In a perspective,' it was pointed out At present, class III antiarrhythmic agents2 are favored increasingly to treat patients with serious tachycardias. Although these agents could be very powerful antiarrhythmics, our investigation suggests that the currently available drugs have electrophysiologic features that render them relatively less effective than an ideal class III agent could be and may even render them proarrhythmic. We review briefly the kinetic effects on action potential duration of current class III agents. Desirable properties for lengthening of action potential duration are derived, and a simple model illustrating the time-and voltagedependent actions of a hypothetical compound is developed. Finally, we apply current knowledge of sodium and potassium channel blockade to illustrate how therapy combining class I and class III effects might prove to be especially effective.

Voltage-gated K ؉ channels control excitability in neuronal and various other tissues. We identified three unique ␣-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.electrically silent subunits ͉ ER retention ͉ heterotetrameric assembly ͉ KCNG3

The electrophysiological properties of HK2 (Kvl.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°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 ~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-HodgkinKatz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher

The antiarrhythmic agent quinidine blocks the human cardiac hKv1.5 channel expressed in mammalian cells at therapeutically relevant concentrations (EC50, 6.2 mumol/L). Mechanistic analysis has suggested that quinidine acts as a cationic open-channel blocker at a site in the internal mouth of the ionic pore and that binding is stabilized by hydrophobic interactions. We tested these hypotheses using site-directed mutagenesis of residues proposed to line the internal mouth of the channel or of nearby residues. Amino acid substitutions in the midsection of S6 (T505I, T505V, T505S, and V512A) reduced the dissociation rate for quinidine, increased the affinity (0.7, 1.5, 3.4, and 1.4 mumol/L, respectively), and preserved both the voltage-dependent open channel-block mechanism and the electrical binding distance (0.19 to 0.22). In contrast, smaller or nonsignificant effects were observed for: deletion of the intracellular C-terminal domain, charge neutralizations in the region immediately C-terminal to S6, elimination of aromatic residues in S6, and mutations at the putative internal turn of the P loop, at the external entrance of the pore, and at sites in the S4S5 linker. The approximately 10-fold increase in affinity with T505I and the reduction of the dissociation rate constant with the mutations that increased affinity are consistent with a hydrophobic stabilization of binding. Moreover, the T505 and V512 residues align on the same side of the putative alpha-helical S6 segment. Taken together, these results localize the hydrophobic binding site for this antiarrhythmic drug in the internal mouth of this human K+ channel and provide molecular support for the open channel-block model and the role of S6 in contributing to the inner pore.

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.