RESUMO
hERG (human ether-a-go-go-related gene) potassium (K+) channels are expressed in a range of tissue types including neuroblastoma cells and the heart, in which hERG K+ current is important for action potential repolarization. Whilst gender differences in cardiac repolarization and the QT interval of the cardiac electrocardiogram are well-established, comparatively little is known about regulation of hERG channels by sex hormones. In this study, whole-cell patch-clamp recordings were made at 37 degrees C from SH-SY5Y human neuroblastoma cells to investigate modulation of endogenous hERG K+ channel current (I(hERG)) by testosterone. Acutely applied testosterone at a physiologically relevant concentration (10 nM) produced a modest (approximately 13-15 %) increase in I(hERG) amplitude, whilst a high concentration (1 microM) slightly decreased I(hERG). The stimulatory effect of testosterone was inhibited by the androgen receptor antagonist flutamide (10 microM) and the PI-3 kinase inhibitor wortmannin (1 microM). Chronic (24 h) application of testosterone also augmented IhERG via flutamide-sensitive receptor activation, without modulation of the current's voltage-dependence. These results demonstrate for the first time that testosterone can stimulate (hERG) K+ channels via activation of classical androgen receptors and implicate PI-3 kinase in the acute response.
Assuntos
Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Testosterona/farmacologia , Antagonistas de Androgênios/farmacologia , Androstadienos/farmacologia , Transporte Axonal , Linhagem Celular , Estimulação Elétrica , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Canais de Potássio Éter-A-Go-Go/agonistas , Flutamida/farmacologia , Humanos , Técnicas de Patch-Clamp , Inibidores de Fosfoinositídeo-3 Quinase , Receptores Androgênicos/efeitos dos fármacos , Temperatura , WortmaninaRESUMO
Spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) appears to play an important role in cardiac sinoatrial node pacemaking. However, comparatively little is known about the role of intracellular Ca(2+) in the atrioventricular node (AVN). Intracellular Ca(2+) was therefore monitored in cells isolated from the rabbit AVN, using fluo-3 in conjunction with confocal microscopy. These cells displayed spontaneous Ca(2+) transients and action potentials. Ca(2+) transients were normally preceded by a small, slow increase (ramp) of intracellular Ca(2+) which was sometimes, but not always, accompanied by Ca(2+) sparks. During the Ca(2+) transient, intracellular [Ca(2+)] increased initially at the cell periphery and propagated inhomogeneously to the cell centre. The rate of spontaneous activity was decreased by ryanodine (1muM) and increased by isoprenaline (500nM); these changes were accompanied by a decrease and increase, respectively, in the slope of the preceding Ca(2+) ramp, with no significant change in Ca(2+) spark characteristics. Rapidly reducing bathing [Na(+)] inhibited spontaneous activity. These findings provide the first information on Ca(2+) handling at the sub-cellular level and link cellular Ca(2+) cycling to the genesis of spontaneous activity in the AVN.
Assuntos
Nó Atrioventricular/citologia , Nó Atrioventricular/metabolismo , Sinalização do Cálcio , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Isoproterenol/farmacologia , Microscopia Confocal , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Receptores Adrenérgicos beta/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , TemperaturaRESUMO
HERG (human ether-à-go-go-related gene) encodes channels responsible for the cardiac rapid delayed rectifier potassium current, I(Kr). This study investigated the effects on HERG channels of doxepin, a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia. Whole-cell patch-clamp recordings were made at 37 degrees C of recombinant HERG channel current (I(HERG)), and of native I(Kr) 'tails' from rabbit ventricular myocytes. Doxepin inhibited I(HERG) with an IC(50) value of 6.5+/-1.4 microM and native I(Kr) with an IC(50) of 4.4+/-0.6 microM. The inhibitory effect on I(HERG) developed rapidly upon membrane depolarization, but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of I(HERG). Neither the S631A nor N588K inactivation-attenuating mutations (of residues located in the channel pore and external S5-Pore linker, respectively) significantly reduced the potency of inhibition. The S6 point mutation Y652A increased the IC(50) for I(HERG) blockade by approximately 4.2-fold; the F656A mutant also attenuated doxepin's action at some concentrations. HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin. Notably, this study also establishes doxepin as an effective inhibitor of mutant (N588K) HERG channels responsible for variant 1 of the short QT syndrome.
Assuntos
Antidepressivos Tricíclicos/farmacologia , Doxepina/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Animais , Células Cultivadas , Canal de Potássio ERG1 , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Técnicas de Patch-Clamp , CoelhosRESUMO
4-Aminopyridine (4-AP) has been used extensively to study transient outward K+ current (ITO,1) in cardiac cells and tissues. We report here inhibition by 4-AP of HERG (the human ether-à-go-go-related gene) K+ channels expressed in a mammalian cell line, at concentrations relevant to those used to study ITO,1. Under voltage clamp, whole cell HERG current (IHERG) tails following commands to +30 mV were blocked with an IC50 of 4.4 +/- 0.5 mM. Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 mM 4-AP inhibited peak IHERG during an applied action potential clamp waveform by ~59 %. It also significantly prolonged action potentials and inhibited resurgent IK tails from guinea-pig isolated ventricular myocytes, which lack an ITO,1. We conclude that by blocking the alpha-subunit of the IKr channel, millimolar concentrations of 4-AP can modulate ventricular repolarisation independently of any action on ITO,1.