RESUMO
Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca2+-activated K+ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K+ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF's four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K+ affinity (â¼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K+, similar to KcsA, but that even a single K+ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K+ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK's S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.
Assuntos
Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Cristalografia por Raios X , Canais de Potássio Ativados por Cálcio de Condutância Alta/isolamento & purificação , Canais de Potássio Ativados por Cálcio de Condutância Alta/ultraestrutura , Methanobacterium , Simulação de Dinâmica Molecular , Potássio/metabolismoRESUMO
Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).
Assuntos
Transferência de Energia , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Medições Luminescentes , Movimento , Oócitos/metabolismo , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Superfamília Shaker de Canais de Potássio , Xenopus laevisRESUMO
The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the proton-binding sites and at neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.
Assuntos
Técnicas Biossensoriais , Proteínas de Escherichia coli/química , Modelos Moleculares , Canais de Potássio/química , Proteínas de Bactérias , Ácido Glutâmico/química , Histidina/química , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Quantitative investigations into functional properties of purified ion channel proteins using standard electrophysiological methods are challenging, in particular for the determination of average ion channel behavior following rapid changes in experimental conditions (e.g., ligand concentration). Here, we describe a method for determining the functional activity of liposome-reconstituted K+ channels using a stopped-flow fluorometric ion flux assay. Channel activity is quantified by measuring the rate of fluorescence decrease of a liposome-encapsulated fluorophore, specifically quenched by thallium ions entering the liposomes via open channels. This method is well suited for studying the lipid bilayer dependence of channel activity, the activation and desensitization kinetics of ligand-dependent K+ channels, and channel modulation by channel agonists, blockers, or other antagonists.
Assuntos
Gorduras/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais de Potássio/metabolismo , Animais , Fluorometria , Humanos , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismoRESUMO
Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Neuropathic pain arises from injury to the nervous system. Conditions associated with neuropathic pain are diverse, and lesions and/or pathological changes in the central nervous system (CNS) or peripheral nervous system (PNS) can frequently, but not always, be identified. It is difficult to treat, with patients often on multiple, different classes of medications, all with appreciable adverse side effect profiles. Consequently, there is a pressing need for the development of new medications. The development of such therapeutics is predicated on a clear understanding of the relevant molecular and cellular processes that contribute to the development, and maintenance, of the neuropathic pain state. One proposed mechanism thought to contribute to the ontogeny of neuropathic pain is altered expression, trafficking, and functioning of ion channels expressed by primary sensory neurons. Here, we will focus on three voltage-gated ion channel families, CaV, HCN, and NaV, first reviewing the preclinical data and then the human data where it exists.
Assuntos
Canais Iônicos/antagonistas & inibidores , Neuralgia/tratamento farmacológico , Sistema Nervoso Periférico/fisiologia , Animais , Canais de Cálcio Tipo N/fisiologia , Canais de Cálcio Tipo T/fisiologia , Compostos Heterocíclicos com 2 Anéis/uso terapêutico , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Canais Iônicos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.8/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.9/fisiologia , Neuralgia/fisiopatologia , Sulfonamidas/uso terapêuticoRESUMO
The process of ion channel gating-opening and closing-involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating.
Assuntos
Proteínas de Bactérias/metabolismo , Lipídeos , Canais de Potássio/metabolismo , Cristalografia por Raios X , Ativação do Canal Iônico , Conformação ProteicaRESUMO
Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K(+) channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca(2+)-activated K(+) channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K(+). Thus, Ca(2+)-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.
Assuntos
Cálcio/metabolismo , Methanobacterium/metabolismo , Canais de Potássio/metabolismo , Cinética , Compostos de Amônio QuaternárioRESUMO
Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. Blockers bind in an aqueous cavity between two putative gates: an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location--an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.
Assuntos
Ativação do Canal Iônico/fisiologia , Methanobacterium/metabolismo , Modelos Moleculares , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Conformação Proteica , Cristalografia por Raios X , Cinética , Compostos de Amônio QuaternárioRESUMO
The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel's response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod-Wyman-Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a "strong" sensor displaying a large shift in pKa between closed and open states, and E118 is a "weak" pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pKa values of the pH-sensing residues was sufficient to capture the effects of all mutations.
Assuntos
Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Prótons , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dados de Sequência Molecular , Mutação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genéticaRESUMO
Voltage-driven activation of Kv channels results from conformational changes of four voltage sensor domains (VSDs) that surround the K(+) selective pore domain. How the VSD helices rearrange during gating is an area of active research. Luminescence resonance energy transfer (LRET) is a powerful spectroscopic ruler uniquely suitable for addressing the conformational trajectory of these helices. Using a geometric analysis of numerous LRET measurements, we were able to estimate LRET probe positions relative to existing structural models. The experimental movement of helix S4 does not support a large 15-20 A transmembrane "paddle-type" movement or a near-zero A vertical "transporter-type" model. Rather, our measurements demonstrate a moderate S4 displacement of 10 +/- 5 A, with a vertical component of 5 +/- 2 A. The S3 segment moves 2 +/- 1 A in the opposite direction and is therefore not moving as an S3-S4 rigid body.