RESUMO
The large-conductance calcium-activated potassium (BKCa) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BKCa channel activator, altering V 1/2 and G max This study investigates CTIBD's activation mechanism, revealing its independence from the Ca2+ and membrane voltage sensing of the BKCa channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer. Key residues (W22, W203, and F266) are important for CTIBD binding, and their replacement with alanine reduces CTIBD-mediated channel activation. The triple-mutant (W22A/W203A/F266A) channel showed the smallest V 1/2 shift with a minimal impact on activation and deactivation kinetics by CTIBD. At the single-channel level, CTIBD treatment was much less effective at increasing P o in the triple mutant, mainly because of a drastically increased dissociation rate compared with the WT. These findings highlight CTIBD's mechanism, offering crucial insights for developing small-molecule treatments for BKCa-related pathophysiological conditions.
Assuntos
Agonistas dos Canais de Cloreto , Microscopia Crioeletrônica , Canais de Potássio Ativados por Cálcio de Condutância Alta , Animais , Humanos , Sítios de Ligação , Cálcio/metabolismo , Células HEK293 , Ativação do Canal Iônico , Cinética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Bicamadas Lipídicas/metabolismo , Mutação , Ligação Proteica , Agonistas dos Canais de Cloreto/química , Agonistas dos Canais de Cloreto/farmacologiaRESUMO
Voltage-gated potassium channels are critical in modulating cellular excitability, with Slo (slowpoke) channels forming a unique family characterized by their large conductance and dual regulation by electrical signals and intracellular messengers. Despite their structural and evolutionary similarities, Slo1 and Slo3 channels exhibit significant differences in their voltage-gating properties. This study investigates the molecular determinants that differentiate the voltage-gating properties of human Slo1 and mouse Slo3 channels. Utilizing Slo1/Slo3 chimeras, we pinpointed the selectivity filter region as a key factor in the Slo3 channel's reduced conductance at negative voltages. The S6 transmembrane (TM) segment was identified as pivotal for the Slo3 channel's biphasic deactivation kinetics at these voltages. Additionally, the S4 and S6 TM segments were found to be responsible for the gradual slope in the Slo3 channel's conductance-voltage relationship, while multiple TM regions appear to be involved in the Slo3 channel's requirement of strong depolarization for activation. Mutations in the Slo1's S5 and S6 TM segments revealed three residues (I233, L302, and M304) that likely play a crucial role in the allosteric coupling between the voltage sensors and the pore gate.
Assuntos
Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Animais , Humanos , Camundongos , Sequência de Aminoácidos , Membrana Celular/metabolismo , Cinética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana , Mutação , Domínios ProteicosRESUMO
Mammalian Ca2+-dependent Slo K+ channels can stably associate with auxiliary γ subunits which fundamentally alter their behavior. By a so far unknown mechanism, the four γ subunits reduce the need for voltage-dependent activation and, thereby, allow Slo to open independently of an action potential. Here, using cryo-EM, we reveal how the transmembrane helix of γ1/LRRC26 binds and presumably stabilizes the activated voltage-sensor domain of Slo1. The activation is further enhanced by an intracellular polybasic stretch which locally changes the charge gradient across the membrane. Our data provide a possible explanation for Slo1 regulation by the four γ subunits and also their different activation efficiencies. This suggests a novel activation mechanism of voltage-gated ion channels by auxiliary subunits.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Subunidades Proteicas , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Animais , Ativação do Canal Iônico , Modelos Moleculares , Células HEK293 , Ligação Proteica , Domínios ProteicosRESUMO
CaV1.2 channels play a fundamental role in the regulation of vascular smooth muscle tone. The aim of the present study was to synthesize morin derivatives bearing the nitrophenyl moiety of dihydropyridine Ca2+ antagonists to increase the flavonoid vasorelaxant activity. The effects of morin and its derivatives were assessed on CaV1.2 and KCa1.1 channels, both in vitro and in silico, as well as on the contractile responses of rat aorta rings. All compounds were effective CaV1.2 channel blockers, positioning in the α1C subunit region where standard blockers bind. Among the four newly synthesized morin derivatives, the penta-acetylated morin-1 was the most efficacious Ca2+ antagonist, presenting a vasorelaxant profile superior to that of the parent compound and, contrary to morin, antagonized also the release of Ca2+ from the sarcoplasmic reticulum; surprisingly, it also stimulated KCa1.1 channel current. Computational analysis demonstrated that morin-1 bound close to the KCa1.1 channel S6 segment. In conclusion, these findings open a new avenue for the synthesis of valuable multi-functional, vasorelaxant morin derivatives capable to target several pathways underpinning the pathogenesis of hypertension.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Flavonoides/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Vasodilatadores/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Canais de Cálcio Tipo L/química , Relação Dose-Resposta a Droga , Flavonoides/administração & dosagem , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Masculino , Simulação de Acoplamento Molecular/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Vasodilatadores/administração & dosagemRESUMO
Coumarins exhibit a wide variety of biological effects, including activities in the cardiovascular system and the aim of this study was to evaluate the vascular therapeutic potential of 7-Hydroxicoumarin (7-HC). The vascular effects induced by 7-HC (0.001 µM-300 µM), were investigated by in vitro approaches using isometric tension measurements in rat superior mesenteric arteries and by in silico assays using Ligand-based analysis. Our results suggest that the vasorelaxant effect of 7-HC seems to rely on potassium channels, notably through large conductance Ca2+-activated K+ (BKCa) channels activation. In fact, 7-HC (300 µM) significantly reduced CaCl2-induced contraction as well as the reduction of intracellular calcium mobilization. However, the relaxation induced by 7-HC was independent of store-operated calcium entry (SOCE). Moreover, in silico analysis suggests that potassium channels have a common binding pocket, where 7-HC may bind and hint that its binding profile is more similar to quinine's than verapamil's. These results are compatible with the inhibition of Ca2+ release from intracellular stores, which is prompted by phenylephrine and caffeine. Taken together, these results demonstrate a therapeutic potential of 7-HC on the cardiovascular system, making it a promising lead compound for the development of drugs useful in the treatment of cardiovascular diseases.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Artérias Mesentéricas/efeitos dos fármacos , Umbeliferonas/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Sinalização do Cálcio/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Masculino , Artérias Mesentéricas/fisiologia , Técnicas de Cultura de Órgãos , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Vasodilatação/fisiologiaRESUMO
Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue 'C-linker'. To determine the linker's role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.
Assuntos
Membrana Celular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/fisiologia , Fenômenos Eletrofisiológicos , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K+ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.
Assuntos
Indóis/antagonistas & inibidores , Indóis/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/efeitos dos fármacos , Animais , Sítios de Ligação , Ativação do Canal Iônico/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Domínios ProteicosRESUMO
Slo1 is a Ca2+- and voltage-activated K+ channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, ß4. Four ß4, each containing two transmembrane helices, encircle Slo1, contacting it through helical interactions inside the membrane. On the extracellular side, ß4 forms a tetrameric crown over the pore. Structures with high and low Ca2+ concentrations show that identical gating conformations occur in the absence and presence of ß4, implying that ß4 serves to modulate the relative stabilities of 'pre-existing' conformations rather than creating new ones. The effects of ß4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Microscopia Crioeletrônica , Humanos , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
In the present study, we report that neolignan1 (Diethyl-4,4'-dihydroxy-8,3'-neolign-7,7'-dien-9,9'-dionate) relaxes the superior mesenteric artery in a concentration dependent manner (pD2 value 5.392⯱â¯0.04; nâ¯=â¯8 for endothelium intact and 5.204⯱â¯0.03; nâ¯=â¯8 for endothelium denuded mesenteric rings, respectively). The relaxation response of neolignan1 was found to be endothelium independent and sensitive to 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-on (ODQ; 1⯵M) and tetraethyl ammonium (TEA; 1â¯mM). In-silico studies showed good LibDock score (92.66) of neolignan1 with BKCa channel and are in well corroboration with ex-vivo study. Further, neolignan1 significantly decreased the systolic blood pressure, diastolic blood pressure and mean arterial pressure in the Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 50â¯mg/kg) treated Wistar rats at the dose of 30 and 100â¯mg/kg given once orally for 15 days. In addition, neolignan1 is well tolerated up to 100â¯mg/kg when given as a repeated dose, once orally for 28 days in Swiss albino mice. Neolignan1 was well absorbed from oral route, reached peak at 4â¯h and eliminated below detection level by 12â¯h after administration. Our present study concludes that neolignan1 produced relaxation in superior mesenteric artery by opening of BKCa channel and produced significant antihypertensive activity in L-NAME treated Wistar rats and was well tolerated by the experimental animal.
Assuntos
Anti-Hipertensivos/farmacologia , Ácidos Cumáricos/farmacologia , Lignanas/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Animais , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacocinética , Ácidos Cumáricos/metabolismo , Ácidos Cumáricos/farmacocinética , Feminino , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Lignanas/metabolismo , Lignanas/farmacocinética , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Ratos , Ratos Wistar , Vasodilatação/efeitos dos fármacosRESUMO
KCNMA1 encodes the large-conductance Ca2+- and voltage-activated K+ (BK) potassium channel α-subunit, and pathogenic gain-of-function variants in this gene have been associated with a dominant form of generalized epilepsy and paroxysmal dyskinesia. Here, we genetically and functionally characterize eight novel loss-of-function (LoF) variants of KCNMA1. Genome or exome sequencing and the participation in the international Matchmaker Exchange effort allowed for the identification of novel KCNMA1 variants. Patch clamping was used to assess functionality of mutant BK channels. The KCNMA1 variants p.(Ser351Tyr), p.(Gly356Arg), p.(Gly375Arg), p.(Asn449fs) and p.(Ile663Val) abolished the BK current, whereas p.(Cys413Tyr) and p.(Pro805Leu) reduced the BK current amplitude and shifted the activation curves toward positive potentials. The p.(Asp984Asn) variant reduced the current amplitude without affecting kinetics. A phenotypic analysis of the patients carrying the recurrent p.(Gly375Arg) de novo missense LoF variant revealed a novel syndromic neurodevelopmental disorder associated with severe developmental delay, visceral and cardiac malformations, connective tissue presentations with arterial involvement, bone dysplasia and characteristic dysmorphic features. Patients with other LoF variants presented with neurological and developmental symptoms including developmental delay, intellectual disability, ataxia, axial hypotonia, cerebral atrophy and speech delay/apraxia/dysarthria. Therefore, LoF KCNMA1 variants are associated with a new syndrome characterized by a broad spectrum of neurological phenotypes and developmental disorders. LoF variants of KCNMA1 cause a new syndrome distinctly different from gain-of-function variants in the same gene.
Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Mutação com Perda de Função , Fenótipo , Alelos , Substituição de Aminoácidos , Fenômenos Eletrofisiológicos , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Recém-Nascido , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Masculino , Mutação de Sentido Incorreto , Linhagem , Domínios Proteicos , Domínios e Motivos de Interação entre ProteínasRESUMO
Membrane depolarization and intracellular Ca2+ promote activation of the large-conductance Ca2+- and voltage-gated (Slo1) big potassium (BK) channel. We examined the physical interactions that stabilize the closed and open conformations of the ion conduction gate of the human Slo1 channel using electrophysiological and computational approaches. The results show that the closed conformation is stabilized by intersubunit ion-ion interactions involving negative residues (E321 and E324) and positive residues (329RKK331) at the cytoplasmic ends of the transmembrane S6 segments ("RKK ring"). When the channel gate is open, the RKK ring is broken and the positive residues instead make electrostatic interactions with nearby membrane lipid oxygen atoms. E321 and E324 are stabilized by water. When the 329RKK331 residues are mutated to hydrophobic amino acids, these residues form even stronger hydrophobic interactions with the lipid tails to promote the open conformation, shifting the voltage dependence of activation to the negative direction by up to 400 mV and stabilizing the selectivity filter region. Thus, the RKK segment forms electrostatic interactions with oxygen atoms from two sources, other amino acid residues (E321/E324), and membrane lipids, depending on the gate status. Each time the channel opens and closes, the aforementioned interactions are formed and broken. This lipid-dependent Slo1 gating may explain how amphipathic signaling molecules and pharmacologically active agents influence the channel activity, and a similar mechanism may be operative in other ion channels.
Assuntos
Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Cálcio/química , Cálcio/metabolismo , Linhagem Celular , Humanos , Magnésio/química , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Mutação , Potássio/química , Potássio/metabolismoRESUMO
Mammalian BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.
Assuntos
Ácido Aspártico/química , Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Sítios de Ligação , Citosol/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Modelos Moleculares , Oócitos/citologia , Oócitos/fisiologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Xenopus laevisRESUMO
Voltage-gated ion channels (VGICs) are associated with hundreds of human diseases. To date, 3D structural models of human VGICs have not been reported. We developed a 3D structural integrity metric to rank the accuracy of all VGIC structures deposited in the PDB. The metric revealed inaccuracies in structural models built from recent single-particle, non-crystalline cryo-electron microscopy maps and enabled the building of highly accurate homology models of human Cav channel α1 subunits at atomic resolution. Human Cav Mendelian mutations mostly located to segments involved in the mechanism of voltage sensing and gating within the 3D structure, with multiple mutations targeting equivalent 3D structural locations despite eliciting distinct clinical phenotypes. The models also revealed that the architecture of the ion selectivity filter is highly conserved from bacteria to humans and between sodium and calcium VGICs.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Modelos Moleculares , Mutação , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Domínios ProteicosRESUMO
BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.
Assuntos
Cálcio/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Domínios Proteicos , Sequência de Aminoácidos , Animais , Aplysia/genética , Aplysia/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Mutação , Técnicas de Patch-Clamp , Homologia de Sequência de AminoácidosRESUMO
For those interested in the machinery of ion channel gating, the Ca2+ and voltage-activated BK K+ channel provides a compelling topic for investigation, by virtue of its dual allosteric regulation by both voltage and intracellular Ca2+ and because its large-single channel conductance facilitates detailed kinetic analysis. Over the years, biophysical analyses have illuminated details of the allosteric regulation of BK channels and revealed insights into the mechanism of BK gating, e.g., inner cavity size and accessibility and voltage sensor-pore coupling. Now the publication of two structures of an Aplysia californica BK channel-one liganded and one metal free-promises to reinvigorate functional studies and interpretation of biophysical results. The new structures confirm some of the previous functional inferences but also suggest new perspectives regarding cooperativity between Ca2+-binding sites and the relationship between voltage- and Ca2+-dependent gating. Here we consider the extent to which the two structures explain previous functional data on pore-domain properties, voltage-sensor motions, and divalent cation binding and activation of the channel.
Assuntos
Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Animais , Aplysia/fisiologia , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genéticaRESUMO
Large conductance voltage- and Ca2+-activated K+ (BKCa) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BKCa channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BKCa channels is the ß1-subunit. We have previously described that BKCa channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the ß1-subunit, but not by the ß2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by ß-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BKCa containing three N-terminal sequences with two ß1-ß2 chimeric constructs containing the extracellular loop of ß1 or ß2, and the transmembrane and cytoplasmic domains of ß2 or ß1, respectively. Both ß chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 µM intracellular Ca2+. However, MSSN showed no shift of the voltage-activation, at the same Ca2+ concentrations. The presence of the extracellular loop of ß1 in the chimera resembled results seen with the full ß1 subunit, suggesting that the extracellular region of ß1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BKCa channels either containing or lacking this poly-serine stretch were leftward shifted by ß1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BKCa channel by ß-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Peptídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Ion channels have been extensively reported as effectors of carbon monoxide (CO). However, the mechanisms of heme-independent CO action are still not known. Because most ion channels are heterologously expressed on human embryonic kidney cells that are cultured in Fe3+-containing media, CO may act as a small and strong iron chelator to disrupt a putative iron bridge in ion channels and thus to tune their activity. In this review CFTR and Slo1 BKCa channels are employed to discuss the possible heme-independent interplay between iron and CO. Our recent studies demonstrated a high-affinity Fe3+ site at the interface between the regulatory domain and intracellular loop 3 of CFTR. Because the binding of Fe3+ to CFTR prevents channel opening, the stimulatory effect of CO on the Cl- and HCO3- currents across the apical membrane of rat distal colon may be due to the release of inhibitive Fe3+ by CO. In contrast, CO repeatedly stimulates the human Slo1 BKCa channel opening, possibly by binding to an unknown iron site, because cyanide prohibits this heme-independent CO stimulation. Here, in silico research on recent structural data of the slo1 BKCa channels indicates two putative binuclear Fe2+-binding motifs in the gating ring in which CO may compete with protein residues to bind to either Fe2+ bowl to disrupt the Fe2+ bridge but not to release Fe2+ from the channel. Thus, these two new regulation models of CO, with iron releasing from and retaining in the ion channel, may have significant and extensive implications for other metalloproteins.
Assuntos
Monóxido de Carbono/metabolismo , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Heme/metabolismo , Ativação do Canal Iônico/fisiologia , Ferro/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Monóxido de Carbono/química , Membrana Celular/química , Regulador de Condutância Transmembrana em Fibrose Cística/química , Humanos , Ferro/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Ligação ProteicaRESUMO
Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Nav channel from American cockroach (designated NavPaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSDI, and a carboxy-terminal domain binds to the III-IV linker. The structure of NavPaS establishes an important foundation for understanding function and disease mechanism of Nav and related voltage-gated calcium channels.
Assuntos
Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/ultraestrutura , Animais , Sequência Conservada , Microscopia Crioeletrônica , Glicosilação , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Modelos Químicos , Periplaneta , Domínios ProteicosRESUMO
Large-conductance Ca2+- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming α subunit (Slo) and 4 types of auxiliary ß subunits or just a pore-forming α subunit. Although multiple N-linked glycosylation sites in the extracellular loop of ß subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp recordings, we demonstrated that 3 sites in the extracellular loop of ß2 subunit are N-glycosylated (N-X-T/S at N88, N96 and N119). Glycosylation of these sites strongly and differentially regulate gating kinetics, outward rectification, toxin sensitivity and physical association between the α and ß2 subunits. We constructed a model and used molecular dynamics (MD) to simulate how the glycosylation facilitates the association of α/ß2 subunits and modulates the dimension of the extracellular cavum above the pore of the channel, ultimately to modify biophysical and pharmacological properties of BK channels. Our results suggest that N-glycosylation of ß2 subunits plays crucial roles in imparting functional heterogeneity of BK channels, and is potentially involved in the pathological phenotypes of carbohydrate metabolic diseases.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Sequência de Aminoácidos , Animais , Charibdotoxina/farmacologia , Glicosilação , Células HEK293 , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Camundongos , Modelos Biológicos , Simulação de Dinâmica Molecular , Fenótipo , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery.