The human ether-a'-go-go related gene (hERG) K+ channel blockade by the investigative selective-serotonin reuptake inhibitor CONA-437: limited dependence on S6 aromatic residues.

Diverse non-cardiac drugs adversely influence cardiac electrophysiology by inhibiting repolarising K(+) currents mediated by channels encoded by the human ether-a-go-go-related gene (hERG). In this study, pharmacological blockade of hERG K(+) channel current (I(hERG)) by a novel investigative serotonin-selective reuptake inhibitor (SSRI), CONA-437, was investigated. Whole-cell patch-clamp measurements of I(hERG) were made from human embryonic kidney (HEK 293) cells expressing wild-type (WT) or mutant forms of the hERG channel. With a step-ramp voltage-command, peak I(hERG) was inhibited with an IC(50) of 1.34 µM at 35 ±1°C; the IC(50) with the same protocol was not significantly different at room temperature. Voltage-command waveform selection had only a modest effect on the potency of I(hERG) block: the IC50 with a ventricular action potential command was 0.72 µM. I(hERG) blockade developed rapidly with time following membrane depolarisation and showed a weak dependence on voltage, accompanied by a shift of ≈ -5 mV in voltage-dependence of activation. There was no significant effect of CONA-437 on voltage-dependence of I(hERG) inactivation, though at some voltages an apparent acceleration of the time-course of inactivation was observed. Significantly, mutation of the S6 aromatic amino acid residues Y652 and F656 had only a modest effect on I(hERG) blockade by CONA-437 (a 3-4 fold shift in affinity). CONA-437 at up to 30 µM had no significant effect on either Nav1.5 sodium channels or L-type calcium channels. In conclusion, the novel SSRI CONA-437 is particularly notable as a gating-dependent hERG channel inhibitor for which neither S6 aromatic amino-acid constituent of the canonical drug binding site on the hERG channel appears obligatory for I(hERG) inhibition to occur.

Mechanism of action potential prolongation by RP 58866 and its active enantiomer, terikalant. Block of the rapidly activating delayed rectifier K+ current, IKr.

BACKGROUND: The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, are reported to selectively block the inward rectifier K+ current, IK1. These drugs have demonstrated efficacy in animal models of cardiac arrhythmias, suggesting that block of IK1 may be a useful antiarrhythmic mechanism. The symmetrical action potential (AP)-prolonging and bradycardic effects of these drugs, however, are inconsistent with a sole effect on IK1. METHODS AND RESULTS: We studied the effects of RP 58866 and terikalant on AP and outward K+ currents in guinea pig ventricular myocytes. RP 58866 and terikalant potently blocked the rapidly activating delayed rectifier K+ current, IKr, with IC50S of 22 and 31 nmol/L, respectively. Block of IK1 was approximately 250-fold less potent; IC50S were 8 and 6 mumol/L, respectively. No significant block of the slowly activating delayed rectifier, IK1, was observed at < or = 10 mumol/L. The phenotypical IKr currents in mouse AT-1 cells and Xenopus oocytes expressing HERG were also blocked 50% by 200 to 250 nmol/L RP 58866 or terikalant, providing further conclusive evidence for potent block of IKr. RP 58866 < or = 1 mumol/L and dofetilide increased AP duration symmetrically, consistent with selective block of IKr. Only higher concentrations (> or = 10 mumol/L) of RP 58866 slowed the rate of AP repolarization and decreased resting membrane potential, consistent with an additional but substantially less potent block of IK1. CONCLUSIONS: These data demonstrate that RP 58866 and terikalant are potent blockers of IKr and prompt a reinterpretation of previous studies that assumed specific block of IK1 by these drugs.

IK of rabbit ventricle is composed of two currents: evidence for IKs.

The delayed rectifier K+ current (IK) in rabbit heart has long been thought to consist of only a single, rapidly activating, dofetilide-sensitive current, IKr. However, we find that IK of rabbit ventricular myocytes actually consists of both rapid and slow components, IKr and IKs, respectively, that can be isolated pharmacologically. Thus, after complete blockade of IKr with dofetilide, the remaining current, IKs, is homogeneous as judged by an envelope of tails test. IKs activates and deactivates slowly, continues to activate during sustained depolarizations, has a half-activation potential of 7.0 +/- 0.8 mV and slope factor of 11.0 +/- 0.7 mV, reverses at -77.2 +/- 1.3 mV (extracellular K+ concentration = 4 mM), is increased by removing extracellular K+, and is enhanced by isoproterenol and stocked by azimilide. Northern analysis demonstrates that the minK (IsK) gene, which encodes a subunit of the channel that underlies the IKs current, is expressed in rabbit heart. Expression of the rabbit protein in Xenopus oocytes elicits a slowly activating, voltage-dependent current, IsK, similar to those expressed previously from mouse, rat, guinea pig, and human genes. The results demonstrate that IKs is present in rabbit ventricle and therefore contributes to cardiac repolarization in this species.

Adrenergic modulation of ultrarapid delayed rectifier K+ current in human atrial myocytes.

The ultrarapid delayed rectifier K+ current (IKur) in human atrial cells appears to correspond to Kv1.5 cloned channels and to play an important role in human atrial repolarization. Kv1.5 channels have consensus sites for phosphorylation by protein kinase A and C, suggesting possible modulation by adrenergic stimulation. The present study was designed to assess the adrenergic regulation of IKur in human atrial myocytes. Isoproterenol increased IKur in a concentration-dependent manner, with significant effects at concentrations as low as 10 nmol/L. The effects of isoproterenol were reversible by washout or by the addition of propranolol (1 mumol/L). Isoproterenol's effects were mimicked by the direct adenylate cyclase stimulator, forskolin, and by the membrane-permeable form of cAMP, 8-bromo cAMP. Isoproterenol had no effect on IKur when the protein kinase A inhibitor peptide, PKI(6-22)amide, was included in the pipette solution; in a separate set of experiments in which isoproterenol alone increased IKur by 45 +/- 9% relative to control, subsequent superfusion with isoproterenol in the presence of the protein kinase inhibitor H-7 failed to alter IKur. In contrast to isoproterenol, phenylephrine (in the presence of propranolol to block beta-adrenegic effects) induced a concentration-dependent inhibition of IKur, with significant effects observed at concentrations as low as 10 mumol/L. The inhibitory actions of phenylephrine were reversed by the addition of prazosin and prevented by coadministration with a highly selective inhibitor of protein kinase C, bisindolylmaleimide. These results indicate that beta-adrenergic stimulation enhances, whereas alpha-adrenergic stimulation inhibits, IKur and suggest that these actions are mediated by protein kinase A and protein kinase C, respectively. The modulation of IKur by adrenergic influences is a potentially novel control mechanism for human atrial repolarization and arrhythmias.

Properties of sodium and potassium currents of cultured adult human atrial myocytes.

Cultured cell systems are valuable for the study of regulation of phenotypic expression, but little is known about the electrophysiological properties of human cardiac tissues in culture. The present studies were designed to determine the feasibility of maintaining human atrial myocytes in primary culture and to assess changes in Na+ (INa) and K+ (Ito, transient outward, and Ikur, ultra-rapid delayed rectifier) currents. Within 24 h of culture, cells assumed an avoid shape, which they maintained for up to 7 days. The voltage dependence, kinetics, and density of INa were unchanged in culture. The activation properties of Ito (kinetics and voltage dependence) were not altered, but Ito density (current normalized to cell capacitance) was reduced and inactivation properties were altered (negative shift in voltage dependence and slowed kinetics) in cultured compared with fresh cells. The absolute current amplitude, kinetics, voltage dependence, and 4-aminopyridine sensitivity of IKur were unchanged, but current density was increased. All changes in ionic currents occurred within 24 h of culture and remained stable for the next 4 days. We conclude that human atrial myocytes can be maintained in primary culture, that the qualitative properties of INa, Ito, and IKur remain constant but that some quantitative changes occur, and that cultured human atrial myocytes may be valuable for studies of the molecular mechanisms and regulation of cardiac channel function in humans.

Alpha-adrenergic control of volume-regulated Cl- currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism.

alpha-Adrenergic stimulation is known to play a role in cardiac arrhythmogenesis and to modulate a variety of cardiac K+ currents. The effects of alpha-adrenergic stimulation on Cl- currents are largely unknown. Many cardiac cell types show a volume-sensitive Cl- current induced by cell swelling (ICl.swell). The present experiments were designed to assess the potential alpha-adrenergic modulation of ICl.swell in rabbit atrial myocytes. ICl.swell was induced with the use of a hypotonic superfusate, under conditions designed to prevent currents carried by K+, Na+, and Ca2+ ions. A basal Cl- current (ICl.b) was observed under isotonic conditions in 128 of 150 cells (85%), had the same dependency on [Cl-]o as ICl.swell, and was reduced by cell shrinkage induced by hypertonic superfusion, suggesting that ICl.b is carried by the same volume-sensitive Cl- conductance as ICl.swell. Phenylephrine produced a concentration-dependent and near-complete inhibition of ICl.b and ICl.swell, with EC50 values of 86 +/- 5 and 72 +/- 7 (mean +/- SEM) mumol/L, respectively, at +20 mV. Norepinephrine (administered in the presence of 1 mumol/L propranolol) also inhibited ICl.b and ICl.swell, with EC50 values of 2.6 +/- 0.1 and 2.8 +/- 0.4 mumol/L, respectively. The concentration-response curve for phenylephrine was shifted significantly (P < .001) to the right by the alpha 1-adrenoceptor antagonist prazosin and by the alpha 1A-receptor antagonists (+)-niguldipine and 5-methylurapidil but was unaltered by the alpha 1B-receptor antagonist chloroethylclonidine (100 mumol/L). Inhibition of protein kinase C (PKC) with staurosporine, H-7, or 18-hour preincubation with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) blocked the effects of phenylephrine on ICl.swell, and the highly selective PKC inhibitor bisindolylmaleimide blocked the effects of norepinephrine on ICl.swell and ICl.b. Both PMA and 1-oleoyl-2-acetylglycerol inhibited ICl.swell in a concentration-dependent fashion. In blinded studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced ICl.swell by 91 +/- 3%; its inactive analogue 4 alpha-PDD had no effect (mean change, 3 +/- 1%). Preincubation with pertussis toxin (PTX) prevented the actions of phenylephrine on ICl.swell, indicating a role for a PTX-sensitive guanine nucleotide-binding (G) protein. We conclude that alpha-adrenergic agonists inhibit volume-sensitive Cl- currents in rabbit atrial cells by interacting with an alpha 1A-adrenoceptor mechanism that is coupled to PKC via a PTX-sensitive G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative mechanisms of 4-aminopyridine-resistant Ito in human and rabbit atrial myocytes.

The cardiac transient outward current (Ito) has been shown in several species to consist of two components: 1) a 4-aminopyridine (4-AP)-sensitive component (Ito1) and 2) a 4-AP-resistant component (Ito2). In rabbits, Ito2 is a Ca(2+)-dependent Cl- current [ICl(Ca)]; similar mechanisms have been suggested to underlie Ito2 in human atrium. We used whole cell patch-clamp techniques to define the mechanism of Ito2 (defined as the component resistant to 5 mM 4-AP) in human atrial myocytes, with parallel experiments performed in rabbit atrial cells. In rabbit atrium, Ito2 activated more slowly than Ito1 and had a bell-shaped current-voltage of Ito with properties similar to Ito2 in the rabbit, and a similar component recorded with pipette K+ replaced by Cs+ was suppressed by the substitution of methanesulfonate for Cl- in the superfusate. In human cells, a 4-AP-resistant Ito2 was recorded at a depolarizing pulse frequency of 1 Hz, but not at 0.1 Hz. Ito2 activated rapidly and inactivated earlier than Ito1, whereas its I-V relation was linear like that of Ito1. Ryanodine had no effect on human atrial Ito. When K(+)-free pipette solutions were used, no Ito was recorded in 30 human atrial myocytes, and external Cl- replacement with methanesulfonate failed to reveal an Ito. In 13 human myocytes, isoproterenol increased ICa but failed to activate an Ito compatible with ICl(Ca). Whereas caffeine suppressed human atrial Ito, it also suppressed Ito1 [in the presence of 200 microM Cd2+ to block ICa and 5 mM intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to buffer intracellular Ca2+] in both human and rabbit atrium, indicating an action unrelated to Ca(2+)-triggered Ca2+ release. In conclusion, we were unable to demonstrate the presence of ICl(Ca) in human atrial myocytes, and the 4-AP-resistant component of Ito appeared to be due to 4-AP unblocking.

Use-dependent effects of the class III antiarrhythmic agent NE-10064 (azimilide) on cardiac repolarization: block of delayed rectifier potassium and L-type calcium currents.

We studied the effects of NE-10064 (azimilide), a new antiarrhythmic agent reported to be a selective blocker of the slowly activating component of the delayed rectifier, IKs. In ferret papillary muscles, NE-10064 increased effective refractory period (ERP) and decreased isometric twitch tension in a concentration-dependent manner (0.3-30 microM). Increases in ERP showed reverse use-dependence, and were greater at 1 than at 3 Hz. In contrast, changes in tension were use dependent, with larger decreases observed at 3 than at 1 Hz. In guinea pig ventricular myocytes, NE-10064 (0.3-3 microM) significantly prolonged action potential duration (APD) at 1 Hz. At 3 Hz, NE-10064 (0.3-1 microM) increased APD only slightly, and at 10 microM decreased APD and the plateau potential. NE-10064 potently blocked the rapidly activating component of the delayed rectifier, IKr (IC50 0.4 microM), and inhibited IKs (IC50 3 microM) with nearly 10-fold less potency. NE-10064 (10 microM) did not block the inward rectifier potassium current (IKl). NE-10064 (10 microM) blocked the L-type calcium current (ICa) in a use-dependent manner; block was greater at 3 than at 1 Hz. We conclude that (a) NE-10064's block of potassium currents is relatively selective for IKr over IKs, (b) NE-10064 inhibits ICa in a use-dependent fashion, and (c) NE-10064's effects on ERP and tension in papillary muscle as well as APD and action potential plateau level in myocytes may be explained by its potassium and calcium channel blocking properties.

Voltage- and time-dependent block by perhexiline of K+ currents in human atrium and in cells expressing a Kv1.5-type cloned channel.

Perhexiline maleate is an antianginal drug that has been shown to have antiarrhythmic effects in humans. To examine whether some of these clinical observations could be caused by block of cardiac K+ channels, we examined the effects of perhexiline on a rapidly activating delayed rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in human embryonic kidney cells as well as a corresponding K+ current (the ultra-rapid delayed rectifier, IKur) in human atrial myocytes. With the use of inside-out macropatches, we found that perhexiline inhibited Kv1.5 current in a time- and voltage-dependent manner with an IC50 value of 1.5 x 10(-6) M at +50 mV. Perhexiline reduced Kv1.5 tail current amplitude and slowed its decay relative to control. These data are consistent with blockade of open channels, probably from the intracellular surface. Perhexiline (3 microM) also blocked IKur in human atrial myocytes. The block that was observed was both time- and voltage-dependent in qualitatively similar ways to block of Kv1.5 channels. However, the time-dependent block of IKur by perhexiline was somewhat slower and its voltage-dependence steeper relative to its effects on Kv1.5. These data indicate that perhexiline blocks both cloned and native human cardiac K+ channels. Blockade of one or more types of voltage-dependent K+ channels may explain some of the electrophysiological effects of perhexiline observed in humans.

Role of chloride currents in repolarizing rabbit atrial myocytes.

Rabbit atrial cells manifest a prominent transient outward K+ current (Ito1), but this current recovers slowly from inactivation and is unlikely to be important at physiological rates (3-5 Hz). Depolarization of rabbit atrial cells also elicits a transient Ca(2+)-dependent outward Cl- current (Ito2). To compare the relative magnitude of these transient outward currents at various rates, we applied whole cell voltage-clamp techniques to isolated rabbit atrial myocytes. Whereas peak Ito1 exceeded Ito2 at slow rates (0.1 Hz), Ito1 was strongly reduced as rate was increased (by 97 +/- 2%, mean +/- SE, at 4 Hz), while Ito2 was slightly reduced (by 28 +/- 4%, 4 Hz). The reversal potential of transient outward tail currents at 0.07 Hz was -49 +/- 9 mV, while at 2.5 Hz the reversal potential became -18 +/- 7 mV (calculated Cl- reversal potential -18 mV). The addition of the Cl- transport blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 150 microM) or the replacement of external Cl- with methanesulfonate inhibited a large part of the transient outward current elicited by depolarization at 4 Hz. DIDS and Cl- replacement increased action potential duration in both single rabbit atrial cells and multicellular rabbit atrial preparations. We conclude that the Ca(2+)-dependent Cl- current is substantially larger than the transient K+ current at physiological rates in the rabbit and is likely to play a more important role in action potential repolarization than the latter current in this tissue in vivo.

Effects of flecainide, quinidine, and 4-aminopyridine on transient outward and ultrarapid delayed rectifier currents in human atrial myocytes.

Antiarrhythmic drugs prevent atrial reentrant arrhythmias by prolonging atrial action potential duration and refractoriness. The ionic mechanisms by which antiarrhythmic drugs alter human atrial repolarization are poorly understood. The present study was designed to assess the concentration-, voltage-, time- and frequency-dependent effects of the antiarrhythmic agents quinidine and flecainide, as well as of the K+ channel blocker 4-aminopyridine, on the calcium-independent transient outward current (Ito1) and the ultrarapid delayed rectifier current (IKur) in isolated human atrial myocytes. Quinidine and flecainide blocked Ito1 at clinically relevant concentrations. Block of Ito1 by quinidine was use and frequency dependent, whereas block by flecainide was frequency independent, and 4-aminopyridine showed use-dependent unblocking. Depolarizing prepulses enhanced flecainide block and reduced 4-aminopyridine block in a fashion suggesting a preferential interaction with the inactivated state for flecainide and with the resting, closed state for 4-aminopyridine. Quinidine block depended on the potential of a depolarizing test pulse in a fashion suggesting open channel block. All three drugs accelerated channel inactivation during depolarization at 1 Hz and failed to block Ito1 during initial current rise, with block appearing with time constants of 6.3 +/- 1.2 msec for flecainide, 14.5 +/- 4.2 msec for quinidine and 3.0 +/- 0.9 msec for 4-aminopyridine at 16 degrees C, suggesting a role for channel opening in block development. Quinidine blocked IKur at clinical concentrations, whereas flecainide had no effect on IKur. Quinidine block of IKur was voltage dependent, with part of the voltage dependence attributable to open-channel block and the remainder compatible with a blocking site within the voltage field at a position subject to 23% of the total electrical field. Quinidine's blocking actions on IKur were similar to those previously reported for block of a cardiac K+ channel clone of the Shaker family (Kv1.5), for which IKur is believed to be the equivalent native current. These results indicate that flecainide and quinidine block Ito1, and quinidine blocks IKur, in human atrial myocytes in a state-dependent fashion. Because drug effects are manifest at clinically relevant concentrations, and Ito1 and IKur have been shown to be potentially important currents in human atrial repolarization, these findings are relevant to understanding the ionic mechanisms underlying the clinical antiarrhythmic properties of these drugs.

Rapid and slow components of delayed rectifier current in human atrial myocytes.

OBJECTIVE: Previous studies in guinea pig heart cells have shown pharmacologically and kinetically distinct components of the classical delayed rectifier current (IK), generally referred to as IKr (rapid IK) and IKs (slow IK). This study was designed to determine whether the human heart contains corresponding components. METHODS: The whole cell voltage clamp technique was used to study IK in single myocytes isolated from human right atrial appendages removed at the time of aortocoronary artery bypass surgery. RESULTS: The activation of IK was best fitted by a biexponential relation, with time constants averaging 204(SEM 20) and 1080(197) ms at +10 mV. IK was inhibited by the specific IKr blocker E-4031 (5 microM), with the drug sensitive and drug resistant components having markedly different kinetic properties. The E-4031 sensitive current activated rapidly, while the drug resistant component activated more slowly, and the activation time courses of E-4031 sensitive and resistant currents paralleled the rapid and slow components of IK between -20 and +50 mV. The E-4031 sensitive component showed strong inward rectification, a half activation voltage (V 1/2) of -14.0(3.3) mV and a slope factor (k) of 6.5(1.5) mV, while the E-4031 resistant current had a linear current-voltage relationship, and values of +19.9(4.2) mV and 12.7(2.5) mV for V 1/2 and k respectively. The envelope of tails analysis showed a time dependent change in IKtail/IKstep under control conditions, and E-4031 strongly reduced the time dependent variation, suggesting that the E-4031 resistant current consisted of one dominant component. CONCLUSIONS: (1) IK in human atrium shows kinetically distinguishable rapid and slow components. (2) These components correspond to E-4031 sensitive and resistant currents. (3) The kinetics and voltage dependence of the rapid (E-4031 sensitive) and slow (E-4031 resistant) components correspond to properties previously described in guinea pig myocytes. These findings have important potential implications for understanding the mechanisms of human atrial repolarisation and its regulation by the autonomic nervous system and antiarrhythmic drugs.

Choline chloride activates time-dependent and time-independent K+ currents in dog atrial myocytes.

Using the whole cell configuration of the patch-clamp technique, we studied the effect of isotonic replacement of bath sodium chloride (NaCl) by choline chloride (ChCl) in dog atrial myocytes. Our results show that ChCl triggered 1) activation of a time-independent background current, characterized by a shift of the holding current in the outward direction at potentials positive to the K+ equilibrium potential (EK), and 2) activation of a time- and voltage-dependent outward current, following depolarizing voltage steps positive to EK. Because the choline-induced current obtained by depolarizing steps exhibited properties similar to the delayed rectifier K+ current (IK), we named it IKCh. The amplitude of IKCh was determined by extracellular ChCl concentration, and this current was generally undetectable in the absence of ChCl. IKCh was not activated by acetylcholine (0.001-1.0 mM) or carbachol (10 microM) and could not be recorded in the absence of ChCl or when external NaCl was replaced by sucrose or tetramethylammonium chloride. IKCh was inhibited by atropine (0.01-1.0 microM) but not by the M1 antagonist pirenzepine (up to 10 microM). This current was carried mainly by K+ and was inhibited by CsCl (120 mM, in the pipette) or barium (1 mM, in the bath). We conclude that in dog atrial myocytes, ChCl activates a background conductance comparable to ACh-dependent K+ current, together with a time-dependent K+ current showing properties similar to IK.

Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents.

Depolarization of human atrial myocytes activates a transient outward current that rapidly inactivates, leaving a sustained outward current after continued depolarization. To evaluate the ionic mechanism underlying this sustained current (Isus), we applied whole-cell voltage-clamp techniques to single myocytes isolated from right atrial specimens obtained from patients undergoing coronary bypass surgery. The magnitude of Isus was constant for up to 10 seconds at +30 mV and was unaffected by 40 mmol/L tetraethylammonium, 100 nmol/L dendrotoxin, 1 mmol/L Ba2+, 0.1 mumol/L atropine, or removal of Cl- in the superfusate. Isus could be distinguished from the 4-aminopyridine (4AP)-sensitive transient outward current (Ito1) by differences in voltage-dependent inactivation (1000-millisecond prepulse to -20 mV reduced Ito1 by 91.7 +/- 0.1% [mean +/- SEM], P < .001, versus 9.4 +/- 0.4% reduction of Isus) and 4AP sensitivity (IC50 for block of Ito1, 1.96 mmol/L; for Isus, 49 mumol/L). Isus activation had a voltage threshold near -30 mV, a half-activation voltage of -4.3 mV, and a slope factor of 8.0 mV. Isus was not inactivated by 1000-millisecond prepulses but was reduced by 16 +/- 8% (P < .05) at a holding potential of -20 mV relative to values at a holding potential of -80 mV. Isus activated very rapidly, with time constants (tau) at 25 degrees C ranging from 18.2 +/- 1.8 to 2.1 +/- 0.2 milliseconds at -10 to +50 mV, two orders of magnitude faster than previously described kinetics of the rapid component of the delayed rectifier K+ current. At 16 degrees C, Isus activation was greatly slowed (tau at +10 mV, 46.7 +/- 4.1 milliseconds; tau at 25 degrees C, 7.1 +/- 0.8 milliseconds; P < .01), and the envelope of tails test was satisfied. The reversal potential of Isus tail currents changed linearly with log [K+]o (slope, 55.3 +/- 2.9 mV per decade), and the fully activated current-voltage relation showed substantial outward rectification. Selective inhibition of Isus with 50 mumol/L 4AP increased human atrial action potential duration by 66 +/- 11% (P < .01). In conclusion, Isus in human atrial myocytes is due to a very rapidly activating delayed rectifier K+ current, which shows limited slow inactivation, is insensitive to tetraethylammonium, Ba2+, and dendrotoxin, and is highly sensitive to 4AP. These properties resemble the characteristics of channels encoded by the Kv1.5 group of cardiac cDNAs and may represent a physiologically significant manifestation of such channels in human atrium.

Mechanism of flecainide's rate-dependent actions on action potential duration in canine atrial tissue.

Increases in action potential duration (APD) caused by most antiarrhythmic drugs are maximal at slow rates and are attenuated during tachycardia, causing decreased action during arrhythmias and maximum effects during sinus rhythm. This property, "reverse use-dependence," limits efficacy and contributes to proarrhythmic potential. We have shown that the class 1c antiarrhythmic drug flecainide increases atrial APD to a greater extent at faster rates and that this property may underlie some of the drug's antiarrhythmic actions. The present studies were designed to evaluate possible underlying ionic mechanisms. Standard whole-cell voltage clamp and microelectrode techniques were used to study ionic currents and action potentials of canine atrial tissue. Flecainide (4.5 microM) increased APD at cycle lengths ranging from 150 to 1000 msec and attenuated the APD shortening that resulted from increased activation rate, resulting in greater APD prolongation at faster rates. The major time-dependent outward current (Ito), was reduced by flecainide in a rate-independent fashion. Flecainide's effect on Ito was due to inhibition of the 4-aminopyridine-sensitive component (Ito1); flecainide did not alter inward calcium current or the calcium-sensitive component of Ito (Ito2). The specific sodium channel blocker tetrodotoxin (1 microM) and the Na+, K(+)-ATP'ase inhibitor ouabain (1 microM) suppressed rate-dependent APD shortening in a fashion similar to flecainide, and both flecainide and ouabain attenuated postoverdrive membrane hyperpolarization. We conclude that the rate-dependence of flecainide's action on APD is not explained by use-dependent changes in outward currents but may be due to sodium channel blockade resulting in decreased sodium loading and reduced Na+, K(+)-ATP'ase stimulation during tachycardia.

Delayed rectifier outward current and repolarization in human atrial myocytes.

Previous work has suggested that the primary time-dependent repolarizing current in human atrium is the transient outward current (Ito), but interventions known to alter the magnitude of the delayed rectifier current (IK) affect atrial electrophysiology and arrhythmias in humans. To explore the potential role of IK in human atrial tissue, we used the whole-cell configuration of the patch-clamp technique to record action potentials and ionic currents in isolated myocytes from human atrium. A delayed outward current was present in the majority of myocytes, activating with a time constant ranging from 348 +/- 61 msec (mean +/- SEM) at -20 mV to 129 +/- 25 msec at +60 mV. The reversal potential of tail currents was linearly related to log [K+]o with a slope of 55 mV per decade, and fully activated tail currents showed inward rectification. The potassium selectivity, kinetics, and voltage dependence were similar to those reported for IK in other cardiac preparations. In cells with both Ito and IK, IK greatly exceeded both components of Ito (Ito1 and Ito2) within 50 msec of a voltage step from -70 to +20 mV. Based on the relative magnitude of Ito and IK, three types of cells could be distinguished: type 1 (58% [73/126] of the cells) displayed a large Ito together with a clear IK, type 2 (13% [17/126] of the cells) displayed only IK, and type 3 (29% [36/126] of the cells) was characterized by a prominent Ito and negligible IK. Consistent differences in action potential morphology were observed, with type 2 cells having a higher plateau and steeper phase 3 slope and type 3 cells showing a triangular action potential and lesser phase 3 slope compared with type 1 cells. We conclude that IK is present in a majority of human atrial myocytes and may play a significant role in their repolarization and that previously observed variability in human atrial action potential morphology may be partially due to differences in the relative magnitude of time-dependent outward currents.

Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current.

In human myocardium, the nature of the K+ currents mediating repolarization of the action potential is still speculative. Delayed rectifier channels have recently been cloned from human myocardium, but it is unclear whether or not these currents are involved in the termination of the cardiac action potential plateau. In intact human atrial myocytes, we have identified a rapid delayed rectifier K+ current with properties and kinetics identical to those expressed by a K+ channel clone (fHK) isolated from human heart and stably incorporated into a human cell line for the first time. The myocyte current amplitude was 3.6 +/- 0.2 pA/pF (at +20 mV, n = 15) and activated with a time constant of 13.1 +/- 2 milliseconds at 0 mV (n = 15). The half-activation potential (V0.5) was -6 +/- 2.5 mV (n = 10) with a slope factor (k) of 8.6 +/- 2.2 (n = 10). The heterologously expressed fHK current amplitude was 136 pA/pF (at +20 mV, n = 9) with an activation time constant of 11.8 +/- 4.6 milliseconds at 0 mV; V0.5 was 4.1 +/- 2.4 mV (mean +/- SEM, n = 8); and k was 7.0. The conductance of single fHK channels was 16.9 picosiemens in 5 mM bath K+. Both native and cloned channel currents inactivated partially during sustained depolarizing pulses. Both currents were blocked by micromolar concentrations of 4-aminopyridine and were relatively insensitive to tetraethylammonium ions and class III antiarrhythmic agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium channel blocking properties of propafenone in rabbit atrial myocytes.

Propafenone, a class 1c antiarrhythmic agent, is known to be a potent blocker of voltage-dependent sodium channels; however, several clinical actions of the drug point toward possible potassium channel blocking capability. The present experiments were designed to assess the extent and potential mechanisms of potassium channel blocking properties of propafenone. Whole-cell voltage-clamp techniques were used to define the actions of propafenone on the transient outward current (Ito), the delayed rectifier current (Ik) and the inward rectifier current (Ik1) in isolated rabbit atrial myocytes. Propafenone blocked all three currents, with the extent of blockade being independent of test potential During depolarizing voltage steps, block of Ito and Ik developed as an exponential function of time, consistent with time-dependent open channel blockade. The rate constant of block onset was concentration dependent. The inactivation of Ito was a monoexponential function of time under control conditions, with a time constant averaging 19.1 +/- 1.3 msec (mean +/- S.E.) at +10 mV. Propafenone accelerated Ito inactivation, resulting in a biexponential process having time constants of 5.1 +/- 0.9 (P < .001 vs. control) and 23.5 +/- 2.0 msec (P = N.S. vs. control) at 5 microM and 3.4 +/- 0.5 (P < .001 vs. control) and 28.5 +/- 4.3 msec (P = N.S.) at 10 microM concentrations, respectively. The rapid phase inactivation time constants were of the same order as time constants for the onset of block (3.1 +/- 0.6 and 1.8 +/- 0.3 msec at 5 and 10 microM respectively), suggesting that the acceleration of Ito inactivation was due to open channel block by the drug. The IC50 for blockade was substantially less for effects on Ik (0.76 microM; 95% confidence limits 0.44-1.30 microM) than for Ito (5.91 microM; 95% confidence limits 4.19-8.33 microM) or Ik1 (7.10; 5.24-9.61 microM). We conclude that 1) propafenone is an efficacious potassium channel blocker; 2) propafenone blockade of time-dependent potassium currents is open-state dependent; and 3) propafenone block of potassium currents is relatively selective for Ik.

Modulation of flecainide's cardiac sodium channel blocking actions by extracellular sodium: a possible cellular mechanism for the action of sodium salts in flecainide cardiotoxicity.

Sodium salts reverse the clinical cardiotoxicity of class 1c antiarrhythmic agents, but the underlying mechanisms are unknown. We studied the modulation of flecainide's action by changes in extracellular sodium concentration ([Na+]e) produced by isotonic substitution of choline for sodium. Increasing [Na+]e by 25 mM attenuated the depressant effects of 3.2 microM flecainide of Vmax in canine cardiac Purkinje fibers, whereas decreasing [Na+]e enhanced drug action. The voltage dependence of Vmax was shifted by flecainide (activation potential for 50% decrease in Vmax, V50: -77.4 +/- 3.5 mV at 3.2 microM flecainide) compared to control (V50: -73.7 +/- 2.8 mV, mean +/- S.D., P < .05). Increasing [Na+]e in the presence of flecainide returned V50 toward control (-75.8 +/- 3.1 mV, P < .05 vs. flecainide at normal [Na+]e). Increased [Na+]e shifted the flecainide concentration-response curve to the right (EC50 19.0 microM) compared to normal (EC50 14.6 microM) and low (EC50 10.8 microM) [Na+]e. [Na+]e modulated the concentration-dependent displacement by flecainide of [3H]batrachotoxin-A-benzoate, with increased [Na+]e shifting the binding curve to the right and decreased [Na+]e shifting it to the left compared to normal [Na+]e. There was a strong linear correlation (r = 0.99) between flecainide's EC50 for Vmax depression and its IC50 for [3H]batrachotoxin-A-benzoate displacement at various [Na+]e. We conclude that [Na+]e modulates flecainide's interaction with the sodium channel. Sodium's ability to displace blocking drug from the sodium channel may underlie the efficacy of sodium salts in treating flecainide toxicity, and could play a similar role in antagonizing cardiotoxicity of other class 1 compounds.

Differences in rate dependence of transient outward current in rabbit and human atrium.

Both human and rabbit atrial cells possess a large 4-aminopyridine-sensitive transient outward current (I(to1)). However, the slow reactivation of this current in rabbits suggests that its role may be limited to very slow heart rates. We used whole cell voltage-clamp recordings to evaluate the rate dependency of I(to1) in rabbit and human atrial myocytes. Our results show that at physiological temperatures in human atrium, I(to1) is rate independent at rates between 0.1 and 4.0 Hz. Peak I(to1) at 4.0 Hz in rabbit was 3.4 +/- 1.4% (mean +/- SE) of current at 0.1 Hz (P < 0.001, n = 8), whereas in humans, peak I(to1) at 4.0 Hz averaged 88.8 +/- 6.1% of the current at 0.1 Hz (P > 0.05, n = 7). These differences were due to marked discrepancies in reactivation time course, which was biexponential with time constants that averaged 650 +/- 159 ms and 8.4 +/- 1.1 s in rabbit (n = 8) compared with a single exponential time constant of 33.6 +/- 6.8 ms (n = 8) in human atrium (both at 30 degrees C). These findings suggest that I(to1) can contribute importantly to atrial repolarization at all physiological heart rates in humans. Furthermore, these results emphasize that there are important interspecies variations in the rate dependence of I(to1), which need to be considered in understanding the physiological and pharmacological regulation of atrial repolarization.