Person:
Gómez García, Ricardo

First Name

Ricardo

Last Name

Gómez García

URI

https://hdl.handle.net/20.500.14352/86357

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Medicina

Department

Farmacología y Toxicología

Area

Farmacología

Identifiers

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Now showing 1 - 10 of 26

Structural basis of drugs that increase cardiac inward rectifier Kir2.1 currents
(Cardiovascular Research, 2014) Gómez García, Ricardo; Caballero Collado, Ricardo; Barana Muñoz, Adriana; Amorós García, Irene; de Palm, Sue Haida; Matamoros, Marcos; Núñez, Mercedes; Pérez Hernández, Marta; Iriepa, Isabel; Tamargo Menéndez, Juan; Delpón Mosquera, María Eva
Aims: We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. Methods and results: Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. Conclusions: Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now.
Interaction of angiotensin II with the angiotensin type 2 receptor inhibits the cardiac transient outward potassium current
(Cardiovascular Research, 2004) Caballero Collado, Ricardo; Gómez García, Ricardo; Moreno, Fernández, Ignacio; Núñez Fernández, Lucía; González, Teresa; Arias, Cristina; Guizy, Miriam; Valenzuela, Carmen; Tamargo Menéndez, Juan; Delpón Mosquera, María Eva
Objective: The Ca2+ -independent transient outward K+ current (Ito) plays a crucial role in shaping the cardiac action potential. In the present study, we examined whether angiotensin II (AngII) regulated the Ito as well as the putative intracellular cascade responsible for the effects. Methods: Ito was recorded in rat ventricular myocytes using the nystatin-perforated patch-clamp configuration. Results: AngII (0.1 microM) inhibited Ito (21.9+/-4.8% at +40 mV), but not the IK1, in a voltage- and time-independent manner. The inhibition decreased at concentrations higher than 1 microM resulting in a bell-shaped dose-response curve (IC50 = 3.1+/-1.5 microM). The blocking effects were abolished in the presence of the type 2 AngII receptor (AT2R) antagonist PD123319, but not in the presence of the selective type 1 AngII receptor (AT1R) antagonist candesartan. Moreover, the selective AT2R agonist CGP42112A completely reproduced the effects of AngII (20.5+/-2.4% of block at +40 mV), indicating that AngII-induced Ito block was mediated via stimulation of AT2R. Furthermore, selective stimulation of AT2R by CGP42112A significantly prolonged the rat atrial action potentials recorded using conventional microelectrode techniques. The AngII-induced inhibition of I(to) was not modified by either Npi-nitro-L-arginine-methyl ester (L-NAME) or eicosatetrayonic acid (ETYA), indicating that neither the nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate (cGMP) system nor the arachidonic acid cascade was implicated in the effects of AngII on Ito. However, the AngII-induced Ito inhibition was completely abolished by the serine/threonine phosphatase type 2A (PP2A) inhibitors, okadaic acid and cantharidin, but not by the inactive analog of okadaic acid, 1-norokadaone. Intracellular application of PP2A decreased Kv4.2 currents recorded in transiently transfected Chinese hamster ovary cells (CHO). Conclusion: These results indicate that AngII activates PP2A through the stimulation of the AT2R, resulting in a decrease of the Ito amplitude.
In humans, chronic atrial fibrillation decreases the transient outward current and ultrarapid component of the delayed rectifier current differentially on each atria and increases the slow component of the delayed rectifier current in both
(Journal of the American College of Cardiology, 2010) Caballero Collado, Ricardo; González de la Fuente, Marta; Gómez García, Ricardo; Barana Muñoz, Adriana; Amorós García, Irene; Dolz Gaitón, Pablo; Osuna, Lourdes; Almendral Garrote, Jesús; Atienza Fernández, Felipe; Fernández-Avilés Díaz, Francisco Jesús; Pita, Ana; Rodríguez Roda, Jorge; Pinto, Ángel; Tamargo Menéndez, Juan; Delpón Mosquera, María Eva
Objectives: The purpose of this study was to compare the voltage-dependent K(+) currents of human cells of the right and left atria and determine whether electrical remodeling produced by chronic atrial fibrillation (CAF) is chamber-specific. Background: Several data point to the existence of interatrial differences in the repolarizing currents. Therefore, it could be possible that CAF-induced electrical remodeling differentially affects voltage-dependent K(+) currents in each atrium. Methods: Currents were recorded using the whole-cell patch-clamp in myocytes from left (LAA) and right atrial appendages (RAA) obtained from sinus rhythm (SR) and CAF patients. Results: In SR, LAA and RAA myocytes were divided in 3 types, according to their main voltage-dependent repolarizing K(+) current. CAF differentially modified the proportion of these 3 types of cells on each atrium. CAF reduced the Ca(2+)-independent 4-aminopyridine-sensitive component of the transient outward current (I(to1)) more markedly in the LAA than in the RAA. Therefore, an atrial right-to-left I(to1) gradient was created by CAF. In contrast, the ultrarapid component of the delayed rectifier current (I(Kur)) was more markedly reduced in the RAA than in the LAA, thus abolishing the atrial right-to-left I(Kur) gradient observed in SR. Importantly, in both atria, CAF increased the slow component of the delayed rectifier current (I(Ks)). Conclusions: Our results demonstrated that in SR there are intra-atrial heterogeneities in the repolarizing currents. CAF decreases I(to1) and I(Kur) differentially in each atrium and increases I(Ks) in both atria, an effect that further promotes re-entry.
Propafenone blocks human cardiac Kir2.x channels by decreasing the negative electrostatic charge in the cytoplasmic pore
(Biochemical Pharmacology, 2013) Amorós García, Irene; Dolz Gaitón, Pablo; Gómez García, Ricardo; Matamoros, Marcos; Barana Muñoz, Adriana; González de la Fuente, Marta; Núñez, Mercedes; Pérez Hernández, Marta; Moraleda, Ignacio; Gálvez Ruano, Enrique; Iriepa, Isabel; Tamargo Menéndez, Juan; Caballero Collado, Ricardo; Delpón Mosquera, María Eva
Human cardiac inward rectifier current (IK1) is generated by Kir2.x channels. Inhibition of IK1 could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (IKir2.x) and the IK1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1μM propafenone inhibited IKir2.x the order of potency being Kir2.3∼IK1>Kir2.2>Kir2.1 channels. Blockade was irrespective of the extracellular K(+) concentration whereas markedly increased when the intracellular K(+) concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K(+) equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K(+) ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels.
Functional effects of a missense mutation in HERG associated with type 2 long QT syndrome
(2011) Amorós García, Irene; Jiménez Jáimez, Juan; Tercedor, Luis; Barana Muñóz, Adriana; Gómez García, Ricardo; González de la Fuente, Marta; Dolz Gaitón, Pablo; Álvarez, Miguel; Martínez Espín, Esther; Lorente, José A.; Melgares, Rafael; Tamargo Menéndez, Juan; Delpón Mosquera, María Eva; Caballero Collado, Ricardo
Background: Long QT syndrome (LQTS) is characterized by a prolonged QT interval that can lead to severe ventricular arrhythmias (torsades de pointes) and sudden death. Congenital LQTS type 2 (LQT2) is due to loss-of-function mutations in the KCNH2 gene encoding Kv11.1 channels responsible for the rapid component of the delayed rectifier current. Objective: The purpose of this study was to determine the functional properties of the LQT2-associated mutation p.E637G found in a Spanish family. Methods: Wild-type (WT) and p.E637G Kv11.1 channels were transiently transfected in Chinese hamster ovary cells, and currents were recorded using the patch-clamp technique. Results: The p.E637G channels lost inward rectification and K(+) selectivity, generating small but measurable slowly activating, noninactivating currents. These important alterations were corrected neither by cotransfection with WT channels nor by incubation at low temperatures or with pharmacological chaperones. As a consequence of its effects on channel gating, the mutation significantly reduced the outward repolarizing current during the action potential (AP), resulting in a marked lengthening of the duration of a simulated human ventricular AP. Conclusion: We have identified and characterized an LQT2-associated mutation that through removal of C-type inactivation and reduction of K(+) selectivity causes the QT prolongation observed in the patients carrying the mutation. Moreover, the results obtained demonstrate the importance of the glutamic acid at position 637 for the inactivation process and K(+) selectivity of Kv11.1 channels.
Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification
(Proceedings of the National Academy of Sciences (PNAS), 2010) Caballero Collado, Ricardo; Dolz Gaitón, Pablo; Gómez García, Ricardo; Amorós García, Irene; Barana Muñoz, Adriana; González de la Fuente, Marta; Osuna, Lourdes; Duarte, Juan; López Izquierdo, Angélica; Moraleda, Ignacio; Gálvez Ruano, Enrique; Sánchez Chapula, José Antonio; Tamargo Menéndez, Juan; Delpón Mosquera, María Eva
Both increase and decrease of cardiac inward rectifier current (I(K1)) are associated with severe cardiac arrhythmias. Flecainide, a widely used antiarrhythmic drug, exhibits ventricular proarrhythmic effects while effectively controlling ventricular arrhythmias associated with mutations in the gene encoding Kir2.1 channels that decrease I(K1) (Andersen syndrome). Here we characterize the electrophysiological and molecular basis of the flecainide-induced increase of the current generated by Kir2.1 channels (I(Kir2.1)) and I(K1) recorded in ventricular myocytes. Flecainide increases outward I(Kir2.1) generated by homotetrameric Kir2.1 channels by decreasing their affinity for intracellular polyamines, which reduces the inward rectification of the current. Flecainide interacts with the HI loop of the cytoplasmic domain of the channel, Cys311 being critical for the effect. This explains why flecainide does not increase I(Kir2.2) and I(Kir2.3), because Kir2.2 and Kir2.3 channels do not exhibit a Cys residue at the equivalent position. We further show that incubation with flecainide increases expression of functional Kir2.1 channels in the membrane, an effect also determined by Cys311. Indeed, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in Andersen syndrome patients. Moreover, our findings provide noteworthy clues about the structural determinants of the C terminus cytoplasmic domain of Kir2.1 channels involved in the control of gating and rectification.
A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family.
(PNAS, 2023) Cámara Checa, Anabel; Perin, Francesca; Rubio Alarcón, Marcos; Dago Requena, María; Rapún, Josu; Cebrián Castillo, Jorge; Gómez García, Ricardo; Tamargo Menéndez, Juan; Caballero Collado, Ricardo; Delpón Mosquera, María Eva
In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.
Nitric oxide inhibits Kv4.3 and human cardiac transient outward potassium current (Ito1)
(Cardiovascular Research, 2008) Gómez García, Ricardo; Macaya Miguel, Carlos; Caballero Collado, Ricardo; López Farre, Antonio José; Tamargo Menéndez, Juan; Delpón Mosquera, María Eva
Aims: Chronic atrial fibrillation (CAF) is characterized by a shortening of the plateau phase of the action potentials (AP) and a decrease in the bioavailability of nitric oxide (NO). In this study, we analysed the effects of NO on Kv4.3 (I(Kv4.3)) and on human transient outward K(+) (I(to1)) currents as well as the signalling pathways responsible for them. We also analysed the expression of NO synthase 3 (NOS3) in patients with CAF. Methods and results: I(Kv4.3) and I(to1) currents were recorded in Chinese hamster ovary cells and in human atrial and mouse ventricular dissociated myocytes using the whole-cell patch clamp. The expression of NOS3 was analysed by western blotting. AP were recorded using conventional microelectrode techniques in mouse atrial preparations. NO and NO donors inhibited I(Kv4.3) and human I(to1) in a concentration- and voltage-dependent manner (IC(50) for NO: 375.0 +/- 48 nM) as a consequence of the activation of adenylate cyclase and the subsequent activation of the cAMP-dependent protein kinase and the serine-threonine phosphatase 2A. The density of the I(to1) recorded in ventricular myocytes from wild-type (WT) and NOS3-deficient mice (NOS3(-/-)) was not significantly different. Furthermore, the duration of atrial AP repolarization in WT and NOS3(-/-) mice was not different. The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atrial AP and lengthened the AP duration measured at 20 and 50% of repolarization of the human atrial CAF-remodelled AP as determined using a mathematical model. However, the expression of NOS3 was not modified in left atrial appendages from CAF patients. Conclusion: Our results suggested that the increase in the atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the I(to1) as a result of the activation of non-canonical enzymatic pathways.
p.D1690N Nav1.5 rescues p.G1748D mutation gating defects in a compound heterozygous Brugada syndrome patient
(Heart Rhythm, 2013) Núñez Fernández, Lucía; Barana Muñoz, Adriana; Amorós García, Irene; González de la Fuente, Marta; Dolz Gaitón, Pablo; Gómez García, Ricardo; Rodríguez García, Isabel; Mosquera, Ignacio; Monserrat, Lorenzo; Delpón Mosquera, María Eva; Caballero Collado, Ricardo; Castro Beirás, Alfonso; Tamargo Menéndez, Juan
Background: We identified 2 compound heterozygous mutations (p.D1690N and p.G1748D) in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) in a proband diagnosed with Brugada syndrome type 1. Furthermore, in the allele encoding the p.D1690N mutation, the p.H558R polymorphism was also detected. Objective: The purpose of this study was to analyze the functional properties of the mutated channels as well as the putative modulator effects produced by the presence of the polymorphism. Methods: Wild-type and mutated human Nav1.5 channels were expressed in Chinese hamster ovary cells and recorded using whole-cell patch-clamp technique. Results: Separately, both p.D1690N and p.G1748D mutations produced a marked reduction in peak Na(+) current density (>80%), mainly due to their limited trafficking toward the membrane. Furthermore, p.G1748D mutation profoundly affected channel gating. Both p.D1690N and p.G1748D produced a marked dominant negative effect when cotransfected with either wild-type or p.H558R channels. Conversely, p.H558R was able to rescue defective trafficking of p.D1690N channels toward the membrane when both polymorphism and mutation were in the same construct. Surprisingly, cotransfection with p.D1690N, either alone or together with the polymorphism (p.H558R-D1690N), completely restored the profound gating defects exhibited by p.G1748D channels but only slightly rescued their trafficking. Conclusions: Our results add further support to the hypothesis that Nav1.5 subunits interact among them before trafficking toward the membrane and suggest that a missense mutation can "rescue" the defective gating produced by another missense mutation.
Pharmacology of cardiac potassium channels
(Cardiovascular Research, 2004) Tamargo Menéndez, Juan; Caballero Collado, Ricardo; Gómez García, Ricardo; Valenzuela, Carmen; Delpón Mosquera, María Eva
Cardiac K+ channels are membrane-spanning proteins that allow the passive movement of K+ ions across the cell membrane along its electrochemical gradient. They regulate the resting membrane potential, the frequency of pacemaker cells and the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters and hormones and class III antiarrhythmic drugs that prolong the action potential duration (APD) and refractoriness and have been found effective to prevent/suppress cardiac arrhythmias. In the human heart, K+ channels include voltage-gated channels, such as the rapidly activating and inactivating transient outward current (Ito1), the ultrarapid (IKur), rapid (IKr) and slow (IKs) components of the delayed rectifier current and the inward rectifier current (IK1), the ligand-gated channels, including the adenosine triphosphate-sensitive (IKATP) and the acetylcholine-activated (IKAch) currents and the leak channels. Changes in the expression of K+ channels explain the regional variations in the morphology and duration of the cardiac action potential among different cardiac regions and are influenced by heart rate, intracellular signalling pathways, drugs and cardiovascular disorders. A progressive number of cardiac and noncardiac drugs block cardiac K+ channels and can cause a marked prolongation of the action potential duration (i.e. an acquired long QT syndrome, LQTS) and a distinct polymorphic ventricular tachycardia termed torsades de pointes. In addition, mutations in the genes encoding IKr (KCNH2/KCNE2) and IKs (KCNQ1/KCNE1) channels have been identified in some types of the congenital long QT syndrome. This review concentrates on the function, molecular determinants, regulation and, particularly, on the mechanism of action of drugs modulating the K+ channels present in the sarcolemma of human cardiac myocytes that contribute to the different phases of the cardiac action potential under physiological and pathological conditions.