Multiple structural domains contribute to voltage-dependent inactivation of rat brain alpha(1E) calcium channels.
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We have investigated the molecular determinants that mediate the differences in voltage-dependent inactivation properties between rapidly inactivating (R-type) alpha(1E) and noninactivating (L-type) alpha(1C) calcium channels. When coexpressed in human embryonic kidney cells with ancillary beta(1b) and alpha(2)-delta subunits, the wild type channels exhibit dramatically different inactivation properties; the half-inactivation potential of alpha(1E) is 45 mV more negative than that observed with alpha(1C), and during a 150-ms test depolarization, alpha(1E) undergoes 65% inactivation compared with only about 15% for alpha(1C). To define the structural determinants that govern these intrinsic differences, we have created a series of chimeric calcium channel alpha(1) subunits that combine the major structural domains of the two wild type channels, and we investigated their voltage-dependent inactivation properties. Each of the four transmembrane domains significantly affected the half-inactivation potential, with domains II and III being most critical. In particular, substitution of alpha(1C) sequence in domains II or III with that of alpha(1E) resulted in 25-mV negative shifts in half-inactivation potential. Similarly, the differences in inactivation rate were predominantly governed by transmembrane domains II and III and to some extent by domain IV. Thus, voltage-dependent inactivation of alpha(1E) channels is a complex process that involves multiple structural domains and possibly a global conformational change in the channel protein. (+info)
The effect of alpha2-delta and other accessory subunits on expression and properties of the calcium channel alpha1G.
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1. The effect has been examined of the accessory alpha2-delta and beta subunits on the properties of alpha1G currents expressed in monkey COS-7 cells and Xenopus oocytes. 2. In immunocytochemical experiments, the co-expression of alpha2-delta increased plasma membrane localization of expressed alpha1G and conversely, the heterologous expression of alpha1G increased immunostaining for endogenous alpha2-delta, suggesting an interaction between the two subunits. 3. Heterologous expression of alpha2-delta together with alpha1G in COS-7 cells increased the amplitude of expressed alpha1G currents by about 2-fold. This finding was confirmed in the Xenopus oocyte expression system. The truncated delta construct did not increase alpha1G current amplitude, or increase its plasma membrane expression. This indicates that it is the exofacial alpha2 domain that is involved in the enhancement by alpha2-delta. 4. Beta1b also produced an increase of functional expression of alpha1G, either in the absence or the presence of heterologously expressed alpha2-delta, whereas the other beta subunits had much smaller effects. 5. None of the accessory subunits had any marked influence on the voltage dependence or kinetics of the expressed alpha1G currents. These results therefore suggest that alpha2-delta and beta1b interact with alpha1G to increase trafficking of, or stabilize, functional alpha1G channels expressed at the plasma membrane. (+info)
State-dependent inactivation of the alpha1G T-type calcium channel.
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We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with +info)
Inadequate ischaemia-selectivity limits the antiarrhythmic efficacy of mibefradil during regional ischaemia and reperfusion in the rat isolated perfused heart.
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1. Mibefradil was compared with (+/-)-verapamil for effects on ischaemia- and reperfusion-induced ventricular fibrillation (VF), and the role of ischaemia-selective L-channel block was examined. Langendorff perfused rat hearts (n=12/group) were used. 2. Neither drug at up to 100 nM reduced the incidence of VF during 30 min regional ischaemia. 300 and 600 nM (+/-)-verapamil abolished VF (P<0. 05); mibefradil was effective only at 600 nM (P<0.05). Reperfusion-induced VF incidence was reduced only by 600 nM (+/-)-verapamil (P<0.05). Both drugs at >/=100 nM increased coronary flow (P<0.05) with a similar potency and maximum effectiveness. 3. In separate hearts perfused with Krebs' solution containing 3 mM K+ (the same as that used for arrhythmia studies) neither drug at up to 600 nM affected ventricular contractility. With K+ raised to 6 mM, (+/-)-verapamil >/=30 nM reduced developed pressure (P<0.05); mibefradil did so only at 600 nM (P<0.05). With K+ raised to 10 mM the effects of (+/-)-verapamil were further increased (P<0.05) and mibefradil became active at >/=100 nM (P<0.05). Likewise both drugs impaired diastolic relaxation, with raised K+ exacerbating the effects and (+/-)-verapamil being more potent and its effects more greatly exacerbated by K+. In contrast, when K+ was normal (3 mM), coronary flow was increased by each drug at >/=30 nM (P<0.05) indicating a marked vascular : myocardial selectivity. 4. In conclusion, mibefradil differed from (+/-)-verapamil in its myocardial effects only in terms of its lower potency. As mibefradil is the more potent T-channel blocker, the T-channel is unlikely to represent the molecular target for these effects. The K+ elevations that occur in the ischaemic milieu determine the ability of both drugs to block myocardial L-channels; this is sufficient to account for the drugs' actions on VF. Neither drug possesses sufficient selectivity for ischaemic myocardium versus blood vessels to permit efficacy (VF suppression without marked vasodilatation) and so inappropriate hypotension is likely to preclude the safe use of mibefradil (or similar analogue) in VF suppression, and explains the lack of clinical effectiveness of (+/-)-verapamil. (+info)
Calcium dynamics underlying pacemaker-like and burst firing oscillations in midbrain dopaminergic neurons: a computational study.
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A mathematical model of midbrain dopamine neurons has been developed to understand the mechanisms underlying two types of calcium-dependent firing patterns that these cells exhibit in vitro. The first is the regular, pacemaker-like firing exhibited in a slice preparation, and the second is a burst firing pattern sometimes exhibited in the presence of apamin. Because both types of oscillations are blocked by nifedipine, we have focused on the slow calcium dynamics underlying these firing modes. The underlying oscillations in membrane potential are best observed when action potentials are blocked by the application of TTX. This converts the regular single-spike firing mode to a slow oscillatory potential (SOP) and apamin-induced bursting to a slow square-wave oscillation. We hypothesize that the SOP results from the interplay between the L-type calcium current (I(Ca,L)) and the apamin-sensitive calcium-activated potassium current (I(K,Ca,SK)). We further hypothesize that the square-wave oscillation results from the alternating voltage activation and calcium inactivation of I(Ca,L). Our model consists of two components: a Hodgkin-Huxley-type membrane model and a fluid compartment model. A material balance on Ca(2+) is provided in the cytosolic fluid compartment, whereas calcium concentration is considered constant in the extracellular compartment. Model parameters were determined using both voltage-clamp and calcium-imaging data from the literature. In addition to modeling the SOP and square-wave oscillations in dopaminergic neurons, the model provides reasonable mimicry of the experimentally observed response of SOPs to TEA application and elongation of the plateau duration of the square-wave oscillations in response to calcium chelation. (+info)
LVA and HVA Ca(2+) currents in ventricular muscle cells of the Lymnaea heart.
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The single-electrode voltage-clamp technique was used to characterize voltage-gated Ca(2+) currents in dissociated Lymnaea heart ventricular cells. In the presence of 30 mM tetraethylammonium (TEA), two distinct Ca(2+) currents could be identified. The first current activated between -70 and -60 mV. It was fully available for activation at potentials more negative than -80 mV. The current was fast to activate and inactivate. The inactivation of the current was voltage dependent. The current was larger when it was carried by Ca(2+) compared with Ba(2+), although changing the permeant ion had no observable effect on the kinetics of the evoked currents. The current was blocked by Co(2+) and La(3+) (1 mM) but was particularly sensitive to Ni(2+) ions ( approximately 50% block with 100 microM Ni(2+)) and insensitive to low doses of the dihydropyridine Ca(2+) channel antagonist, nifedipine. All these properties classify this current as a member of the low-voltage-activated (LVA) T-type family of Ca(2+) currents. The activation threshold of the current (-70 mV) suggests that it has a role in pacemaking and action potential generation. Muscle contractions were first seen at -50 mV, indicating that this current might supply some of the Ca(2+) necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between -40 and -30 mV and was fully available for activation at potentials more negative than -60 mV. This current was also fast to activate and with Ca(2+) as the permeant ion, inactivated completely during the 200-ms voltage step. Substitution of Ba(2+) for Ca(2+) increased the amplitude of the current and significantly slowed the rate of inactivation. The inactivation of this current appeared to be current rather than voltage dependent. This current was blocked by Co(2+) and La(3+) ions (1 mM) but was sensitive to micromolar concentrations of nifedipine ( approximately 50% block 10 microM nifedipine) that were ineffective at blocking the LVA current. These properties characterize this current as a L-type Ca(2+) current. The voltage sensitivity of this current suggests that it is also important in generating the spontaneous action potentials, and in providing some of the Ca(2+) necessary for excitation-contraction coupling. These data provide the first detailed description of the voltage-dependent Ca(2+) currents present in the heart muscle cells of an invertebrate and indicate that pacemaking in the molluscan heart has some similarities with that of the mammalian heart. (+info)
The T-type Ca(2+) channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by tachycardia-induced atrial remodeling in dogs.
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BACKGROUND: Ca(2+) overload is believed to play a role in tachycardia-induced atrial electrophysiological remodeling. L-type Ca(2+) channel blockers attenuate effective refractory period (ERP) changes caused by 24 hours of atrial tachycardia but may not substantially alter atrial fibrillation (AF) inducibility. This study assessed the effects of the T-type Ca(2+) channel blocker mibefradil on tachycardia-induced atrial remodeling. METHODS AND RESULTS: Dogs subjected to rapid atrial pacing (400 bpm) for 7 days were treated with mibefradil (100 mg/d, n=8) or matching placebo (n=10) in blinded fashion. Radiofrequency ablation of atrioventricular conduction and ventricular pacing were used to control ventricular rate. Placebo dogs showed significant decreases in atrial ERP (76+/-5 ms at a cycle length of 300 ms) and increases in ERP heterogeneity (27.7+/-2.4%), AF duration (414+/-232 seconds), and AF inducibility by single extrastimuli (41+/-10% of sites) compared with 10 unpaced control dogs (ERP 114+/-3 ms, ERP heterogeneity 13.8+/-0.9%, AF duration 7+/-3 seconds, AF inducibility 1.9+/-1.0% of sites). The changes caused by atrial tachycardia were strongly attenuated in mibefradil dogs, with ERPs averaging 102+/-7 ms, ERP heterogeneity 18.8+/-1.4%, AF duration 3+/-1 seconds, and AF inducibility 9.6+/-4.0% of sites. Among mibefradil-treated dogs, ERP, AF duration, and inducibility correlated with plasma drug concentration. Acute mibefradil administration did not alter ERP or AF. CONCLUSIONS: Mibefradil, a drug with strong T-type Ca(2+) channel blocking properties, prevents AF-promoting electrophysiological remodeling by atrial tachycardia. These findings have important potential implications for the mechanisms of tachycardia-induced atrial remodeling and demonstrate the feasibility of preventing electrical remodeling caused by several days of atrial tachycardia. (+info)
Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H.
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Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents. (+info)