RESUMO
Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.
Assuntos
Acetilcolina/farmacologia , Agonistas do Receptor A1 de Adenosina/farmacologia , Adenosina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canais de Potássio/metabolismo , Receptor A1 de Adenosina/metabolismo , Animais , Feminino , Cobaias , Masculino , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismoRESUMO
Riluzole is an anticonvulsant drug also used to treat the amyotrophic lateral sclerosis and major depressive disorder. This compound has antiglutamatergic activity and is an important multichannel blocker. However, little is known about its actions on the Kv4.2 channels, the molecular correlate of the A-type K+ current (IA) and the fast transient outward current (Itof). Here, we investigated the effects of riluzole on Kv4.2 channels transiently expressed in HEK-293 cells. Riluzole inhibited Kv4.2 channels with an IC50 of 190 ± 14 µM and the effect was voltage- and frequency-independent. The activation rate of the current (at +50 mV) was not affected by the drug, nor the voltage dependence of channel activation, but the inactivation rate was accelerated by 100 and 300 µM riluzole. When Kv4.2 channels were maintained at the closed state, riluzole incubation induced a tonic current inhibition. In addition, riluzole significantly shifted the voltage dependence of inactivation to hyperpolarized potentials without affecting the recovery from inactivation. In the presence of the drug, the closed-state inactivation was significantly accelerated, and the percentage of inactivated channels was increased. Altogether, our findings indicate that riluzole inhibits Kv4.2 channels mainly affecting the closed and closed-inactivated states.
Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Riluzol/farmacologia , Canais de Potássio Shal/antagonistas & inibidores , Células HEK293 , Humanos , Ativação do Canal Iônico , Potenciais da Membrana , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Fatores de TempoRESUMO
The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.
Assuntos
Potenciais de Ação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Miócitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animais , Gatos , Células Cultivadas , Feminino , Átrios do Coração/citologia , Magnésio/metabolismo , Masculino , Miócitos Cardíacos/fisiologia , Poliaminas/metabolismoRESUMO
Amitriptyline (AMIT) is a compound widely prescribed for psychiatric and non-psychiatric conditions including depression, migraine, chronic pain, and anorexia. However, AMIT has been associated with risks of cardiac arrhythmia and sudden death since it can induce prolongation of the QT interval on the surface electrocardiogram and torsade de pointes ventricular arrhythmia. These complications have been attributed to the inhibition of the rapid delayed rectifier potassium current (IKr). The slow delayed rectifier potassium current (IKs) is the main repolarizing cardiac current when IKr is compromised and it has an important role in cardiac repolarization at fast heart rates induced by an elevated sympathetic tone. Therefore, we sought to characterize the effects of AMIT on Kv7.1/KCNE1 and homomeric Kv7.1 channels expressed in HEK-293H cells. Homomeric Kv7.1 and Kv7.1/KCNE1 channels were inhibited by AMIT in a concentration-dependent manner with IC50 values of 8.8⯱â¯2.1⯵M and 2.5⯱â¯0.8⯵M, respectively. This effect was voltage-independent for both homomeric Kv7.1 and Kv7.1/KCNE1 channels. Moreover, mutation of residues located on the P-loop and S6 domain along with molecular docking, suggest that T312, I337 and F340 are the most important molecular determinants for AMIT-Kv7.1 channel interaction. Our experimental findings and modeling suggest that AMIT preferentially blocks the open state of Kv7.1/KCNE1 channels by interacting with specific residues that were previously reported to be important for binding of other compounds, such as chromanol 293B and the benzodiazepine L7.
Assuntos
Amitriptilina/farmacologia , Canal de Potássio KCNQ1/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Potenciais de Ação , Amitriptilina/química , Antidepressivos Tricíclicos/química , Antidepressivos Tricíclicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Canal de Potássio KCNQ1/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Conformação ProteicaRESUMO
Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.
Assuntos
Pentamidina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Sítios de Ligação , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Pentamidina/metabolismo , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Conformação ProteicaRESUMO
Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3µM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Quinacrina/farmacologia , Animais , Astrócitos/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp/métodos , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Quinacrina/metabolismo , RatosRESUMO
Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5µM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7µM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.
Assuntos
Cloroquina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/química , Sítios de Ligação , Cloroquina/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Simulação de Acoplamento Molecular , Porosidade , Bloqueadores dos Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Conformação ProteicaRESUMO
Recently, it has been shown that G protein-coupled receptors (GPCRs) display intrinsic voltage sensitivity. We reported that the voltage sensitivity of M2 muscarinic receptor (M2R) is also ligand specific. Here, we provide additional evidence to understand the mechanism underlying the ligand-specific voltage sensitivity of the M2R. Using ACh, pilocarpine (Pilo), and bethanechol (Beth), we evaluated the agonist-specific effects of voltage by measuring the ACh-activated K(+) current (I KACh) in feline and rabbit atrial myocytes and in HEK-293 cells expressing M2R-Kir3.1/Kir3.4. The activation of I KACh by the muscarinic agonist Beth was voltage insensitive, suggesting that the voltage-induced conformational changes in M2R do not modify its affinity for this agonist. Moreover, deactivation of the Beth-evoked I KACh was voltage insensitive. By contrast, deactivation of the ACh-induced I KACh was significantly slower at -100 mV than at +50 mV, while an opposite effect was observed when I KACh was activated by Pilo. These findings are consistent with the voltage affinity pattern observed for these three agonists. Our findings suggest that independent of how voltage disturbs the receptor binding site, the voltage dependence of the signaling pathway is ultimately determined by the agonist. These observations emphasize the pharmacological potential to regulate the M2R-parasympathetic associated cardiac function and also other cellular signaling pathways by exploiting the voltage-dependent properties of GPCRs.
Assuntos
Acetilcolina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Receptor Muscarínico M2/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Gatos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Coelhos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
INTRODUCTION: Voltage- and state-dependent blocks are important mechanisms by which drugs affect voltage-gated ionic channels. However, spontaneous (i.e. drug-free) time-dependent changes in the activation and inactivation of hERG and Na(+) channels have been reported when using conventional whole-cell patch-clamp in HEK-293 cells. METHODS: hERG channels were heterologously expressed in HEK-293 cells and in Xenopus laevis oocytes. hERG current (IhERG) was recorded using both conventional and perforated whole-cell patch-clamp (HEK-293 cells), and two microelectrode voltage-clamp (Xenopus oocytes) in drug-free solution, and in the presence of the drug trazodone. RESULTS: In conventional whole-cell setup, we observed a spontaneous time-dependent hyperpolarizing shift in the activation curve of IhERG. Conversely, in perforated patch whole-cell (HEK-293 cells) or in two microelectrode voltage-clamp (Xenopus oocytes) activation curves of IhERG were very stable for periods ~50min. Voltage-dependent inactivation of IhERG was not significantly altered in the three voltage clamp configurations tested. When comparing voltage- and state-dependent effects of the antidepressant drug trazodone on IhERG, similar changes between the three voltage clamp configurations were observed as under drug-free conditions. DISCUSSION: The comparative analysis performed in this work showed that only under conventional whole-cell voltage-clamp conditions, a leftward shift in the activation curve of IhERG occurred, both in the presence and absence of drugs. These spontaneous time-dependent changes in the voltage activation gate of IhERG are a potential confounder in pharmacological studies on hERG channels expressed in HEK-293 cells.
Assuntos
Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Trazodona/farmacologia , Animais , Antidepressivos de Segunda Geração , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Oócitos , Fatores de Tempo , Xenopus laevisRESUMO
The human intestinal pathogen Giardia lamblia is a flagellated unicellular protozoan with pronounced medical and biological relevance. However, the basic physiology of Giardia trophozoites has been sparsely studied, especially the electrical and ionic properties of their cellular membrane which are virtually unknown. In this study, we were able to record and characterize the macroscopic ionic currents of Giardia trophozoite membrane by electrophysiological methods of the patch clamp technique. Giardia trophozoites showed a high current density (â¼600 pA/pF at -140 mV) that was activated upon hyperpolarization. This current was carried by a chloride-selective channel (I Cl-G) and it was the most important determinant of the membrane potential in Giardia trophozoites. Moreover, this conductance was able to carry other halide anions and the sequence of permeability was Br(-) > Cl(-) ≈ I(-) â« F(-). Besides the voltage-dependent inward-rectifying nature of I Cl-G, its activation and deactivation kinetics were comparable to those observed in ClC-2 channels. Extracellular pH modified the voltage-dependent properties of I Cl-G, shifting the activation curve from a V 1/2 = -79 ± 1 mV (pH 7.4) to -93 ± 2 mV (pH 8.4) and -112 ± 2 mV (pH 5.4). Furthermore, the maximal amplitude of I Cl-G measured at -100 mV showed dependence to external pH in a bell-shaped fashion reported only for ClC-2 channels. Therefore, our results suggest that I Cl-G possesses several functional properties similar to the mammalian ClC-2 channels.
Assuntos
Potenciais de Ação , Canais de Cloreto/metabolismo , Giardia lamblia/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Canais de Cloro CLC-2 , Cloretos/metabolismo , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/fisiologia , Potenciais da Membrana , Trofozoítos/fisiologiaRESUMO
Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 â¼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.
Assuntos
Miócitos Cardíacos/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Células HEK293 , Humanos , Quinacrina/farmacologiaRESUMO
We characterized the properties of the voltage-dependent K(+) currents I (to), I (Kr), and I (Ks) in isolated feline sino-atrial node (SAN) myocytes. I (to) activated rapidly and then inactivated with a single exponential and voltage-independent time course. Recovery from inactivation of I (to) followed a single exponential time course with τ = 21.1 ± 2.5 ms, at -80 mV. Steady-state inactivation relationship showed a V½ of inactivation at -47.9 ± 2.3 mV. These biophysical properties are similar to the fast I (to) phenotype of other mammals. I (Kr) exhibited typical negative slope conductance at test potentials > 0 mV and slow deactivation. I (Ks) activated very slowly. The functional contribution of I (to), I (Kr), and I (Ks) to the sustained pacemaking activity of feline SAN myocytes was analyzed. Similar to other mammals, I (to) underlies the initial repolarization phase of the SAN action potential, whereas I (Kr) and I (Ks) mediate repolarization back to the maximal diastolic potential. I (Kr) and I (Ks) also contribute to diastolic depolarization because of their slow deactivation kinetics. The I (Kr) specific blocker E-4031 and the I (Ks) blocker HMR 1556 significantly increased action potential duration, but had negligible effects on the maximum diastolic potential and only modest effects on the frequency of spontaneous activity, suggesting that each one of these two currents itself is capable of supporting action potential repolarization in the feline sinus node.
Assuntos
Células Musculares/metabolismo , Miocárdio/citologia , Canais de Potássio/fisiologia , Nó Sinoatrial/citologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Gatos , Células Cultivadas , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Técnicas de Patch-Clamp , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Nó Sinoatrial/fisiologiaRESUMO
Carvedilol, a ß- and α-adrenoceptor blocker, is used to treat congestive heart failure, mild to moderate hypertension, and myocardial infarction. It has been proposed to block K(ATP) channels by binding to the bundle crossing region at a domain including cysteine at position 166, and thereby plugging the pore region. However, carvedilol was reported not to affect Kir2.1 channels, which lack 166 Cys. Here, we demonstrate that carvedilol inhibits Kir2.3 carried current by an alternative mechanism. Carvedilol inhibited Kir2.3 channels with at least 100 fold higher potency (IC(50)=0.49 µM) compared to that for Kir2.1 (IC(50)>50 µM). Kir2.3 channel inhibition was concentration-dependent and voltage-independent. Increasing Kir2.3 channel affinity for PIP(2), by a I213L point mutation, decreased the inhibitory effect of carvedilol more than twentyfold (IC(50)=11.1 µM). In the presence of exogenous PIP(2), Kir2.3 channel inhibition by carvedilol was strongly reduced (80 vs. 2% current inhibition). These results suggest that carvedilol, as other cationic amphiphilic drugs, inhibits Kir2.3 channels by interfering with the PIP(2)-channel interaction.
Assuntos
Carbazóis/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Propanolaminas/farmacologia , Carvedilol , Células HEK293 , Humanos , Mutação Puntual , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ligação Proteica/efeitos dos fármacosRESUMO
Pilocarpine is a nonspecific agonist of muscarinic receptors which was recently found to activate the M(2) receptor subtype in a voltage-dependent manner. The purpose of our study was to investigate the role of the acetylcholine (muscarinic)-activated K(+) current (I (KACh)) on the negative chronotropic effect of pilocarpine in rabbit sinoatrial node. In multicellular preparations, we studied the effect of pilocarpine on spontaneous action potentials. In isolated myocytes, using the patch clamp technique, we studied the effects of pilocarpine on I (KACh). Pilocarpine produced a decrease in spontaneous frequency, hyperpolarization of the maximum diastolic potential, and a decrease in the diastolic depolarization rate. These effects were partially antagonized by tertiapin Q. Cesium and calyculin A in the presence of tertiapin Q partially prevented the effects of pilocarpine. In isolated myocytes, pilocarpine activated the muscarinic potassium current, I (KACh) in a voltage-dependent manner. In conclusion, the negative chronotropic effects of pilocarpine on the sinatrial node could be mainly explained by activation of I (KACh).
Assuntos
Acetilcolina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Pilocarpina/farmacologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Nó Sinoatrial/efeitos dos fármacos , Animais , Venenos de Abelha/farmacologia , Césio/metabolismo , Inibidores Enzimáticos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Toxinas Marinhas , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oxazóis/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Coelhos , Nó Sinoatrial/fisiologiaRESUMO
The endocannabinoid, N-arachidonoylethanolamine (anandamide; AEA) is known to interact with voltage-gated K(+) (Kv) channels in a cannabinoid receptor-independent manner. AEA modulates the functional properties of Kv channels, converting channels with slowly inactivating current into apparent fast inactivation. In this study, we characterize the mechanism of action and binding site for AEA on Kv1.5 channels expressed on HEK-293 cells using the patch-clamp techniques. AEA exhibited high-potency block (IC(50) approximately 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial beta-branching amino acids, Val505 and Ile508 within the S6 domain. Both residues face toward the central cavity and constitute a motif that forms a hydrophobic ring around the ion conduction pathway. This hydrophobic ring motif may be a critical determinant of cannabinoid receptor-independent AEA modulation in other K(+) channel families.
Assuntos
Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Canal de Potássio Kv1.5/genética , Alcamidas Poli-Insaturadas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Clonagem Molecular , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Endocanabinoides , Humanos , Rim , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/fisiologia , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K(+) current I(K1) and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I(K1). The voltage- and K(+)-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I(K1) even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.
Assuntos
Cloroquina/metabolismo , Cloroquina/farmacologia , Bloqueadores dos Canais de Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Antimaláricos/síntese química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Sítios de Ligação/genética , Linhagem Celular , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/síntese química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Propriedades de Superfície , TransfecçãoRESUMO
Many drugs block delayed rectifier K+ channels and prolong the cardiac action potential duration. Here we investigate the molecular mechanisms of voltage-dependent block of human ether-a-go-go-related gene (HERG) K+ channels expressed in cells HEK-293 and Xenopus oocytes by maprotiline. The IC50 determined at 0 mV on HERG expressed HEK-293 cell and oocytes was 5.2 and 23.7 microM, respectively. Block of HERG expressed in oocytes by maprotiline was enhanced by progressive membrane depolarization and accompanied by a negative shift in the voltage dependence of channel activation. The potency of maprotiline was reduced 7-fold by point mutation of a key aromatic residue (F656T) and 3-fold for Y652A, both located in the S6 domain. The mutation Y652A inverted the voltage dependence of HERG channel block by maprotiline. Together, these results suggest that voltage-dependent block of HERG results from gating dependent changes in the accessibility of Y652, a critical component of the drug binding site.
Assuntos
Antidepressivos de Segunda Geração/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Maprotilina/farmacologia , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Canal de Potássio ERG1 , Estimulação Elétrica , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/fisiologia , Feminino , Expressão Gênica , Humanos , Potenciais da Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Mutação Puntual , XenopusRESUMO
Berberine prolongs the duration of cardiac action potentials without affecting resting membrane potential or action potential amplitude. Controversy exists regarding whether berberine exerts this action by preferential block of different components of the delayed rectifying potassium current, I(Kr) and I(Ks). Here we have studied the effects of berberine on hERG (I(Kr)) and KCNQ1/KCNE1 (I(Ks)) channels expressed in HEK-293 cells and Xenopus oocytes. In HEK-293 cells, the IC50 for berberine was 3.1 +/- 0.5 microM on hERG compared with 11 +/- 4% decreases on KCNQ1/KCNE1 channels by 100 microM berberine. Likewise in oocytes, hERG channels were more sensitive to block by berberine (IC50 = 80 +/- 5 microM) compared with KCNQ1/KCNE1 channels (approximately 20% block at 300 microM). hERG block was markedly increased by membrane depolarization. Mutation to Ala of Y652 or F656 located on the S6 domain, or V625 located at the base of the pore helix of hERG decreased sensitivity to block by berberine. An inactivation-deficient mutant hERG channel (G628C/S631C) was also blocked by berberine. Together these findings indicate that berberine preferentially blocks the open state of hERG channels by interacting with specific residues that were previously reported to be important for binding of more potent antagonists.
Assuntos
Berberina/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Canal de Potássio KCNQ1/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , XenopusRESUMO
Halofantrine is an antimalarial drug developed as a treatment of P. falciparum resistant to chloroquine. However, halofantrine can also induce long QT syndrome (LQTS) and torsades de pointes, a potentially life-threatening ventricular arrhythmia. Drug-induced LQTS is usually caused by block of the human ether-a-go-go-related gene (HERG) channels that conduct the rapid delayed rectifier K(+) current, I(Kr), in the heart. Here we show that halofantrine preferentially blocks open and inactivated HERG channels heterologously expressed in Xenopus laevis oocytes. The half-maximal inhibitory concentration (IC(50)) for block of wild-type (WT) HERG was 1.0 microM. As we reported previously for other HERG channel blockers, the potency of halofantrine was reduced by mutation to Ala of aromatic residues (Y652, F656) located in the S6 domain, or a Val (V625) located in the pore helix. Halofantrine at a concentration 10 microM did not affect the transient outward potassium channel, Kv4.3, the slow delayed rectifier potassium channel, KvLQT1+minK and inward rectifier potassium channel, Kir2.1. An inactivation deficient mutant (G628C/S631C HERG) was only slightly less sensitive (IC(50)=2.0 microM). The rate of block onset by halofantrine at 0 mV was used to estimate the apparent association (k(on)) and dissociation (k(off)) rate constants for drug binding. For WT and G628C/S631C HERG, k(on) was similar (0.0114 and 0.0163 M(-1)/s(-1) respectively). In contrast, k(off) was significantly faster for G628C/S631C (0.357 s(-1)) than WT (0.155 s(-1)), and explains the observed decrease in drug potency for the inactivation-deficient mutant channel. We conclude that halofantrine requires channels to open before it can gain access to its binding site located in the central cavity of the HERG channel.
Assuntos
Fenantrenos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Xenopus laevisRESUMO
BACKGROUND: Organophosphorus, DDT, and pyrethroid insecticides are potent neurotoxic compounds. In addition, serious cardiovascular toxic manifestations such as ventricular arrhythmias have been reported. The objective of the present work was to study possible cardiac electrophysiologic effects of the type II pyrethroid, deltamethrin. METHODS: The effects of deltamethrin were studied in enzymatically isolated cat ventricular myocytes. Whole cell, patch-clamp technique was used in current- and voltage-clamp modes. RESULTS: Deltamethrin significantly increased action potential duration. Under voltage-clamp conditions, the most striking effects of deltamethrin were on sodium current. Pyrethroid induced a sustained component of the sodium current and caused a negative shift in current-voltage relation for peak current. The drug also slightly inhibited delayed rectifying outward potassium current. CONCLUSIONS: Pyrethroid type II, deltamethrin, increased action potential duration due to modulation of sodium current. This effect of deltamethrin can be potentially arrhythmogenic because it can induce Q-T prolongation of ECG and ventricular arrhythmias of the torsade-de-pointes type.