With regard to currently available class III agents, although their class III effect may reduce the likelihood of tachycardia initiation, their reverse use-dependent prolongation of action potential duration reduces their effectiveness during tachycardias and may even render them proarrhythmic, especially after long diastolic intervals. In contrast, agents that exhibit normal use-dependent prolongation of refractoriness hold great promise: While having relatively less effects on the normal heart beat, they could induce self-termination of a tachycardia. Prolongation of refractoriness can be achieved by lengthening of action potential duration and delaying recovery of excitability. Combination of these drug actions may yield important clinical applications.

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4-aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

BackgroundBupivacaine is a potent local anesthetic widely used for prolonged local and regional anesthesia. However, accidental intravascular injection of bupivacaine can produce severe arrhythmias and cardiac depression. Although used clinically as a racemic mixture,S(−)-bupivacaine appears less toxic than theR(+)-enantiomer despite at least equal potency for local anesthesia. If theR(+)-enantiomer is more potent in blocking cardiac sodium channels, then theS(−)-enantiomer could be used with less chance of cardiovascular toxicity. Therefore, we tested whether such stereoselectivity existed in the bupivacaine affinity for the cardiac sodium channel.Methods and ResultsThe inhibitory effects on the cardiac sodium current (I_Na) of 10 μmol/LR(+)- andS(−)-bupivacaine were investigated by use of the whole-cell voltage clamp technique in isolated guinea pig ventricular myocytes. Both enantiomers produced similar but limited levels of tonic block (6% and 8%). During long depolarizations (5 seconds at 0 mV),R(+)-bupivacaine induced a significantly larger inhibition of I_Na: 72±2% versus 58±3% for theS(−)-enantiomer (P<.01). Development of block was slow, but its rate was faster forR(+)-bupivacaine [time constant, 1.84±0.16 versus 2.56±0.26 seconds for theS(−)-enantiomer,P<.05]. The voltage dependence of the availability of the Na⁺current was shifted to more hyperpolarizing potentials compared with the control;R(+)-bupivacaine induced a larger shift thanS(−)-bupivacaine (37±2 versus 30±2 mV,P<.05). These data indicate stereoselective interactions with the inactivated state. In addition, both enantiomers induced substantial use-dependent block during 2.5-Hz pulse trains with medium (100-ms) and short (10-ms) depolarizations but without stereoselective difference. A stepwise approach was used to model these experimental results and to derive apparent affinities and rate constants. We initially assumed that bupivacaine interacted only with the rested and inactivated states of the Na⁺channel. The apparent affinities of the inactivated state forS(−)- andR(+)-bupivacaine were 4.8 and 2.9 μmol/L, respectively. With the derived binding and unbinding rate constants, this model reproduced the stereoselective block during long depolarizations but failed to predict the use-dependent block induced by trains of short (10-ms) depolarizations. To account for the observed use-dependent interactions, it was necessary to include interactions with the activated state, which resulted in adequate reproduction of the experimental results. The apparent affinities of the activated or open state forS(−)- andR(+)-bupivacaine were 4.3 and 3.3 μmol/L, respectively.ConclusionsBoth the large level of pulse-dependent block and the failure of the pure inactivated-state block model indicate that bupivacaine interacts with the activated (or open) state of the cardiac sodium channel in addition to its block of the inactivated state. The bupivacaine-induced block of the inactivated state of the Na⁺channel displayed stereoselectivity, withR(+)-bupivacaine interacting faster and more potently. Both enantiomers also bind with high affinity to the activated or open state of the channel, but this interaction did not display stereoselectivity, although the binding to the activated or open state was faster forS(−)- than forR(+)-bupivacaine. The higher potency ofR(+)-bupivacaine to block the inactivated state of the cardiac Na⁺channel may explain its higher toxicity because of the large contribution of the inactivated-state block during the plateau phase of the cardiac action potential. These results would support the use of theS(−)-enantiomer to reduce cardiac toxicity.

Voltage-gated K ⁺ channels control excitability in neuronal and various other tissues. We identified three unique α-subunits of voltage-gated K ⁺ -channels in the human genome. Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1. Although they have all of the hallmarks of voltage-gated K ⁺ channel subunits, they did not produce K ⁺ currents when expressed in mammalian cells. Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum. Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1. Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents. This heteromerization was confirmed by co-immunoprecipitation. The Kv2 subfamily consists of only two members and uses interaction with “silent subunits” to diversify its function. Including the subunits described here, the “silent subunits” represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.

Recent advances in molecular biology have had a major impact on our understanding of the biophysical and molecular properties of ion channels. This review is focused on cardiac potassium channels which, in general, serve to control and limit cardiac excitability. Approximately 60 K+ channel subunits have been cloned to date. The (evolutionary) oldest potassium channel subunits consist of two transmembrane (Tm) segments with an intervening pore-loop (P). Channels formed by four 2Tm-1P subunits generally function as inwardly rectifying K(+)-selective channels (KirX.Y): they conduct substantial current near the resting potential but carry little or no current at depolarized potentials. The inward rectifier IK1 and the ligand-gated KATP and KACh channels are composed of such subunits. The second major class of K+ channel subunits consists of six transmembrane segments (S1-S6). The S5-P-S6 section resembles the 2Tm-1P subunit, and the additional membrane-spanning segments (especially the charged S4 segment) endow these 6Tm-1P channels with voltage-dependent gating. For both major families, four subunits assemble into a homo- or heterotetrameric channel, subject to specific subunit-subunit interactions. The 6Tm-1P channels are closed at the resting potential, but activate at different rates upon depolarization to carry sustained or transient outward currents (the latter due to inactivation by different mechanisms). Cardiac cells typically display at least one transient outward current and several delayed rectifiers to control the duration of the action potential. The molecular basis for each of these currents is formed by subunits that belong to different Kvx.y subfamilies and alternative splicing can contribute further to the diversity in native cells. These subunits display distinct pharmacological properties and drug-binding sites have been identified. Additional subunits have evolved by concatenation of two 2Tm-1P subunits (4Tm-2P); dimers of such subunits yield voltage-independent leak channels. A special class of 6Tm-1P subunits encodes the 'funny' pacemaker current which activates upon hyperpolarization and carries both Na+ and K+ ions. The regional heterogeneity of K+ currents and action potential duration is explained by the heterogeneity of subunit expression, and significant changes in expression occur in cardiac disease, most frequently a reduction. This electrical remodelling may also be important for novel antiarrhythmic therapeutic strategies. The recent crystallization of a 2Tm-1P channel enhances the outlook for more refined molecular approaches.

The long QT-related arrhythmia torsades de pointes (TdP) can arise with mutations in HERG and during treatment with drugs that block cardiac I Kr , the current encoded by HERG. Multiple test systems have been used to assess drug block of I Kr . This study evaluated the I Kr blocking potency of a series of antiarrhythmics associated with a range of clinical risks of TdP in two such systems: mouse AT-1 cells (in which I Kr is the major repolarizing current) and Ltk cells transiently transfected with HERG (n = 4–10 cells per drug). For each compound, the concentration required to produce 50% block of I Kr or HERG tail currents (IC 50 ) was determined. There was an excellent correlation (r = 0.98, p < 10 −5 ) between values obtained in the two systems. However, the relation between the liability of a drug to cause TdP appeared dissociated from I Kr blocking potency. Quinidine, dofetilide, ibutilide, procainamide, and disopyramide are all associated with TdP, but only the first three were potent blockers (IC 50 ≤ 1 μM), whereas procainamide and disopyramide were not (IC 50 > 50 μM). Conversely, verapamil and amiodarone, drugs not associated with TdP, were also blockers (IC 50 ≤ 1 μM). We conclude that I Kr blocking potency can be readily assessed in either AT-1 cells or systems in which HERG is heterologously expressed. However, not all drugs causing TdP are potent I Kr blockers, and I Kr block is not necessarily associated with TdP. Other properties of these drugs, therefore, contribute to their propensity to cause TdP.