds identified in this study are suggested to act via the same site and mechanism of action as previously studied COOH-group compounds, such as polyunsaturated fatty acids and resin acids, but distinct from sites for several other types of potassium channel-activating compounds.
Assuntos
Ativação do Canal Iônico , Animais , Ativação do Canal Iônico/efeitos dos fármacos , Superfamília Shaker de Canais de Potássio/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ2/agonistas , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canal de Potássio KCNQ3/metabolismo , Humanos , Xenopus laevisRESUMO
A recent study investigated the correlation between telmisartan (TEL) exposure and Alzheimer's disease (AD) risk among African Americans (AAs) and European Americans. Their findings indicated that moderate-to-high TEL exposure was linked to a decreased incidence of AD among AAs. These results suggest a potential association between TEL and a reduced risk of AD specifically within the AA population. Here, we investigated the effects of TEL, either alone or in combination with ranolazine (Ran) or dapagliflozin (Dapa), on voltage-gated Na + currents ( INa ) in Neuro-2a cells. TEL, primarily used for treating hypertension and cardiovascular disorders, showed a stimulatory effect on INa , while Ran and Dapa reversed this stimulation. In Neuro-2a cells, we demonstrated that with exposure to TEL, the transient ( INa(T) ) and late ( INa(L) ) components of INa were differentially stimulated with effective EC 50 's of 16.9 and 3.1 µM, respectively. The research implies that TEL's impact on INa might be associated with enhanced neuronal excitability. This study highlights the complex interplay between TEL, Ran, and Dapa on INa and their potential implications for AD, emphasizing the need for further investigation to understand the mechanisms involved.
Assuntos
Acetanilidas , Compostos Benzidrílicos , Benzimidazóis , Benzoatos , Glucosídeos , Neuroblastoma , Piperazinas , Ranolazina , Telmisartan , Telmisartan/farmacologia , Telmisartan/uso terapêutico , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Ranolazina/farmacologia , Ranolazina/uso terapêutico , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Linhagem Celular Tumoral , Animais , Acetanilidas/farmacologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Camundongos , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
Automated patch clamp recording is a valuable technique in drug discovery and the study of ion channels. It allows for the precise measurement and manipulation of channel currents, providing insights into their function and modulation by drugs or other compounds. The melanocortin 4 receptor (MC4-R) is a G protein-coupled receptor (GPCR) crucial to appetite regulation, energy balance, and body weight. MC4-R signaling is complex and involves interactions with other receptors and neuropeptides in the appetite-regulating circuitry. MC4-Rs, like other GPCRs, are known to modulate ion channels such as Kir7.1, an inward rectifier potassium channel, in response to ligand binding. This modulation is critical for controlling ion flow across the cell membrane, which can influence membrane potential, excitability, and neurotransmission. The MC4-R is the target for the anti-obesity drug Imcivree. However, this drug is known to lack optimal potency and also has side effects. Using high-throughput techniques for studying the MC4-R/Kir7.1 complex allows researchers to rapidly screen many compounds or conditions, aiding the development of drugs that target this system. Additionally, automated patch clamp recording of this receptor-channel complex and its ligands can provide valuable functional and pharmacological insights supporting the development of novel therapeutic strategies. This approach can be generalized to other GPCR-gated ion channel functional complexes, potentially accelerating the pace of research in different fields with the promise to uncover previously unknown aspects of receptor-ion channel interactions.
Assuntos
Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização , Receptor Tipo 4 de Melanocortina , Técnicas de Patch-Clamp/métodos , Animais , Humanos , Receptor Tipo 4 de Melanocortina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293RESUMO
Studies on the interaction sites of peptide toxins and ion channels typically involve site-directed mutations in toxins. However, natural mutant toxins exist among them, offering insights into how the evolutionary process has conserved crucial sequences for activities and molecular target selection. In this study, we present a comparative investigation using electrophysiological approaches and computational analysis between two alpha toxins from evolutionarily close scorpion species of the genus Tityus, namely, Tst3 and Ts3 from T. stigmurus and T. serrulatus, respectively. These toxins exhibit three natural substitutions near the C-terminal region, which is directly involved in the interaction between alpha toxins and Nav channels. Additionally, we characterized the activity of the Tst3 toxin on Nav1.1-Nav1.7 channels. The three natural changes between the toxins did not alter sensitivity to Nav1.4, maintaining similar intensities regarding their ability to alter opening probabilities, delay fast inactivation, and induce persistent currents. Computational analysis demonstrated a preference for the down conformation of VSD4 and a shift in the conformational equilibrium towards this state. This illustrates that the sequence of these toxins retained the necessary information, even with alterations in the interaction site region. Through electrophysiological and computational analyses, screening of the Tst3 toxin on sodium isoform revealed its classification as a classic α-NaTx with a broad spectrum of activity. It effectively delays fast inactivation across all tested isoforms. Structural analysis of molecular energetics at the interface of the VSD4-Tst3 complex further confirmed this effect.
Assuntos
Venenos de Escorpião , Escorpiões , Venenos de Escorpião/química , Venenos de Escorpião/genética , Animais , Brasil , Humanos , Xenopus laevis , Ativação do Canal Iônico/efeitos dos fármacos , Sequência de Aminoácidos , Animais PeçonhentosRESUMO
Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca2+-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins.
Assuntos
Adenosina Difosfato Ribose , Cálcio , Evolução Molecular , Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Adenosina Difosfato Ribose/metabolismo , Humanos , Animais , Cálcio/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Sítios de Ligação , Filogenia , Pirofosfatases/metabolismo , Pirofosfatases/genéticaRESUMO
Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Canais de Cálcio Tipo L , Células Cromafins , Glucose , Animais , Células Cromafins/metabolismo , Células Cromafins/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Ratos , Glucose/metabolismo , Glucose/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Trifosfato de Adenosina/metabolismo , Ribonucleotídeos/farmacologia , Pirimidinas/farmacologia , Cálcio/metabolismo , Pirazóis/farmacologia , Células Cultivadas , Ratos Wistar , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.
Assuntos
Ativação do Canal Iônico , Canais de Cátion TRPM , Temperatura , Humanos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sítios de Ligação , Cálcio/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Vanadatos/química , Vanadatos/farmacologia , Vanadatos/metabolismoRESUMO
Cannabidiol (CBD), the main non-psychotropic phytocannabinoid produced by the Cannabis sativa plant, blocks a variety of cardiac ion channels. We aimed to identify whether CBD regulated the cardiac pacemaker channel or the hyperpolarization-activated cyclic nucleotide-gated channel (HCN4). HCN4 channels are important for the generation of the action potential in the sinoatrial node of the heart and increased heart rate in response to ß-adrenergic stimulation. HCN4 channels were expressed in HEK 293T cells, and the effect of CBD application was examined using a whole-cell patch clamp. We found that CBD depolarized the V1/2 of activation in holo-HCN4 channels, with an EC50 of 1.6 µM, without changing the current density. CBD also sped activation kinetics by approximately threefold. CBD potentiation of HCN4 channels occurred via binding to the closed state of the channel. We found that CBD's mechanism of action was distinct from cAMP, as CBD also potentiated apo-HCN4 channels. The addition of an exogenous PIP2 analog did not alter the ability of CBD to potentiate HCN4 channels, suggesting that CBD also acts using a unique mechanism from the known HCN4 potentiator PIP2. Lastly, to gain insight into CBD's mechanism of action, computational modeling and targeted mutagenesis were used to predict that CBD binds to a lipid-binding pocket at the C-terminus of the voltage sensor. CBD represents the first FDA-approved drug to potentiate HCN4 channels, and our findings suggest a novel starting point for drug development targeting HCN4 channels.