Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Electrophysiological and pharmacological correspondence between Kv4.2 current and rat cardiac transient outward current.

OBJECTIVE: The transient outward current (ITO) plays an important role in early repolarization and overall time course of the cardiac action potential. At least two K+ channel alpha-subunits cloned from cardiac tissue (Kv1.4 and Kv4.2) encode rapidly inactivating channels. The goal of this study was to determine functional and pharmacological properties of Kv4.2 expressed in mammalian cells, especially those that would differentiate between both isoforms in comparison to native ITO. METHODS: Both Kv4.2 and Kv1.4 isoforms were stably expressed in mouse L-cell lines, and expressed currents were studied using whole-cell voltage clamp techniques. RESULTS: The expressed Kv4.2 currents displayed fast inactivation with a half-inactivation potential of -41 mV. Recovery from inactivation was rapid (tau recov = 160 ms at -90 mV) and strongly voltage-dependent. Flecainide (10 microM) had minimal effects on Kv1.4 currents, but reduced Kv4.2 peak current by 53% and increased the apparent rate of inactivation consistent with open channel block. Quinidine (10-20 microM) reduced the peak current and accelerated the apparent rate of inactivation in both isoforms. The Kv4.2 current displayed use-dependent unblock in the presence of 4-AP. CONCLUSIONS: The functional properties of Kv4.2, especially the flecainide sensitivity, resemble those of ITO in rat (and human) myocytes better than those of Kv1.4. These results provide the necessary functional support for the hypothesis that Kv4.2 is a major isoform contributing to cardiac ITO, consistent with independent biochemical and molecular evidence that indicates that Kv4.2 is readily detected in rat myocytes.

Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding.

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.

Determinants of antiarrhythmic drug action. Electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel.

The molecular basis of antiarrhythmic drug action is still poorly understood. We recently reported that block of the human cardiac hKv1.5 channel by quinidine displayed similarity with internal quaternary ammonium block of squid and Shaker potassium channels. To gain further insight into the molecular determinants of the affinity and the stereoselectivity of antiarrhythmic drug action, we studied the effects of quinine (a diastereomer of quinidine), clofilium (a quaternary ammonium class III agent), and tetrapentylammonium (TPeA, a biophysical reference probe for the internal quaternary ammonium binding site). For all compounds, block was voltage dependent, with a steep increase over the voltage range of channel opening and a superimposed weaker voltage dependence at more positive potentials. The latter electrostatic component was similar for all drugs, consistent with a binding reaction sensing approximately 20% of the transmembrane electrical field. Clofilium and TPeA displayed a higher apparent affinity (0.15 and 0.28 mumol/L, respectively), and quinine displayed a lower one (21 mumol/L) compared with quinidine (6.2 mumol/L). Block development upon depolarization was time dependent for clofilium and TPeA but slow compared with quinidine. A time-dependent component was difficult to resolve for quinine, but the time course of deactivating tail currents was slower than in the control condition. The resulting crossover phenomenon was also observed for the quaternary drugs. Compared with TPeA alone, the combined application of quinine and TPeA resulted in a reduced current that decayed slower, consistent with competition.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of highly conserved C-terminal sequences in the Kv1 K+ channel sub-family does not prevent expression of currents with wild-type characteristics.

The C-terminal regions of Kv1 K+ channels show little conservation between isoforms except for the last four C-terminal residues, (E/L)TDV, which are well conserved from Drosophila to man. Deletions of the 4, 16, and 57 C-terminal residues of the human Kv1.5 channel did not affect whole cell current amplitude, midpoint of activation, degree of inactivation, or activation kinetics following expression in mouse L-cells. Similar results were obtained with the rat Kv1.1 channel. Therefore, the conserved (E/L)TDV motif, and most of the C-terminal amino acids, are not required for Kv1 channel expression.