Mendelian inheritance, linkage and genotypic disequilibrium in microsatellite loci isolated from Hymenaea courbaril (Leguminosae).

The Neotropical tree Hymenaea courbaril, locally known as Jatobá, is a valuable source of lumber and also produces comestible and medicinal fruit. We characterized Mendelian inheritance, linkage and genotypic disequilibrium at nine microsatellite loci isolated from H. courbaril, in order to determine if they would provide accurate estimates of population genetic parameters of this important Amazon species. The study was made on 250 open-pollinated offspring originated from 14 seed trees. Only one of nine loci presented significant deviation from the expected Mendelian segregation (1:1). Genotypic disequilibrium between pairwise loci was investigated based on samples from 55 adult and 56 juvenile trees. No genetic linkage between any paired loci was observed. After Bonferroni's corrections for multiple tests, we found no evidence of genotypic disequilibrium between pairs of loci. We conclude that this set of loci can be used for genetic diversity/ structure, mating system, gene flow, and parentage analyses in H. courbaril populations.

Pathways of HERG inactivation.

The rapid, repolarizing K(+) current in cardiomyocytes (I(Kr)) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene (HERG) expresses a macroscopic current virtually identical to I(Kr), but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at -100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K(+) solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.

Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family.

Low voltage-activated Ca2+ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, alpha1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels alpha1G and alpha1H and to the high voltage-activated channels formed by alpha1Ebeta3. The alpha1I channels opened after small depolarizations of the membrane similar to alpha1G and alpha1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The alpha1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that alpha1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

Molecular characterization of a neuronal low-voltage-activated T-type calcium channel.

The molecular diversity of voltage-activated calcium channels was established by studies showing that channels could be distinguished by their voltage-dependence, deactivation and single-channel conductance. Low-voltage-activated channels are called 'T' type because their currents are both transient (owing to fast inactivation) and tiny (owing to small conductance). T-type channels are thought to be involved in pacemaker activity, low-threshold calcium spikes, neuronal oscillations and resonance, and rebound burst firing. Here we report the identification of a neuronal T-type channel. Our cloning strategy began with an analysis of Genbank sequences defined as sharing homology with calcium channels. We sequenced an expressed sequence tag (EST), then used it to clone a full-length complementary DNA from rat brain. Northern blot analysis indicated that this gene is expressed predominantly in brain, in particular the amygdala, cerebellum and thalamus. We mapped the human gene to chromosome 17q22, and the mouse gene to chromosome 11. Functional expression of the channel was measured in Xenopus oocytes. Based on the channel's distinctive voltage dependence, slow deactivation kinetics, and 7.5-pS single-channel conductance, we conclude that this channel is a low-voltage-activated T-type calcium channel.

Interactions of the nonsedating antihistamine loratadine with a Kv1.5-type potassium channel cloned from human heart.

The use of nonsedating antihistamines may, on rare occasions, be associated with cardiac arrhythmias. This could be due to blockade of voltage-dependent K+ channels in the heart, leading to a prolongation in repolarization in the human myocardium. For this reason, we examined the effects of the nonsedating antihistamine loratadine on a rapidly activating delayed-rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in HEK 293 cells or mouse Ltk- cells. Using patch-clamp electrophysiology, we found that loratadine blocked Kv1.5 current measured from inside-out membrane patches at concentrations of > or = 100 nM, resulting in an IC50 value of 808 nM at +50 mV. The drug enhanced the rate of Kv1.5 current decay, and block was enhanced at membrane potentials near threshold relative to higher potentials. Loratadine did not alter the kinetics of Kv1.5 current activation or deactivation. Unitary Kv1.5 currents were recorded in cell-attached patches. At the single-channel level, the main effect of loratadine was to reduce the mean probability of opening of Kv1.5. This effect of loratadine was achieved by a reduced number of openings in bursts and burst duration. Finally, loratadine (10 microM) failed to inhibit HERG K+ channel currents expressed in Xenopus laevis oocytes. It is concluded that loratadine is an effective blocker of Kv1.5 that interacts with an activated state or states of the channel. This interaction suggests a potential for loratadine to alter cardiac excitability in vivo.

Molecular physiology and pharmacology of HERG. Single-channel currents and block by dofetilide.

BACKGROUND: The human ether-a-go-go-related gene (HERG) is one locus for the hereditary long-QT syndrome. A hypothesis is that HERG produces the repolarizing cardiac potassium current IKr with the consequence that mutations in HERG prolong the QT interval by reducing IKr. The elementary properties of HERG are unknown, and as a test of the hypothesis that HERG produces IKr, we compared their elementary properties. METHODS AND RESULTS: We injected HERG cRNA into Xenopus oocytes and measured currents from single channels or current variance from the noise produced by ensembles of channels recorded from macro patches. Single-channel conductance was dependent on the extracellular potassium concentration ([K]o). At physiological [K]o, it was 2 picosiemens (pS), and at 100 mmol/L [K]o, it was 10 pS. Openings occurred in bursts with a mean duration of 26 ms at -100 mV. Mean open time was 3.2 ms and closed times were 1.0 and 26 ms. In excised macro patches, HERG currents were blocked by the class III antiarrhythmic drug dofetilide, with an IC50 of 35 nmol/L. Dofetilide block was slow and greatly attenuated at positive potentials at which HERG rectifies. CONCLUSIONS: The microscopic physiology of HERG and IKr is similar, consistent with HERG being an important component of IKr. The pharmacology is also similar; dofetilide appears to primarily block activated channels and has a much lower affinity for closed and inactivated channels.

Mapping the block of a cloned human inward rectifier potassium channel by dofetilide.

Dofetilide, a methanesulfonanilide derivative, is a potent class III antiarrhythmic drug. Like other members of this class of K+ channel blockers, the sites in the channel to which the drug binds are unknown, although high and low affinity binding has been reported in cardiomyocytes. The most sensitive K+ channel target for dofetilide seems to be IKr, the rapid component of the repolarizing delayed rectifier K+ current. However, block of other K+ channels occurs at higher concentrations and is of special interest in regard to toxicity. Recently, we have demonstrated that hIRK, a cloned inward rectifier K+ channel (IRK) isolated from human atrium and expressed heterologously in Xenopus oocytes, is blocked by dofetilide. We report the localization of a site that is critical for dofetilide block in hIRK. We used chimeric constructs between hIRK and ROMK1, a related inward rectifier that is drug resistant. Substitution of hIRK-M2, the second putative transmembrane spanning segment of IRKs, with ROMK1-M2 increased unblocking of dofetilide by 10-20-fold in hIRK. Site-directed mutagenesis further pinpointed the effects to a single hydrophobic residue (I177) in M2. A reduction in hydrophobicity by the point mutation I177C increased recovery from block > 10-fold (1.17 sec in wild-type to 0.112 sec at -80 mV at physiological K+ concentrations), leading us to suggest that hydrophobic interactions are essential for dofetilide block in hIRK. A similar mechanism may explain dofetilide block in other ion channels, including IKr.

Voltage-dependent inactivation in a cardiac-skeletal chimeric calcium channel.

The loci for inactivation in calcium channel proteins are unknown. Mechanisms for inactivation may be distributed across Ca2+ channel subunits and appear to be complex, multiple and interacting. We took advantage of the properties of chimeras, constructed between cardiac (H4) and skeletal muscle (Sk4) calcium channel alpha 1 subunits to study the molecular mechanism of inactivation in L-type calcium channels. Sk1H3, a chimeric construct of these two L-type calcium channels, was expressed in Xenopus oocytes in the absence of auxiliary subunits. Sk1H3 incorporated repeat I from skeletal muscle alpha 1 and repeats II, III, IV from heart alpha 1 subunit. Sk1H3 inactivated faster (tau = 300 ms) and more fully than the wild-type H4 with Ba2+ ions as the charge carrier. Thus, inactivation of Sk1H3 was 90% complete after a 5-s conditioning pulse at +20 mV while inactivation of H4 was only 37% complete. Sk1H3 inactivation also developed at more negative potentials with E0.5 = -15 mV as compared to E0.5 = -5 mV for H4. In the presence of external calcium ions, the extent of inactivation significantly increased from 37 to 83% for H4 while inactivation of Sk1H3 was only slightly increased. Inactivation with Ba2+ as the charge carrier was confirmed at the single- channel level where averaged single-channel ensembles showed a similar rate of inactivation. Collectively, these observations demonstrate that Sk1H3 inactivation appears to have a prominent voltage-dependent component. Whether Sk1H3 inactivation involves interactions within repeat I alone or interactions between repeat I and site(s) located in the three other repeats of the alpha 1 subunit has yet to be determined.

T-type and N-type calcium channels of Xenopus oocytes: evidence for specific interactions with beta subunits.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit.

Voltage-sensitive Ca2+ channels are multisubunit complexes that include, among others, a large alpha 1 subunit, which by itself is sufficient to form a channel. Several alpha 1 genes encoding L-, N-, and P-type Ca2+ channels have been cloned. These alpha 1 genes share a high degree of sequence homology in the putative transmembrane regions, but vary substantially in the putative intracellular loops and the flanking amino and carboxyl termini. In the present study, we investigated the functional roles of the 665-amino acid long carboxyl terminus of a cardiac alpha 1 by constructing deletion mutants. Expression in Xenopus oocytes of delta C1856, delta C1733, and delta C1700, which lack from 307 to 472 amino acids at the carboxyl terminus, led to inward Ba2+ currents that were 4- to 6-fold greater than observed with the 2171-amino acid long wild type alpha 1. Ionic currents increased without a change in the amount of charge moved during voltage-dependent gating, suggesting that the increase in ionic currents was not due to an increase in the number of channels that were expressed. Single channel analysis revealed an unaltered unitary conductance. Thus, removal of up to 70% of the carboxyl terminus increased current density by facilitating the coupling between the voltage-dependent gating and channel opening, leading to an increased opening probability of the channel.

Variability of the QTc interval: impact on defining drug effect and low-frequency cardiac event.

Prolongation of the QT interval corrected for heart rate (QTc) can lead to the development of torsades de pointes, a life-threatening form of polymorphic ventricular tachycardia. However, the QTc interval duration exhibits a high degree of spontaneous variability and is not necessarily a direct predictor of the risk of torsades. This observation holds implications for the assessment of the potential proarrhythmic effects of noncardiac pharmacologic agents. To date, the antihistamine terfenadine is the only noncardiac drug that has undergone a comprehensive and systematic evaluation related to the consequences of its causing QTc prolongation. The results suggest that QTc prolongation resulting solely from terfenadine at clinical doses does not have an important impact on clinically relevant endpoints. The risk of serious ventricular arrhythmias with terfenadine using epidemiologic data is the same or less than that associated with traditional first-generation antihistamines. The risk of a clinical cardiac event (QTc prolongation, ventricular arrhythmias, syncope, or sudden death) with terfenadine is similar to that of other antihistamines. Factors associated with increased risk in patients taking terfenadine include significant liver disease, hypokalemia, overdose, and concomitant administration of ketoconazole-like agents or erythromycin; use of terfenadine is relatively contraindicated in these settings. No increased risk of serious arrhythmias has been confirmed in conjunction with the use of terfenadine in patients with cardiac disease.

Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel.

The skeletal muscle dihydropyridine receptor/Ca2+ channel is composed of five protein components (alpha 1, alpha 2 delta, beta, and gamma). Only two such components, alpha 1 and alpha 2, have been identified in heart. The present study reports the cloning and expression of a novel beta gene that is expressed in heart, lung, and brain. Coexpression of this beta with a cardiac alpha 1 in Xenopus oocytes causes the following changes in Ca2+ channel activity: it increases peak currents, accelerates activation kinetics, and shifts the current-voltage relationship toward more hyperpolarized potentials. It also increases dihydropyridine binding to alpha 1 in COS cells. These results indicate that the cardiac L-type Ca2+ channel has a similar subunit structure as in skeletal muscle, and provides evidence for the modulatory role of the beta subunit.

Parathyroid hormone: an endogenous modulator of cardiac calcium channels.

We have tested the effects of the active 1-34 amino acid sequence of rat parathyroid hormone (PTH) on Ca2+ channel activity in neonatal rat ventricular cells. Rat PTH (30 pM to 10 nM) increased depolarization-induced Ca2+ influx into these cells, an effect that was abolished by 1 microM nifedipine. The 1-34 amino acid sequence of bovine PTH also stimulated Ca2+ influx in control cells but not in cells pretreated with cholera toxin. Rat PTH also elevated adenosine 3',5'-cyclic monophosphate accumulation in these ventricular myocytes. Whole cell voltage-clamp recordings confirmed a stimulatory effect of rat PTH on cardiac L-type Ca2+ channels. Cell-attached single channel recordings revealed an increase in the probability of channel opening as the primary mechanism for the enhancement of Ca2+ current. Taken together these results suggest an important role for PTH as an endogenous modulator of cardiac L-type Ca2+ channels.

A new site for the activation of cardiac calcium channels defined by the nondihydropyridine FPL 64176.

We examined the effects of a new ligand, FPLnM-64176, on L-type Ca++ channels in cardiac tissue. FPL 64176 (10-1 microM) enhanced Ca++ influx into neonatal rat ventricular myocytes, a response which was blocked by nifedipine. FPL 64176 had no effect on [3H]PN200-110 binding in rat ventricular membranes, but dramatically increased L-type Ca++ channel current amplitude. FPL 64176 (1 microM) slowed both the activation and the inactivation kinetics of the L-channel in neonatal rat ventricular cells. We also noted a hyperpolarizing shift in the threshold and peak potential of the Ca++ channel current-voltage relationship in response to the compound. Additionally, the binding site for FPL 64176 appeared to be located on the extracellular face of the channel. We conclude that FPL 64176 is a potent new activator of L-type Ca++ channels with a novel mechanism and site of action.

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.

Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.

[3H]PN200-110 binding in a fibroblast cell line transformed with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel.

We examined the binding of the 1,4-dihydropyridine (DHP) [3H]PN200-110 to membranes from a fibroblast cell line transfected with the alpha 1 subunit (DHP receptor) of the L-type Ca2+ channel from rabbit skeletal muscle. Binding site affinity (KD) and density (Bmax) were 1.16 +/- 0.31 nM and 142 +/- 17 fmoles/mg protein, respectively. This affinity corresponded closely with that observed in native skeletal muscle. The Ca2+ channel antagonists diltiazem and MDL 12,330A stimulated [3H]PN200-110 binding in a dose-dependent manner while flunarizine, quinacrine and trifluoperazine inhibited binding. Surprisingly, D600 also stimulated [3H]PN200-110 binding in a dose-dependent and stereoselective manner. It is concluded that the fibroblast cells used in this study provide a unique system for interactions of the Ca2+ channel ligands with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel.

Angiotensin II modulates cardiac Na+ channels in neonatal rat.

Since chronic congestive heart failure syndromes are associated with both elevated circulating levels of angiotensin II and potentially lethal ventricular tachyarrhythmias, we investigated the effect of angiotensin II on voltage-dependent cardiac Na+ currents. Single-channel Na+ currents in neonatal rat ventricular myocytes were studied using the patch clamp method in the cell-attached mode. Angiotensin II applied outside the patch increased the frequency of opening and rates of activation and inactivation of single-channel Na+ currents within the patch. These effects were mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and were prevented by prior incubation with TPA. Therefore, we propose that angiotensin II modulates cardiac Na+ currents by a cytoplasmic second messenger, perhaps protein kinase C, and this may predispose toward arrhythmia.

Induction of calcium currents by the expression of the alpha 1-subunit of the dihydropyridine receptor from skeletal muscle.

The dihydropyridine (DHP) receptor purified from skeletal muscle comprises five protein subunits (alpha 1, alpha 2, beta, gamma and delta) and produces Ca2+ currents that are blocked by DHPs. Cloning of the alpha 1- and alpha 2-subunits, the former affinity-labelled by DHP, has shown that the alpha 1-subunit is expressed in skeletal muscle alone, whereas the alpha 2- and delta- subunits are also expressed in other tissues. Although the transient expression of the alpha 1-subunit in myoblasts from dysgenic mice (but not in oocytes) has been demonstrated, the use of these expression systems to determine the function of the alpha 1- subunit is complicated by the presence of endogenous Ca2+ currents, which may reflect the constitutive expression of proteins similar to the alpha 2-, beta-, gamma- and/or delta-subunits. We therefore selected a cell line which has no Ca2+ currents or alpha 2- subunit, and probably no delta-subunit for stable transformation with complementary DNA of the alpha 1- subunit. The transformed cells express DHP-sensitive, voltage-gated Ca2+ channels, indicating that the minimum structure of these channels is at most an alpha 1 beta gamma complex and possibly an alpha 1- subunit alone.

Nonmodal gating of cardiac calcium channels as revealed by dihydropyridines.

The hypothesis that dihydropyridine (DHP)-sensitive calcium channels have three distinct modes of gating has been examined. The major prediction is that the relative frequencies among modes depend on DHP concentration while the kinetics within a mode do not. We tested this by studying whole-cell and single-channel calcium currents in neonatal rat and adult guinea pig cardiac myocytes in different concentrations of several DHPs. In the absence of DHPs calcium currents declined with time but the kinetics, which are the focus of this study, were unchanged. Open-time frequency distributions had insignificant numbers of prolonged openings and were well fit by single tau's. Agonist DHP stereoisomers produced concentration-dependent changes in whole-cell tail current tau's. The frequency distribution of single calcium channel current open times became biexponential and the tau's were concentration dependent. The average number of openings per trace of channels with customary open times increased with increases in DHP concentration. Latencies to first opening for the customary openings and for prolonged openings were shorter in the presence of DHPs. A second larger conductance is another important feature of DHP-bound single calcium channels. Thus DHPs not only caused prolonged openings; they produced numerous changes in the kinetics of customary openings and increased channel conductance. It follows that these effects of DHPs do not support the hypothesis of modal gating of calcium channels. The mode model is not the only model excluded by the results; models in which DHPs are allowed to act only or mainly on open states are excluded, as are models in which the effects are restricted to inactivated states. We suggest a different type of model in which cooperative binding of DHPs at two sites produces the essential changes in kinetics and conductance.