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1.
J Gen Physiol ; 156(3)2024 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-38294435

RESUMEN

The ion-conducting IKs channel complex, important in cardiac repolarization and arrhythmias, comprises tetramers of KCNQ1 α-subunits along with 1-4 KCNE1 accessory subunits and calmodulin regulatory molecules. The E160R mutation in individual KCNQ1 subunits was used to prevent activation of voltage sensors and allow direct determination of transition rate data from complexes opening with a fixed number of 1, 2, or 4 activatable voltage sensors. Markov models were used to test the suitability of sequential versus allosteric models of IKs activation by comparing simulations with experimental steady-state and transient activation kinetics, voltage-sensor fluorescence from channels with two or four activatable domains, and limiting slope currents at negative potentials. Sequential Hodgkin-Huxley-type models approximately describe IKs currents but cannot explain an activation delay in channels with only one activatable subunit or the hyperpolarizing shift in the conductance-voltage relationship with more activatable voltage sensors. Incorporating two voltage sensor activation steps in sequential models and a concerted step in opening via rates derived from fluorescence measurements improves models but does not resolve fundamental differences with experimental data. Limiting slope current data that show the opening of channels at negative potentials and very low open probability are better simulated using allosteric models of activation with one transition per voltage sensor, which implies that movement of all four sensors is not required for IKs conductance. Tiered allosteric models with two activating transitions per voltage sensor can fully account for IKs current and fluorescence activation kinetics in constructs with different numbers of activatable voltage sensors.


Asunto(s)
Calmodulina , Canal de Potasio KCNQ1 , Regulación Alostérica , Corazón , Cinética
2.
Elife ; 122023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37707495

RESUMEN

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKs currents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.


Asunto(s)
Calmodulina , Canal de Potasio KCNQ1 , Animales , Canal de Potasio KCNQ1/genética , Calmodulina/genética , Corazón , Frecuencia Cardíaca , Factores Inmunológicos , Mamíferos
3.
J Gen Physiol ; 155(5)2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-36809486

RESUMEN

KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.


Asunto(s)
Canal de Potasio KCNQ1 , Canales de Potasio con Entrada de Voltaje , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Simulación de Dinámica Molecular , Oocitos/metabolismo , Técnicas de Placa-Clamp
4.
Nat Commun ; 13(1): 3760, 2022 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-35768468

RESUMEN

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K+ recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca2+/Calmodulin, with and without the KCNQ1 channel activator, ML277. A single binding site for ML277 was identified, localized to a pocket lined by the S4-S5 linker, S5 and S6 helices of two separate subunits. Several pocket residues are not conserved in other KCNQ isoforms, explaining specificity. MD simulations and point mutations support this binding location for ML277 in open and closed channels and reveal that prevention of inactivation is an important component of the activator effect. Our work provides direction for therapeutic intervention targeting KCNQ1 loss of function pathologies including long QT interval syndrome and seizures.


Asunto(s)
Canal de Potasio KCNQ1 , Síndrome de QT Prolongado , Piperidinas , Tiazoles , Compuestos de Tosilo , Animales , Canal de Potasio KCNQ1/metabolismo , Síndrome de QT Prolongado/tratamiento farmacológico , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Mutación , Piperidinas/farmacología , Tiazoles/farmacología , Compuestos de Tosilo/farmacología , Xenopus
5.
J Physiol ; 600(3): 603-622, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34881429

RESUMEN

G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+ , while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+ , suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+ -selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q-sensitive K+ current and the second a TPN-Q-resistant Li+ current. The results show that a novel Li+ -permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix, S148F and T151A also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations. KEY POINTS: G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important roles in controlling excitation of cells in various organs, such as the brain and the heart. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited mutants of Kir3.2 and Kir3.4. Here we show that a novel Na+ , Li+ -permeable and blocker-resistant pathway exists in an inherited mutant, Kir3.2 G156S, in addition to the conventional ion conducting pathway formed by the selectivity filter (SF). Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations.


Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Proteínas de Unión al GTP , Animales , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Ratones , Mutación , Oocitos/fisiología
6.
J Gen Physiol ; 153(12)2021 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-34636894

RESUMEN

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell-derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.


Asunto(s)
Células Madre Pluripotentes Inducidas , Canales de Potasio con Entrada de Voltaje , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Cinética , Miocitos Cardíacos/metabolismo , Piperidinas , Canales de Potasio con Entrada de Voltaje/metabolismo , Tiazoles , Compuestos de Tosilo
7.
Annu Rev Pharmacol Toxicol ; 61: 381-400, 2021 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-32667860

RESUMEN

Kv7 channels (Kv7.1-7.5) are voltage-gated K+ channels that can be modulated by five ß-subunits (KCNE1-5). Kv7.1-KCNE1 channels produce the slow-delayed rectifying K+ current, IKs, which is important during the repolarization phase of the cardiac action potential. Kv7.2-7.5 are predominantly neuronally expressed and constitute the muscarinic M-current and control the resting membrane potential in neurons. Kv7.1 produces drastically different currents as a result of modulation by KCNE subunits. This flexibility allows the Kv7.1 channel to have many roles depending on location and assembly partners. The pharmacological sensitivity of Kv7.1 channels differs from that of Kv7.2-7.5 and is largely dependent upon the number of ß-subunits present in the channel complex. As a result, the development of pharmaceuticals targeting Kv7.1 is problematic. This review discusses the roles and the mechanisms by which different signaling pathways affect Kv7.1 and KCNE channels and could potentially provide different ways of targeting the channel.


Asunto(s)
Canal de Potasio KCNQ1 , Canales de Potasio con Entrada de Voltaje , Potenciales de Acción , Humanos , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Unión Proteica , Transducción de Señal
8.
Front Physiol ; 11: 504, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32581825

RESUMEN

The IKs channel complex is formed by the co-assembly of Kv7.1 (KCNQ1), a voltage-gated potassium channel, with its ß-subunit, KCNE1 and the association of numerous accessory regulatory molecules such as PIP2, calmodulin, and yotiao. As a result, the IKs potassium current shows kinetic and regulatory flexibility, which not only allows IKs to fulfill physiological roles as disparate as cardiac repolarization and the maintenance of endolymph K+ homeostasis, but also to cause significant disease when it malfunctions. Here, we review new areas of understanding in the assembly, kinetics of activation and inactivation, voltage-sensor pore coupling, unitary events and regulation of this important ion channel complex, all of which have been given further impetus by the recent solution of cryo-EM structural representations of KCNQ1 alone and KCNQ1+KCNE3. Recently, the stoichiometric ratio of KCNE1 to KCNQ1 subunits has been confirmed to be variable up to a ratio of 4:4, rather than fixed at 2:4, and we will review the results and new methodologies that support this conclusion. Significant advances have been made in understanding differences between KCNQ1 and IKs gating using voltage clamp fluorimetry and mutational analysis to illuminate voltage sensor activation and inactivation, and the relationship between voltage sensor translation and pore domain opening. We now understand that the KCNQ1 pore can open with different permeabilities and conductance when the voltage sensor is in partially or fully activated positions, and the ability to make robust single channel recordings from IKs channels has also revealed the complicated pore subconductance architecture during these opening steps, during inactivation, and regulation by 1-4 associated KCNE1 subunits. Experiments placing mutations into individual voltage sensors to drastically change voltage dependence or prevent their movement altogether have demonstrated that the activation of KCNQ1 alone and IKs can best be explained using allosteric models of channel gating. Finally, we discuss how the intrinsic gating properties of KCNQ1 and IKs are highly modulated through the impact of intracellular signaling molecules and co-factors such as PIP2, protein kinase A, calmodulin and ATP, all of which modulate IKs current kinetics and contribute to diverse IKs channel complex function.

9.
Mol Pharmacol ; 97(2): 132-144, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31722973

RESUMEN

The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (I Ks ), which is partly responsible for cardiomyocyte repolarization and physiologic shortening of the cardiac action potential. Mefenamic acid, a nonsteroidal anti-inflammatory drug, has been identified as an I Ks activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid's effect on I Ks Using whole-cell patch clamp, we show that mefenamic acid enhances I Ks activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating I Ks current into an almost linear current with instantaneous onset and slowed tail current decay, sensitive to the I Ks blocker (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide (HMR1556). Both single channels, which reveal no change in the maximum conductance, and whole-cell studies, which reveal a dramatically altered conductance-voltage relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the I Ks channel and shifts channel gating kinetics toward more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose and frequency dependence of mefenamic acid action on I Ks channels. Mutational analysis showed that mefenamic acid's effect on I Ks required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all I Ks channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT: The channels which generate the cardiac slow delayed rectifier K+ current (I Ks ) are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by I Ks in heartbeat regulation, enhancing I Ks current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most I Ks activators, unfortunately, only work on KCNQ1 alone and not the physiologically relevant I Ks channel. We have demonstrated that mefenamic acid can enhance I Ks in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.


Asunto(s)
Antiarrítmicos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ácido Mefenámico/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Potasio/metabolismo , Animales , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/fisiopatología , Relación Dosis-Respuesta a Droga , Fibroblastos , Células HEK293 , Frecuencia Cardíaca/fisiología , Humanos , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Miocardio/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/agonistas , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos
10.
Proc Natl Acad Sci U S A ; 116(16): 7879-7888, 2019 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-30918124

RESUMEN

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance-voltage (G-V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

11.
Biophys J ; 115(9): 1731-1740, 2018 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-30314657

RESUMEN

The delayed potassium rectifier current, IKs, is assembled from tetramers of KCNQ1 and varying numbers of KCNE1 accessory subunits in addition to calmodulin. This channel complex is important in the response of the cardiac action potential to sympathetic stimulation, during which IKs is enhanced. This is likely due to channels opening more quickly, more often, and to greater sublevel amplitudes during adrenergic stimulation. KCNQ1 alone is unresponsive to cyclic adenosine monophosphate (cAMP), and thus KCNE1 is required for a functional effect of protein kinase A phosphorylation. Here, we investigate the effect that KCNE1 has on the response to 8-4-chlorophenylthio (CPT)-cAMP, a membrane-permeable cAMP analog, by varying the number of KCNE1 subunits present using fusion constructs of IKs with either one (EQQQQ) or two (EQQ) KCNE1 subunits in the channel complex with KCNQ1. These experiments use both whole-cell and single-channel recording techniques. EQQ (2:4, E1:Q1) shows a significant shift in V1/2 of activation from 10.4 mV ± 2.2 in control to -2.7 mV ± 1.2 (p-value: 0.0024). EQQQQ (1:4, E1:Q1) shows a smaller change in response to 8-CPT-cAMP, 6.3 mV ± 2.3 to -3.2 mV ± 3.0 (p-value: 0.0435). As the number of KCNE1 subunits is reduced, the shift in the V1/2 of activation becomes smaller. At the single-channel level, a similar graded change in subconductance occupancy and channel activity is seen in response to 8-CPT-cAMP: the less E1, the smaller the response. However, both constructs show a significant reduction of a similar magnitude in the first latency to opening (EQQ control: 0.90 s ± 0.07 to 0.71 s ± 0.06, p-value: 0.0032 and EQQQQ control: 0.94 s ± 0.09 to 0.56 s ± 0.07, p-value < 0.0001). This suggests that there are both E1-dependent and E1-independent effects of 8-CPT-cAMP on the channel.


Asunto(s)
AMP Cíclico/farmacología , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , AMP Cíclico/análogos & derivados , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos
12.
Channels (Austin) ; 12(1): 276-283, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30027808

RESUMEN

The IKs current is important in the heart's response to sympathetic stimulation. ß-adrenergic stimulation increases the amount of IKs and creates a repolarization reserve that shortens the cardiac action potential duration. We have recently shown that 8-CPT-cAMP, a membrane-permeable cAMP analog, changes the channel kinetics and causes it to open more quickly and more often, as well as to higher subconductance levels, which produces an increase in the IKs current. The mechanism proposed to underlie these kinetic changes is increased activation of the voltage sensors. The present study extends our previous work and shows detailed subconductance analysis of the effects of 8-CPT-cAMP on an enhanced gating mutant (S209F) and on a double pseudo-phosphorylated IKs channel (S27D/S92D). 8-CPT-cAMP still produced kinetic changes in S209F + KCNE1, further enhancing gating, while S27D/S92D + KCNE1 showed no significant response to 8-CPT-cAMP, suggesting that these last two mutations fully recapitulate the effect of channel phosphorylation by cAMP.


Asunto(s)
AMP Cíclico/metabolismo , Canales de Potasio/metabolismo , Animales , Células Cultivadas , Cinética , Ratones , Mutación , Fosforilación , Canales de Potasio/genética
13.
Nat Commun ; 8(1): 1730, 2017 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-29167462

RESUMEN

In voltage-activated ion channels, voltage sensor (VSD) activation induces pore opening via VSD-pore coupling. Previous studies show that the pore in KCNQ1 channels opens when the VSD activates to both intermediate and fully activated states, resulting in the intermediate open (IO) and activated open (AO) states, respectively. It is also well known that accompanying KCNQ1 channel opening, the ionic current is suppressed by a rapid process called inactivation. Here we show that inactivation of KCNQ1 channels derives from the different mechanisms of the VSD-pore coupling that lead to the IO and AO states, respectively. When the VSD activates from the intermediate state to the activated state, the VSD-pore coupling has less efficacy in opening the pore, producing inactivation. These results indicate that different mechanisms, other than the canonical VSD-pore coupling, are at work in voltage-dependent ion channel activation.


Asunto(s)
Canal de Potasio KCNQ1/antagonistas & inhibidores , Canal de Potasio KCNQ1/metabolismo , Sustitución de Aminoácidos , Animales , Femenino , Humanos , Activación del Canal Iónico , Canal de Potasio KCNQ1/genética , Cinética , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Potenciales de la Membrana , Ratones , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
14.
Biophys J ; 113(2): 415-425, 2017 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-28746852

RESUMEN

The slow delayed rectifier potassium current (IKs) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 α-subunits and up to four KCNE1 ß-subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p-Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.


Asunto(s)
Canales de Potasio con Entrada de Voltaje/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Aminoácidos/efectos de la radiación , Animales , Benzofenonas/química , Benzofenonas/efectos de la radiación , Línea Celular , Humanos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Mutación , Técnicas de Placa-Clamp , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/efectos de la radiación , Procesos Fotoquímicos , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/efectos de la radiación , Conformación Proteica/efectos de la radiación , Dominios Proteicos , Rayos Ultravioleta
15.
J Gen Physiol ; 149(8): 781-798, 2017 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-28687606

RESUMEN

The delayed potassium rectifier current, IKs , is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During ß-adrenergic stimulation, 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance-voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.


Asunto(s)
AMP Cíclico/metabolismo , Activación del Canal Iónico , Canal de Potasio KCNQ1/metabolismo , Sustitución de Aminoácidos , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/genética , Ratones
16.
Mol Pharmacol ; 90(2): 80-95, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27193582

RESUMEN

The increasing prevalence of influenza viruses with resistance to approved antivirals highlights the need for new anti-influenza therapeutics. Here we describe the functional properties of hexamethylene amiloride (HMA)-derived compounds that inhibit the wild-type and adamantane-resistant forms of the influenza A M2 ion channel. For example, 6-(azepan-1-yl)-N-carbamimidoylnicotinamide ( 9: ) inhibits amantadine-sensitive M2 currents with 3- to 6-fold greater potency than amantadine or HMA (IC50 = 0.2 vs. 0.6 and 1.3 µM, respectively). Compound 9: competes with amantadine for M2 inhibition, and molecular docking simulations suggest that 9: binds at site(s) that overlap with amantadine binding. In addition, tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 27: ) acts both on adamantane-sensitive and a resistant M2 variant encoding a serine to asparagine 31 mutation (S31N) with improved efficacy over amantadine and HMA (IC50 = 0.6 µM and 4.4 µM, respectively). Whereas 9: inhibited in vitro replication of influenza virus encoding wild-type M2 (EC50 = 2.3 µM), both 27: and tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 26: ) preferentially inhibited viruses encoding M2(S31N) (respective EC50 = 18.0 and 1.5 µM). This finding indicates that HMA derivatives can be designed to inhibit viruses with resistance to amantadine. Our study highlights the potential of HMA derivatives as inhibitors of drug-resistant influenza M2 ion channels.


Asunto(s)
Amilorida/análogos & derivados , Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/metabolismo , Proteínas de la Matriz Viral/antagonistas & inhibidores , Amantadina/farmacología , Amilorida/síntesis química , Amilorida/química , Amilorida/farmacología , Animales , Antivirales/química , Muerte Celular/efectos de los fármacos , Línea Celular , Guanidinas/farmacología , Humanos , Concentración de Iones de Hidrógeno , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Simulación del Acoplamiento Molecular , Técnicas de Placa-Clamp , Proteínas de la Matriz Viral/metabolismo
17.
J Physiol ; 594(17): 4901-15, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27062501

RESUMEN

KEY POINTS: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP-activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited Kv 1.5 channels in pulmonary arterial myocytes. AMPK activation by 5-aminoimidazole-4-carboxamide riboside, A769662 or C13 attenuated Kv 1.5 currents in pulmonary arterial myocytes, and this effect was non-additive with respect to Kv 1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human Kv 1.5 channels in cell-free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing Kv 1.5. These results suggest that AMPK is the primary mediator of reductions in Kv 1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP-activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1-dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte Kv currents were also markedly inhibited upon AMPK activation by A769662, 5-aminoimidazole-4-carboxamide riboside and C13 and by intracellular dialysis from a patch-pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2ß2γ1 or α1ß1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK-sensitive K(+) currents, which were also blocked by the selective Kv 1.5 channel inhibitor diphenyl phosphine oxide-1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv 1.5 channels were phosphorylated by AMPK in cell-free assays, and K(+) currents carried by Kv 1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates Kv channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of Kv 1.5 and/or an associated protein.


Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Hipoxia/fisiopatología , Canal de Potasio Kv1.5/fisiología , Mitocondrias/metabolismo , Células Musculares/fisiología , Animales , Células HEK293 , Humanos , Masculino , Fosforilación Oxidativa , Arteria Pulmonar/citología , Ratas Sprague-Dawley
18.
Elife ; 52016 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-26802629

RESUMEN

Cardiac repolarization is determined in part by the slow delayed rectifier current (IKs), through the tetrameric voltage-gated ion channel, KCNQ1, and its ß-subunit, KCNE1. The stoichiometry between α and ß-subunits has been controversial with studies reporting either a strict 2 KCNE1:4 KCNQ1 or a variable ratio up to 4:4. We used IKs fusion proteins linking KCNE1 to one (EQ), two (EQQ) or four (EQQQQ) KCNQ1 subunits, to reproduce compulsory 4:4, 2:4 or 1:4 stoichiometries. Whole cell and single-channel recordings showed EQQ and EQQQQ to have increasingly hyperpolarized activation, reduced conductance, and shorter first latency of opening compared to EQ - all abolished by the addition of KCNE1. As well, using a UV-crosslinking unnatural amino acid in KCNE1, we found EQQQQ and EQQ crosslinking rates to be progressively slowed compared to KCNQ1, which demonstrates that no intrinsic mechanism limits the association of up to four ß-subunits within the IKs complex.


Asunto(s)
Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Subunidades de Proteína/análisis , Humanos
19.
Heart Rhythm ; 12(2): 386-94, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25444851

RESUMEN

BACKGROUND: The slowly activating delayed rectifier current IKs participates in cardiac repolarization, particularly at high heart rates, and mutations in this K(+) channel complex underlie long QT syndrome (LQTS) types 1 and 5. OBJECTIVE: The purpose of this study was to determine biophysical mechanisms of LQT1 through single-channel kinetic analysis of IKs carrying LQT1 mutations in the S3 transmembrane region of the pore-forming subunit KCNQ1. METHODS: We analyzed cell-attached recordings from mammalian cells in which a single active KCNQ1 (wild type or mutant) and KCNE1 complex could be detected. RESULTS: The S3 mutants of KCNQ1 studied (D202H, I204F, V205M, and S209F), with the exception of S209F, all led to a reduction in channel activity through distinct kinetic mechanisms. D202H, I204F, and V205M showed decreased open probability (Po) compared with wild type (0.07, 0.04, and 0.12 vs 0.2); increased first latency from 1.66 to >2 seconds at +60 mV (I204F, V205M); variable-to-severe reductions in open dwell times (≥50% in V205M); stabilization of closed states (D202H); and an inability of channels to reach full conductance levels (V205M, I204F). S209F is a kinetic gain-of-function mutation with a high Po (0.40) and long open-state dwell times. CONCLUSION: S3 mutations in KCNQ1 cause diverse kinetic defects in I(Ks), affecting opening and closing properties, and can account for LQT1 phenotypes.


Asunto(s)
ADN/genética , Canal de Potasio KCNQ1/genética , Mutación , Miocardio/patología , Síndrome de Romano-Ward/genética , Animales , Células Cultivadas , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Canal de Potasio KCNQ1/metabolismo , Ratones Transgénicos , Miocardio/metabolismo , Fenotipo , Síndrome de Romano-Ward/metabolismo , Síndrome de Romano-Ward/patología
20.
Proc Natl Acad Sci U S A ; 110(11): E996-1005, 2013 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-23431135

RESUMEN

Coassembly of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) with potassium voltage-gated channel, Isk-related family, member 1 (KCNE1) the delayed rectifier potassium channel I(Ks). Its slow activation is critically important for membrane repolarization and for abbreviating the cardiac action potential, especially during sympathetic activation and at high heart rates. Mutations in either gene can cause long QT syndrome, which can lead to fatal arrhythmias. To understand better the elementary behavior of this slowly activating channel complex, we quantitatively analyzed direct measurements of single-channel I(Ks). Single-channel recordings from transiently transfected mouse ltk(-) cells confirm a channel that has long latency periods to opening (1.67 ± 0.073 s at +60 mV) but that flickers rapidly between multiple open and closed states in non-deactivating bursts at positive membrane potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall open probability of only ∼0.15 after 4 s under our recording conditions. The mean single-channel conductance was determined to be 3.2 pS, but unlike any other known wild-type human potassium channel, long-lived subconductance levels coupled to activation are a key feature of both the activation and deactivation time courses of the conducting channel complex. Up to five conducting levels ranging from 0.13 to 0.66 pA could be identified in single-channel recordings at 60 mV. Fast closings and overt subconductance behavior of the wild-type I(Ks) channel required modification of existing Markov models to include these features of channel behavior.


Asunto(s)
Canal de Potasio KCNQ1/metabolismo , Potenciales de la Membrana/fisiología , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Potasio/metabolismo , Animales , Línea Celular , Humanos , Canal de Potasio KCNQ1/genética , Ratones , Proteínas Musculares/genética
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