Stereoselective interactions of the enantiomers of chromanol 293B with human voltage-gated potassium channels.

Selective inhibitors of the slow component of the cardiac delayed rectifier K(+) current, I(Ks), are of interest as novel class III antiarrhythmic agents and as tools for studying the physiologic roles of the I(Ks) current. Racemic chromanol 293B is an inhibitor of both native I(Ks) and its putative molecular counterpart, the KvLQT1+minK ion channel complex. We synthesized the (+)-[3S,4R] and (-)-[3R,4S] enantiomers of chromanol 293B using chiral intermediates of known absolute configuration and determined their relative potency to block recombinant human K(+) channels that form the basis for the major repolarizing K(+) currents in human heart, including KvLQT1+minK, human ether-a-go-go-related gene product (hERG), Kv1.5, and Kv4.3, corresponding to the slow (I(Ks)), rapid (I(Kr)), and ultrarapid (I(Kur)) delayed rectifier currents and the transient outward current (I(To)), respectively. K(+) channels were expressed in mammalian cells and currents were recorded using the whole-cell patch-clamp technique. We found that the physicochemical properties and relative potency of the enantiomers differed from those reported previously, with (-)-[3R,4S]293B nearly 7-fold more potent in block of KvLQT1+minK than (+)-[3S,4R]293B, indicating that the original stereochemical assignments were reversed. K(+) current inhibition by (-)-293B was selective for KvLQT1+minK over hERG, whereas the stereospecificity of block for KvLQT1+minK and Kv1.5 was preserved, with (-)-293B more potent than (+)-293B for both channel complexes. We conclude that the (-)-[3R,4S] enantiomer of chromanol 293B is a selective inhibitor of KvLQT1+minK and therefore a useful tool for studying I(Ks).

Cardiovascular disease and arrhythmias: unique risks in women.

Women have higher risks for symptomatic arrhythmias and sudden death than men. A significantly higher resting heart rate and longer QT interval on electrocardiogram may be factors that predispose women to a serious form of ventricular arrhythmia known as torsades de pointes. Recent studies have demonstrated hormonal effects on the expression of cardiac potassium and calcium ion channels, indicating their possible regulatory role in the modulation of cardiac repolarization and QT interval. These results demonstrate a need for greater awareness and further research into the mechanistic differences between men and women with respect to arrhythmia and cardiovascular disease.

HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >>+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.

Charged amino acids near the pore entrance influence ion-conduction of a human L-type cardiac calcium channel.

Voltage-dependent L-type Ca2+ channels form highly selective pores for Ca2+ ions in the membranes of excitable cells. We investigated the functional role of negatively charged residues, within or near the selectivity region, in ion permeation of a human cardiac L-type Ca2+ channel. Glutamates in each of the four repeats, and an aspartate in repeat IV, were substituted with positively charged lysine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Block by Ca2+ and Mg2 of inward Li+ currents through the channels was used to assess the effects of amino acid substitutions on high-affinity divalent cation binding. The rank order of IC50's for Ca2+ block of I(Li) was: E677K > E1086K > E334K > E1387K > D1391K > or approximately wild-type. The order of IC50's for Mg2+ block of I(Li) indicated differential involvement of the same residues in Mg2+ binding: E 1387K > E334K > E1086K > E677K > D 1391K = wild-type. Mutants E1387K and D1391K effectively permeated Ba2+, but exhibited a decreased single-channel conductance. The unitary current amplitude carried by Na+, in the absence of external divalent cations, was slightly decreased in the E1387K mutant but not in the D1391K mutant. The results confirm that each of the four glutamates participate unequally in high-affinity Ca2+ binding. Additionally, our results indicate that these glutamate residues participate in Mg2+ binding. The glutamate at position 1387 may be only peripherally involved in the formation of a high-affinity Ca2+ -binding site but is central to a Mg2+ binding site accessible from the external side of the pore. The aspartate at position 1391 is most likely located just external to the selectivity region.

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

Alteration of channel characteristics by exchange of pore-forming regions between two structurally related Ca2+ channels.

Several types of structurally homologous high voltage-gated Ca2+ channels (L-, P- and N-type) have been identified via biochemical, pharmacological and electrophysiological techniques. Among these channels, the cardiac L-type and the brain BI-2 Ca2+ channel display significantly different biophysical properties. The BI-2 channel exhibits more rapid voltage-dependent current activation and inactivation and smaller single-channel conductance compared to the L-type Ca2+ channel. To examine the molecular basis for the functional differences between the two structurally related Ca2+ channels, we measured macroscopic and single-channel currents from oocytes injected with wild-type and various chimeric channel alpha 1 subunit cRNAs. The results show that a chimeric channel in which the segment between S5-SS2 in repeat IV of the cardiac L-type Ca2+ channel, was replaced by the corresponding region of the BI-2 channel, exhibited macroscopic current activation and inactivation time-courses and single-channel conductance, characteristic of the BI-2 Ca2+ channel. The voltage-dependence of steady-state inactivation was not affected by the replacement. Chimeras, in which the SS2-S6 segment in repeat III or IV of the cardiac channel was replaced by the corresponding BI-2 sequence, exhibited altered macroscopic current kinetics without changes in single-channel conductance. These results suggest that part of the S5-SS2 segment plays a critical role in determining voltage-dependent current activation and inactivation and single-channel conductance and that the SS2-S6 segment may control voltage-dependent kinetics of the Ca2+ channel.

Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel.

Voltage-dependent L-type Ca2+ channels select for Ca2+ and other divalent cations by high-affinity Ca2+ binding and ion-ion interactions in the permeation pathway. We have recently identified a series of highly conserved glutamate residues, located within the SS2 segments of each of the four repeats of the human heart Ca2+ channel alpha 1 subunit, as major determinants of ion selectivity of the channel. To further investigate the functional role of these glutamate residues in ion permeation, we have individually neutralized the glutamic acids in repeats II and IV by substitution with alanine or glutamine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Apparent affinity for external Ca2+ was assessed by measuring the block by Ca2+ of inward Li+ currents through the Ca2+ channels. Mutations reducing net negative charge at these positions resulted in a 10-fold reduction in the affinity of the channel for external Ca2+. Single-channel conductance was measured with either divalent or monovalent cations as the charge carriers. Substitution of glutamic acid at position 677 with either alanine or glutamine increased single-channel conductance, whereas the same substitutions at position 1387 resulted in a decrease in single-channel conductance with Ba2+ as the charge carrier. In contrast, the unitary current amplitude carried by Na+, in the absence of external divalent cations, was not altered by these mutations. The results suggest that these conserved glutamate residues participate in high-affinity Ca2+ binding within the pore. The different effects on Ba2+ permeation indicate an asymmetric arrangement of the glutamate residues between repeats II and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular localization of regions in the L-type calcium channel critical for dihydropyridine action.

Sensitivity to dihydropyridines (DHPs) is a distinct characteristic that differentiates L-type Ca2+ channels from T-, N-, and P-type Ca2+ channels. To identify regions necessary for the functional effects of DHPs, chimeric Ca2+ channels were constructed in which portions of motif III or motif IV of a DHP-insensitive brain Ca2+ channel, BI-2, were introduced into the DHP-sensitive cardiac L-type Ca2+ channel. The resultant chimeric Ca2+ channels were expressed in Xenopus oocytes, and the effects of a DHP agonist and antagonist were studied. The results show that the linker region between S5 and S6 in motif IV of the L-type Ca2+ channel is a major site for DHP action. The DHP agonist and antagonist molecules interact with distinct sites on the alpha 1 subunit of the L-type Ca2+ channel. The data further show that the SS2-S6 region of motif III is not involved in DHP action but may be an important structural component of inactivation.

Differential contribution by conserved glutamate residues to an ion-selectivity site in the L-type Ca2+ channel pore.

In voltage-gated cation channels, it is thought that residues responsible for ion-selectivity are located within the pore-lining SS1-SS2 segments. In this study, we compared the ion permeation properties of mutant calcium channels in which highly conserved glutamate residues, located at analogous positions in the SS2 regions of all four motifs, were individually replaced. All of the mutants exhibited a loss of selectivity for divalent over monovalent cations. However, the permeation properties of the individual mutants varied in a position dependent manner. The results provide strong evidence that these glutamate residues, positioned at equivalent locations in the aligned sequences, play significantly different roles in forming the selectivity barrier of the calcium channel, and are probably arranged in an asymmetrical manner inside the ion-conducting pore.

Block of transient outward-type cloned cardiac K+ channel currents by quinidine.

The antiarrhythmic drug quinidine has been shown to block several types of K+ channel currents in cardiac preparations including the transient outward current (Ito). To characterize the molecular mechanism of quinidine block, a cloned Ito-type cardiac K+ channel (RHK1) was expressed in Xenopus oocytes, and drug effects were examined on whole-cell and single-channel currents. Extracellular application of quinidine reduced whole-cell RHK1 current amplitude in a concentration-dependent manner. The block was voltage dependent, with an IC50 of 1.69 mM at 0 mV, and the value decreased to 875 microM at +60 mV. Quinidine significantly slowed the current inactivation time course during voltage-clamp pulses without changing the rate of activation or the steady-state inactivation. To test the channel-state dependence of quinidine block, the cells were "rested" in the presence of quinidine (500 microM) for 2 to 3 minutes before applying depolarizing pulses to +60 mV. During the first pulse, the current inactivation rate was slower than control, but the peak current was only reduced by less than 5%. Subsequent pulses reduced the peak current amplitude to approximately 50% of control. These results suggest that quinidine blocks the open channel and that the drug must first dissociate before the channel can close, thereby causing a crossover in current tracings. In measurements of single-channel current from cell-attached patches, open time was reduced by quinidine in a concentration-dependent manner. Single-channel current amplitude was not altered by quinidine. Application of quinidine to the intracellular side of inside-out patches had an effect similar to that obtained from cell-attached patches but at 10-fold lower concentrations. External quinidine may therefore have to pass into or through the cell membrane to reach its blocking site.

Molecular localization of ion selectivity sites within the pore of a human L-type cardiac calcium channel.

A highly conserved position of negatively charged amino acids is present in the SS2 segments of the S5-S6 linker regions among calcium channels. We report here that replacing Glu residues at this position alters the ion selectivity of the human cardiac calcium channel. Substituting Glu334 in motif I or Glu1086 in motif III with Lys produced mutant calcium channels that permeated sodium ions 10-fold more effectively than barium ions. More conservative changes such as substitution of Glu1086 with Gln or substitution of Glu1387 with Ala also increased sodium permeation through the mutant calcium channels. Sodium currents through the mutant calcium channels could be modulated by dihydropyridines and blocked by external divalent cations. These results suggest that Glu334, Glu1086, and Glu1387 are part of a ring of glutamate residues formed in the pore-lining SS1-SS2 region and are critical in determining ion selectively and permeability of a human cardiac calcium channel.

Charge movements via the cardiac Na,K-ATPase.

The voltage dependence of transient and steady-state pump currents was examined in guinea pig ventricular myocytes to investigate mechanisms of charge translocation by the Na,K-ATPase. Na/K pump current was determined at approximately 36 degrees C as strophanthidin-sensitive whole-cell current in myocytes voltage clamped and internally dialyzed via wide tipped pipettes containing a pipette perfusion device. External Na ions diminished stationary pump current during forward Na/K cycling in a voltage dependent manner, the inhibition becoming stronger upon hyperpolarization. When Na,K-ATPase activity was restricted to Na translocation steps, stationary pump current was prevented but voltage pulses still elicited large transient pump currents which could be abolished by oligomycin B (> or = 2 micrograms/ml). The transients arose instantaneously on stepping the voltage, and decayed with voltage-dependent approximately single exponential time courses. The decay rates, and their high temperature sensitivity (approximately 200 s-1 at 0 mV at 36 degrees C; approximately 40 s-1 at 20 degrees C), suggest that the charge movements were limited by a conformational change associated with Na deocclusion. Those rates varied asymmetrically with voltage, changing little at positive voltages but increasing roughly exponentially with hyperpolarization (e-fold/approximately 80 mV). Lowering the extracellular [Na] ([Na]o) slowed the relaxation of charge movement at negative potentials but had little effect at positive potentials, and so shifted the rate constant-voltage curve to the left. The implied dependence on [Na]o of the backward rate constant governing pump charge movement accounts satisfactorily for the observed [Na]o sensitivity of stationary outward Na/K pump current, and indicates that the voltage-dependent step somehow involves the release of Na ions to the external medium. However, no strophanthidin-sensitive current was seen, at saturating external [K], when Na,K-ATPase activity was limited to K translocation steps by complete withdrawal of Na ions. But, at very low [Na]o, a weak negative slope appeared in the stationary pump current-voltage relationship at subsaturating, but not at saturating, external [K], indicating an increased apparent affinity for external K at more negative potentials. The results support the existence of a high field access channel through which extracellular Na and K ions must pass before interacting with their binding sites deep within the Na,K-ATPase molecule.

Voltage dependence of transient and steady-state Na/K pump currents in myocytes.

Experiments are reviewed here in which Na/K pump current was determined as strophanthidin-sensitive current in guinea-pig ventricular myocytes, voltage-clamped and internally-dialyzed via wide-tipped pipettes. In the presence of 150 mM extracellular [Na], both outward and inward pump current, during forward and reverse Na/K exchange respectively, were strongly voltage dependent. But reduction of external [Na] to 1.5 mM severely attenuated the voltage sensitivity of outward Na/K pump current. Voltage jumps elicited large transient pump currents during forward or reverse Na/K exchange, or when pump activity was restricted to Na translocation steps, but not when pumps were presumably engaged in K/K exchange. These findings indicate that Na translocation, but not K translocation, involves net charge movement through the membrane field, and that both forward and reverse Na/K transport cycles are rate-limited not by that voltage-sensitive step but by a subsequent voltage-insensitive step.

Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes.

In heart cells, cyclic AMP-dependent protein kinase (PKA) regulates calcium- and potassium-ion current by phosphorylating the ion channels or closely associated regulatory proteins. We report here that isoprenaline induced large chloride-ion currents in voltage-clamped, internally-dialysed myocytes from guinea-pig ventricles. The Cl- current could be activated by intracellular dialysis with cAMP or the catalytic subunit of PKA, indicating regulation by phosphorylation. In approximately symmetrical solutions of high Cl- concentration, the macroscopic cardiac Cl- current showed little rectification, unlike the single-channel current in PKA-regulated Cl- channels of airway epithelial cells. But, like epithelial Cl- -channel currents, the cardiac Cl- current was sensitive to the distilbene,4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS). In the absence of kinase activation, cardiac sarcolemmal Cl- conductance was negligible. During beta-adrenergic stimulation of the heart, this novel Cl- conductance should accelerate action-potential repolarization and so protect impulse propagation in the face of the possibly arrhythmogenic increases in heart rate and in calcium entry into the cells.

Potassium translocation by the Na+/K+ pump is voltage insensitive.

The voltage dependence of steady and transient changes in Na+/K+ pump current, in response to step changes in membrane potential, was investigated in guinea pig ventricular myocytes voltage clamped and internally dialyzed under experimental conditions designed to support four separate modes of Na+/K+ pump activity. Voltage jumps elicited transient pump currents when the pump cycle was running forward or backward, or when pumps were limited to Na+ translocation, but not when they were made to carry out K+/K+ exchange. This result indicates that K+ translocation involves no net charge movement across the membrane field and is therefore voltage insensitive. The transient pump currents seen during Na+/K+ transport demonstrate that both forward and reverse pump cycles are rate limited not by the voltage-dependent step but by a voltage-independent step, probably K+ translocation. These findings severely constrain kinetic models of Na+/K+ pump activity.

Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential.

Under certain conditions, spontaneous release of Ca2+ from the sarcoplasmic reticulum occurs in resting mammalian myocardium. In single rat ventricular myocytes, such spontaneous Ca2+ release appears localized rather than homogeneous. When the increase in cytosolic Ca2+ is present in a single locus within a cell, it causes a small depolarization, which, at the normal resting potential, is subthreshold for generating an action potential. However, when spontaneous Ca2+ release occurs simultaneously at more than a single discrete locus, the resultant sarcolemmal depolarization is augmented to levels that can induce an action potential, even when this depolarization begins at the normal resting membrane potential. Thus, the synchronous occurrence of multifocal localized increases in cytosolic Ca2+ due to spontaneous Ca2+ release from the sarcoplasmic reticulum within ventricular myocytes is a mechanism for "abnormal automaticity."

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium.

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.

Passive membrane properties of isolated feline ventricular myocytes.

Membrane properties of adult mammalian cardiac muscle are difficult to define mainly because of experimental complications arising from complex packing of myocytes in the tissue matrix. Isolated feline myocytes were used in the present study to avoid these complications. The objectives of this study were to define the functional relationship between passive unidirectional transmembrane potassium (K+) fluxes, membrane permeability to K+ (PK), and membrane K+ (Ko) dependency of this relationship. Passive (ouabain-insensitive) components of unidirectional K+ fluxes were measured with 42K, and membrane potential (Em) and membrane (slope) conductance (gm) were measured with electrophysiological techniques. Myocytes studied in solutions with 5 mM K+o had normal resting potentials (-81 +/- 1 mV). The input resistance and membrane time constant were 2.72 +/- 0.47 X 10(-7) omega and 7.01 +/- 1.0 ms, respectively. When K+o was lowered Em hyperpolarized, input resistance (Ri) increased, and K+ fluxes decreased. When K+o was increased Em depolarized, Ri decreased, and K+ fluxes increased. These data were combined to determine whether K+ fluxes obey the independence principle and to calculate PK and gK. The results obtained support the idea that 1) unidirectional K+ fluxes do not obey the independence principle, 2) PK is much greater than the membrane permeability to other ions, and 3) the gK calculated from passive K+ fluxes was similar to the gm measured electrically (at all K+o's tested).

Hypertrophy without contractile dysfunction after reversal of pressure overload in the cat.

To determine whether a causal relationship exists between the myocardial hypertrophy and the deficits in myosin ATPase activity and contractile function that have been associated with chronic, experimental pressure overload, we studied contractile function, myosin ATPase, and isomyosin pattern in a model of severe pressure overload in which the pressure overload was surgically relieved but hypertrophy persisted. Severe hypertrophy, contractile dysfunction, and pump failure were produced in the cat right ventricle by tight pulmonary arterial banding. In two groups of cats banded for 2- and 7-wk periods, right ventricular mass doubled, and contractile function was severely depressed compared with controls. In another group of cats subjected to severe right ventricular pressure overload for 4 wk, pressure overload was reversed by removal of the pulmonary arterial band. After a 4-wk recovery period for this group, right ventricular mass remained markedly increased, but contractile function had returned to normal. Changes in neither isomyosin composition nor myosin ATPase activity were found regardless of contractile function. Thus, following reversal of a right ventricular pressure overload severe enough to cause pump failure and a twofold increase in right ventricular mass, muscle contractile function can return to normal even when severe hypertrophy persists. Furthermore, changes in myosin isozyme composition or ATPase activity do not explain the changes in contractile function.