RESUMEN
Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/fisiología , Potasio/metabolismo , Electrofisiología , Humanos , Cinética , Simulación de Dinámica Molecular , Canales de Potasio de Dominio Poro en Tándem/genéticaRESUMEN
Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l, which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn2+-sensitive H+ fluxes in PMCs that regulate intracellular pH in a Na+/HCO3--independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H+ flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH.
Asunto(s)
Biomineralización , Calcificación Fisiológica/fisiología , Homeostasis , Canales Iónicos/metabolismo , Larva/fisiología , Protones , Erizos de Mar/fisiología , Animales , Transporte Biológico , Concentración de Iones de Hidrógeno , Canales Iónicos/genética , Análisis de la Célula Individual , TranscriptomaRESUMEN
Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Humanos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Células Endoteliales/metabolismo , Enfermedades Neuroinflamatorias , Encéfalo/metabolismo , Moléculas de Adhesión Celular/metabolismoRESUMEN
Two-pore domain K+ (K2P) channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via an SF-mediated gating mechanism, but we found here that only very strong nonphysiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mm). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Cationes Monovalentes/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico , Simulación de Dinámica Molecular , Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Conformación Proteica , Conformación Proteica en Hélice alfa , Rubidio/metabolismo , XenopusRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.
Asunto(s)
Corazón/fisiología , Activación del Canal Iónico , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Marcapaso Artificial , Animales , Proteínas Portadoras/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Células HeLa , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ratones , Ratones Noqueados , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte Vesicular , Xenopus laevis , Pez CebraRESUMEN
Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Sitios de Unión , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mutación , Porosidad , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/genética , Conformación Proteica , Compuestos de Amonio Cuaternario/farmacología , RatasRESUMEN
Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.
Asunto(s)
Canal de Potasio Kv1.5/antagonistas & inhibidores , Canal de Potasio Kv1.5/química , Ficusina/química , Ficusina/farmacología , Canal de Potasio Kv1.5/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The time course of inactivation of voltage-activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvbeta subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce 'open-channel block'. Open-channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid-induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio con Entrada de Voltaje/metabolismo , Edición de ARN , Tetraetilamonio/farmacología , Animales , Ácido Araquidónico/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Mutación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Unión Proteica , Ratas , Xenopus laevisRESUMEN
The two-pore domain potassium (K2P) channels TREK-1 and TREK-2 link neuronal excitability to a variety of stimuli including mechanical force, lipids, temperature and phosphorylation. This regulation involves the C-terminus as a polymodal stimulus sensor and the selectivity filter (SF) as channel gate. Using crystallographic up- and down-state structures of TREK-2 as a template for full atomistic molecular dynamics (MD) simulations, we reveal that the SF in down-state undergoes inactivation via conformational changes, while the up-state structure maintains a stable and conductive SF. This suggests an atomistic mechanism for the low channel activity previously assigned to the down state, but not evident from the crystal structure. Furthermore, experimentally by using (de-)phosphorylation mimics and chemically attaching lipid tethers to the proximal C-terminus (pCt), we confirm the hypothesis that moving the pCt towards the membrane induces the up-state. Based on MD simulations, we propose two gating pathways by which movement of the pCt controls the stability (i.e., conductivity) of the filter gate. Together, these findings provide atomistic insights into the SF gating mechanism and the physiological regulation of TREK channels by phosphorylation.
Asunto(s)
Activación del Canal Iónico , Simulación de Dinámica Molecular , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/genética , Humanos , Fosforilación , Dominios Proteicos , Citosol/metabolismo , Animales , Células HEK293 , Cristalografía por Rayos XRESUMEN
Two-pore domain K+ (K2P) channel activity was previously thought to be controlled primarily via a selectivity filter (SF) gate. However, recent crystal structures of TASK-1 and TASK-2 revealed a lower gate at the cytoplasmic pore entrance. Here, we report functional evidence of such a lower gate in the K2P channel K2P17.1 (TALK-2, TASK-4). We identified compounds (drugs and lipids) and mutations that opened the lower gate allowing the fast modification of pore cysteine residues. Surprisingly, stimuli that directly target the SF gate (i.e., pHe., Rb+ permeation, membrane depolarization) also opened the cytoplasmic gate. Reciprocally, opening of the lower gate reduced the electric work to open the SF via voltage driven ion binding. Therefore, it appears that the SF is so rigidly locked into the TALK-2 protein structure that changes in ion occupancy can pry open a distant lower gate and, vice versa, opening of the lower gate concurrently promote SF gate opening. This concept might extent to other K+ channels that contain two gates (e.g., voltage-gated K+ channels) for which such a positive gate coupling has been suggested, but so far not directly demonstrated.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/genética , Humanos , Citoplasma/metabolismo , Animales , Mutación , Células HEK293 , Xenopus laevis , Iones/metabolismoRESUMEN
Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Anticuerpos de Dominio Único , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Humanos , Cristalografía por Rayos X , Animales , Microscopía por Crioelectrón , Células HEK293 , Modelos MolecularesRESUMEN
The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP2 content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a "ligand-gated" K+ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K+], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Animales , Línea Celular , Electrofisiología , Activación del Canal Iónico/genética , Canales Iónicos/genética , Canales Iónicos/metabolismo , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Mutación , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/fisiologíaRESUMEN
Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.
Asunto(s)
Anomalías Múltiples/genética , Mutación Missense , Canales de Potasio de Rectificación Interna/genética , Animales , Ataxia , Línea Celular , Epilepsia , Pérdida Auditiva Sensorineural , Humanos , Enfermedades Renales , Túbulos Renales Distales/patología , Ratones , Ratones Endogámicos C57BL , Canales de Potasio de Rectificación Interna/análisis , Síndrome , TransfecciónRESUMEN
Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.
Asunto(s)
Benzamidas/farmacología , Bencenoacetamidas/farmacología , Proteínas del Tejido Nervioso/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/química , Potasio/química , Alanina/química , Animales , Benzamidas/química , Bencenoacetamidas/química , Sitios de Unión , ADN Complementario/metabolismo , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Mutagénesis , Mutagénesis Sitio-Dirigida , Oocitos/citología , Técnicas de Placa-Clamp , Conformación Proteica , Xenopus laevisRESUMEN
Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.
Asunto(s)
Potenciales de Acción/fisiología , Neuronas Colinérgicas/fisiología , Transducción de Señal/fisiología , Sueño/fisiología , Tálamo/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Neuronas Colinérgicas/efectos de los fármacos , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Expresión Génica/genética , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Guanosina Trifosfato/antagonistas & inhibidores , Guanosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Núcleos Talámicos Laterales/citología , Núcleos Talámicos Laterales/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Proteínas del Tejido Nervioso , Oxotremorina/análogos & derivados , Oxotremorina/farmacología , Técnicas de Placa-Clamp , Fosfolipasa C beta/antagonistas & inhibidores , Fosfolipasa C beta/genética , Fosfolipasa C beta/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Ratas , Ratas Long-Evans , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Transducción de Señal/efectos de los fármacos , Tálamo/citología , Tionucleótidos/farmacologíaRESUMEN
Work over the past three decades has greatly advanced our understanding of the regulation of Kir K+ channels by polyanionic lipids of the phosphoinositide (e.g., PIP2) and fatty acid metabolism (e.g., oleoyl-CoA). However, comparatively little is known regarding the regulation of the K2P channel family by phosphoinositides and by long-chain fatty acid-CoA esters, such as oleoyl-CoA. We screened 12 mammalian K2P channels and report effects of polyanionic lipids on all tested channels. We observed activation of members of the TREK, TALK, and THIK subfamilies, with the strongest activation by PIP2 for TRAAK and the strongest activation by oleoyl-CoA for TALK-2. By contrast, we observed inhibition for members of the TASK and TRESK subfamilies. Our results reveal that TASK-2 channels have both activatory and inhibitory PIP2 sites with different affinities. Finally, we provided evidence that PIP2 inhibition of TASK-1 and TASK-3 channels is mediated by closure of the recently identified lower X-gate as critical mutations within the gate (i.e., L244A, R245A) prevent PIP2-induced inhibition. Our findings establish that K+ channels of the K2P family are highly sensitive to polyanionic lipids, extending our knowledge of the mechanisms of lipid regulation and implicating the metabolism of these lipids as possible effector pathways to regulate K2P channel activity.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Animales , Ácidos Grasos , Metabolismo de los Lípidos , Fosfatidilinositoles , Canales de Potasio de Dominio Poro en Tándem/metabolismoRESUMEN
Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the 'X-gate', a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies.
Asunto(s)
Mutación con Ganancia de Función , Síndromes de la Apnea del Sueño , Niño , Discapacidades del Desarrollo , Humanos , Mutación/genética , Proteínas del Tejido Nervioso , Canales de Potasio de Dominio Poro en Tándem , Síndromes de la Apnea del Sueño/genéticaRESUMEN
Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.
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Secuencias Hélice-Asa-Hélice/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/fisiología , Alanina/genética , Alanina/metabolismo , Animales , Estimulación Eléctrica/métodos , Femenino , Secuencias Hélice-Asa-Hélice/genética , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Lisina/genética , Lisina/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Microinyecciones/métodos , Modelos Biológicos , Modelos Moleculares , Mutagénesis/fisiología , Oocitos , Técnicas de Placa-Clamp/métodos , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacología , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Conformación Proteica , Ratas , XenopusRESUMEN
BACKGROUND: 5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5HD + CoA + ATP --> 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K(ATP) (sarcK(ATP)) channel activity. METHODS: Single sarcK(ATP) channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK(ATP) channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A. RESULTS: 5-HD inhibited (IC(50) approximately 30 microM) K(ATP) channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 microM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 microM coenzyme A (CoA) inhibited sarcK(ATP) channels. Using giant inside-out patches, which are weakly sensitive to "contaminating" CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK(ATP) channel activation by either metabolic inhibition or rilmakalim. GENERAL SIGNIFICANCE: SarcK(ATP) channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at "directly" inhibiting sarcK(ATP) and mitoK(ATP) channels. However, in intact cells, 5-HD fails to inhibit sarcK(ATP) channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.
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Coenzima A/farmacología , Ácidos Decanoicos/farmacología , Hidroxiácidos/farmacología , Canales KATP/fisiología , Sarcolema/fisiología , Acetato CoA Ligasa/metabolismo , Acetato CoA Ligasa/farmacología , Animales , Ventrículos Cardíacos/efectos de los fármacos , Canales KATP/antagonistas & inhibidores , Canales KATP/efectos de los fármacos , Ratones , Células Musculares/efectos de los fármacos , Células Musculares/fisiología , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Canales de Potasio de Rectificación Interna/fisiología , RatasRESUMEN
The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.