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1.
Elife ; 122024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38465747

RESUMO

Voltage-gated sodium channels (Naáµ¥) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naáµ¥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naáµ¥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naáµ¥1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naáµ¥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naáµ¥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naáµ¥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naáµ¥-related diseases.


Assuntos
Ativação do Canal Iônico , Canais de Sódio Disparados por Voltagem , Ativação do Canal Iônico/fisiologia , Domínios Proteicos , Canais Iônicos , Sítios de Ligação
2.
Protein Sci ; 33(4): e4965, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501596

RESUMO

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.


Assuntos
Proteínas de Escherichia coli , Canais Iônicos , Canais Iônicos/genética , Canais Iônicos/química , Canais Iônicos/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas de Escherichia coli/química , Constrição
3.
Proc Natl Acad Sci U S A ; 121(9): e2315132121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38377199

RESUMO

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Ligantes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , AMP Cíclico/metabolismo , Fenômenos Biofísicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo
4.
Nat Commun ; 15(1): 1803, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413583

RESUMO

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit ß1-ß2 and M2-M3 loops, with agonist binding re-positioning a key ß1-ß2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9' hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.


Assuntos
Receptores Nicotínicos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Ativação do Canal Iônico/fisiologia , Conformação Molecular , Receptores Colinérgicos/metabolismo , Músculos/metabolismo
5.
Int J Mol Sci ; 25(3)2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38338830

RESUMO

This review paper delves into the current body of evidence, offering a thorough analysis of the impact of large-conductance Ca2+-activated K+ (BKCa or BK) channels on the electrical dynamics of the heart. Alterations in the activity of BKCa channels, responsible for the generation of the overall magnitude of Ca2+-activated K+ current at the whole-cell level, occur through allosteric mechanisms. The collaborative interplay between membrane depolarization and heightened intracellular Ca2+ ion concentrations collectively contribute to the activation of BKCa channels. Although fully developed mammalian cardiac cells do not exhibit functional expression of these ion channels, evidence suggests their presence in cardiac fibroblasts that surround and potentially establish close connections with neighboring cardiac cells. When cardiac cells form close associations with fibroblasts, the high single-ion conductance of these channels, approximately ranging from 150 to 250 pS, can result in the random depolarization of the adjacent cardiac cell membranes. While cardiac fibroblasts are typically electrically non-excitable, their prevalence within heart tissue increases, particularly in the context of aging myocardial infarction or atrial fibrillation. This augmented presence of BKCa channels' conductance holds the potential to amplify the excitability of cardiac cell membranes through effective electrical coupling between fibroblasts and cardiomyocytes. In this scenario, this heightened excitability may contribute to the onset of cardiac arrhythmias. Moreover, it is worth noting that the substances influencing the activity of these BKCa channels might influence cardiac electrical activity as well. Taken together, the BKCa channel activity residing in cardiac fibroblasts may contribute to cardiac electrical function occurring in vivo.


Assuntos
Fibroblastos , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Células Cultivadas , Ativação do Canal Iônico , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Cálcio/metabolismo , Mamíferos/metabolismo
6.
BMC Biol ; 22(1): 29, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38317233

RESUMO

BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.


Assuntos
Canal de Potássio ERG1 , Ativação do Canal Iônico , Humanos , Sequência de Aminoácidos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/genética , Mutação , Nucleotídeos Cíclicos , Canal de Potássio ERG1/genética
7.
J Physiol ; 602(2): 333-354, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38186087

RESUMO

Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o- bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl- channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po ) of wild-type CFTR by ≥75% at ATP (1 mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100 nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5-1 µM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. KEY POINTS: Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF). This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR-targeted therapy ivacaftor. Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP-dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease. Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild-type levels, but concurrently reduced current flow through open channels, particularly those of S1159F-CFTR. Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR-targeted therapies to fully restore channel function.


Assuntos
Fibrose Cística , Quinolonas , Cricetinae , Animais , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células CHO , Cricetulus , Aminoácidos , Ativação do Canal Iônico , Aminofenóis/farmacologia , Trifosfato de Adenosina/metabolismo
8.
Nat Commun ; 15(1): 843, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38287019

RESUMO

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Conformação Proteica em alfa-Hélice , Nucleotídeos Cíclicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo
9.
Annu Rev Physiol ; 86: 357-377, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-37931166

RESUMO

A handful of biological proton-selective ion channels exist. Some open at positive or negative membrane potentials, others open at low or high pH, and some are light activated. This review focuses on common features that result from the unique properties of protons. Proton conduction through water or proteins differs qualitatively from that of all other ions. Extraordinary proton selectivity is needed to ensure that protons permeate and other ions do not. Proton selectivity arises from a proton pathway comprising a hydrogen-bonded chain that typically includes at least one titratable amino acid side chain. The enormously diverse functions of proton channels in disparate regions of the phylogenetic tree can be summarized by considering the chemical and electrical consequences of proton flux across membranes. This review discusses examples of cells in which proton efflux serves to increase pHi, decrease pHo, control the membrane potential, generate action potentials, or compensate transmembrane movement of electrical charge.


Assuntos
Ativação do Canal Iônico , Prótons , Humanos , Ativação do Canal Iônico/fisiologia , Concentração de Íons de Hidrogênio , Filogenia , Canais Iônicos/metabolismo
10.
J Chem Inf Model ; 64(2): 555-562, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38159289

RESUMO

In this work, we propose a methodology based on Monte Carlo Markov chains to explore the parameter space of kinetic models for ion channels. The methodology allows the detection of potential parameter sets of a model that are compatible with experimentally obtained whole-cell currents, which could remain hidden when methods focus on obtaining the parameters that provide the best fit. To show its implementation and utility, we considered a four-state kinetic model proposed in the literature to describe the activation of the voltage-gated proton channel (Hv1), Biophysical Journal, 2014, 107, 1564. In that work, a set of values for the rate transitions that describe the channel kinetics at different intracellular H+ concentration (pHi) were obtained by the Simplex method. With our approach, we find that, in fact, there is more than one parameter set for each pHi, which renders the same open probability temporal course within the experimental error margin for all of the considered voltages. The large differences that we obtained for the values of some rate constants among the different solutions show that there is more than one possible interpretation of this channel behavior as a function of pHi. We also simulated a proposed new experimental condition where it is possible to observe that different sets of parameters yield different results. Our study highlights the importance of a comprehensive analysis of parameter space in kinetic models and the utility of the proposed methodology for finding potential solutions.


Assuntos
Ativação do Canal Iônico , Canais Iônicos , Ativação do Canal Iônico/fisiologia , Cadeias de Markov , Canais Iônicos/metabolismo , Concentração de Íons de Hidrogênio , Prótons , Cinética , Modelos Biológicos
11.
Sci Adv ; 9(49): eadj5539, 2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-38064553

RESUMO

Eukaryotic voltage-gated K+ channels have been extensively studied, but the structural bases for some of their most salient functional features remain to be established. C-type inactivation, for example, is an auto-inhibitory mechanism that confers temporal resolution to their signal-firing activity. In a recent breakthrough, studies of a mutant of Shaker that is prone to inactivate indicated that this process entails a dilation of the selectivity filter, the narrowest part of the ion conduction pathway. Here, we report an atomic-resolution cryo-electron microscopy structure that demonstrates that the wild-type channel can also adopt this dilated state. All-atom simulations corroborate this conformation is congruent with the electrophysiological characteristics of the C-type inactivated state, namely, residual K+ conductance and altered ion specificity, and help rationalize why inactivation is accelerated or impeded by certain mutations. In summary, this study establishes the molecular basis for an important self-regulatory mechanism in eukaryotic K+ channels, laying a solid foundation for further studies.


Assuntos
Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Microscopia Crioeletrônica , Dilatação , Ativação do Canal Iônico/fisiologia
12.
Int J Mol Sci ; 24(23)2023 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-38068977

RESUMO

Ion channels exhibit gating behavior, fluctuating between open and closed states, with the transmembrane voltage serving as one of the essential regulators of this process. Voltage gating is a fundamental functional aspect underlying the regulation of ion-selective, mostly α-helical, channels primarily found in excitable cell membranes. In contrast, there exists another group of larger, and less selective, ß-barrel channels of a different origin, which are not directly associated with cell excitability. Remarkably, these channels can also undergo closing, or "gating", induced by sufficiently strong electric fields. Once the field is removed, the channels reopen, preserving a memory of the gating process. In this study, we explored the hypothesis that the voltage-induced closure of the ß-barrel channels can be seen as a form of reversible protein denaturation by the high electric fields applied in model membranes experiments-typically exceeding twenty million volts per meter-rather than a manifestation of functional gating. Here, we focused on the bacterial outer membrane channel OmpF reconstituted into planar lipid bilayers and analyzed various characteristics of the closing-opening process that support this idea. Specifically, we considered the nearly symmetric response to voltages of both polarities, the presence of multiple closed states, the stabilization of the open conformation in channel clusters, the long-term gating memory, and the Hofmeister effects in closing kinetics. Furthermore, we contemplate the evolutionary aspect of the phenomenon, proposing that the field-induced denaturation of membrane proteins might have served as a starting point for their development into amazing molecular machines such as voltage-gated channels of nerve and muscle cells.


Assuntos
Ativação do Canal Iônico , Bicamadas Lipídicas , Ativação do Canal Iônico/fisiologia , Bicamadas Lipídicas/metabolismo , Canais Iônicos/metabolismo , Membrana Celular/metabolismo , Eletricidade
13.
Nat Commun ; 14(1): 7835, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38114487

RESUMO

Understanding voltage-gated sodium (Nav) channels is significant since they generate action potential. Nav channels consist of a pore domain (PD) and a voltage sensor domain (VSD). All resolved Nav structures in different gating states have VSDs that tightly interact with PDs; however, it is unclear whether VSDs attach to PDs during gating under physiological conditions. Here, we reconstituted three different voltage-dependent NavAb, which is cloned from Arcobacter butzleri, into a lipid membrane and observed their structural dynamics by high-speed atomic force microscopy on a sub-second timescale in the steady state. Surprisingly, VSDs dissociated from PDs in the mutant in the resting state and further dimerized to form cross-links between channels. This dimerization would occur at a realistic channel density, offering a potential explanation for the facilitation of positive cooperativity of channel activity in the rising phase of the action potential.


Assuntos
Ativação do Canal Iônico , Sódio , Potenciais de Ação , Membranas
14.
J Mol Evol ; 91(6): 819-836, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37955698

RESUMO

The voltage-sensing domain (VSD) is a module capable of responding to changes in the membrane potential through conformational changes and facilitating electromechanical coupling to open a pore gate, activate proton permeation pathways, or promote enzymatic activity in some membrane-anchored phosphatases. To carry out these functions, this module acts cooperatively through conformational changes. The VSD is formed by four transmembrane segments (S1-S4) but the S4 segment is critical since it carries positively charged residues, mainly Arg or Lys, which require an aqueous environment for its proper function. The discovery of this module in voltage-gated ion channels (VGICs), proton channels (Hv1), and voltage sensor-containing phosphatases (VSPs) has expanded our understanding of the principle of modularity in the voltage-sensing mechanism of these proteins. Here, by sequence comparison and the evaluation of the relationship between sequence composition, intrinsic flexibility, and structural analysis in 14 selected representatives of these three major protein groups, we report five interesting differences in the folding patterns of the VSD both in prokaryotes and eukaryotes. Our main findings indicate that this module is highly conserved throughout the evolutionary scale, however: (1) segments S1 to S3 in eukaryotes are significantly more hydrophobic than those present in prokaryotes; (2) the S4 segment has retained its hydrophilic character; (3) in eukaryotes the extramembranous linkers are significantly larger and more flexible in comparison with those present in prokaryotes; (4) the sensors present in the kHv1 proton channel and the ciVSP phosphatase, both of eukaryotic origin, exhibit relationships of flexibility and folding patterns very close to the typical ones found in prokaryotic voltage sensors; and (5) archaeal channels KvAP and MVP have flexibility profiles which are clearly contrasting in the S3-S4 region, which could explain their divergent activation mechanisms. Finally, to elucidate the obscure origins of this module, we show further evidence for a possible connection between voltage sensors and TolQ proteins.


Assuntos
Ativação do Canal Iônico , Prótons , Ativação do Canal Iônico/fisiologia , Monoéster Fosfórico Hidrolases/genética
15.
EMBO J ; 42(24): e115030, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37984335

RESUMO

Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca2+ -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.


Assuntos
Canais de Cloreto , Ativação do Canal Iônico , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Anoctamina-1/genética , Anoctamina-1/química , Anoctamina-1/metabolismo , Ligantes , Microscopia Crioeletrônica , Sítios de Ligação , Cálcio/metabolismo
16.
Mol Cell ; 83(24): 4555-4569.e4, 2023 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-38035882

RESUMO

Modulation of large conductance intracellular ligand-activated potassium (BK) channel family (Slo1-3) by auxiliary subunits allows diverse physiological functions in excitable and non-excitable cells. Cryoelectron microscopy (cryo-EM) structures of voltage-gated potassium (Kv) channel complexes have provided insights into how voltage sensitivity is modulated by auxiliary subunits. However, the modulation mechanisms of BK channels, particularly as ligand-activated ion channels, remain unknown. Slo1 is a Ca2+-activated and voltage-gated BK channel and is expressed in neurons, muscle cells, and epithelial cells. Using cryo-EM and electrophysiology, we show that the LRRC26-γ1 subunit modulates not only voltage but also Ca2+ sensitivity of Homo sapiens Slo1. LRRC26 stabilizes the active conformation of voltage-senor domains of Slo1 by an extracellularly S4-locking mechanism. Furthermore, it also stabilizes the active conformation of Ca2+-sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes.


Assuntos
Cálcio , Canais de Potássio Ativados por Cálcio de Condutância Alta , Humanos , Cálcio/metabolismo , Microscopia Crioeletrônica , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Ligantes , Potássio , Regulação Alostérica
17.
Int J Mol Sci ; 24(19)2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37833868

RESUMO

Ion channels play an important role in the cellular functions of various organ systems, such as the nervous, cardiovascular, immune, and endocrine systems, and are potential therapeutic targets for treatments of their dysfunctions, via 'channelopathy' [...].


Assuntos
Canalopatias , Canais Iônicos , Humanos , Canais Iônicos/metabolismo , Ativação do Canal Iônico/fisiologia , Transdução de Sinais , Coração
18.
Commun Biol ; 6(1): 1094, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37891195

RESUMO

Transmembrane ion channels frequently regulate ion permeation by forming bundle crossing of the pore-lining helices when deactivated. The resulting physical constriction is believed to serve as the de facto gate that imposes the major free energy barrier to ion permeation. Intriguingly, many ion channels also contain highly hydrophobic inner pores enclosed by bundle crossing, which can undergo spontaneous dewetting and give rise to a "vapor barrier" to block ion flow even in the absence of physical constriction. Using atomistic simulations, we show that hydrophobic gating and bundle-crossing mechanisms co-exist and complement one and another in the human TRPV4 channel. In particular, a single hydrophilic mutation in the lower pore can increase pore hydration and reduce the ion permeation free energy barrier by about half without affecting the bundle crossing. We believe that hydrophobic gating may play a key role in other bundle-crossing ion channels with hydrophobic inner pores.


Assuntos
Ativação do Canal Iônico , Canais de Cátion TRPV , Humanos , Ativação do Canal Iônico/fisiologia , Canais de Cátion TRPV/genética , Mutação , Estrutura Secundária de Proteína , Interações Hidrofóbicas e Hidrofílicas
19.
Nat Commun ; 14(1): 6595, 2023 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-37852983

RESUMO

Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Lipídeos de Membrana , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo
20.
J Biol Phys ; 49(4): 393-413, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37851173

RESUMO

The membrane potential of a cell (Vm) regulates several physiological processes. The voltage sensor domain (VSD) is a region that confers voltage sensitivity to different types of transmembrane proteins such as the following: voltage-gated ion channels, the voltage-sensing phosphatase (Ci-VSP), and the sperm-specific Na+/H+ exchanger (sNHE). VSDs contain four transmembrane segments (S1-S4) and several positively charged amino acids in S4, which are essential for the voltage sensitivity of the protein. Generally, in response to changes of the Vm, the positive residues of S4 displace along the plasma membrane without generating ionic currents through this domain. However, some native (e.g., Hv1 channel) and mutants of VSDs produce ionic currents. These gating pore currents are usually observed in VSDs that lack one or more of the conserved positively charged amino acids in S4. The gating pore currents can also be induced by the isolation of a VSD from the rest of the protein domains. In this review, we summarize gating pore currents from all families of proteins with VSDs with classification into three cases: (1) pathological, (2) physiological, and (3) artificial currents. We reinforce the model in which the position of S4 that lacks the positively charged amino acid determines the voltage dependency of the gating pore current of all VSDs independent of protein families.


Assuntos
Ativação do Canal Iônico , Sêmen , Masculino , Humanos , Ativação do Canal Iônico/fisiologia , Domínios Proteicos , Potenciais da Membrana , Aminoácidos
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