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.

The mechanism of inhibition of Na⁺channels by lidocaine has been suggested to involve low-affinity binding to rested states and high-affinity binding to the inactivated state of the channel, implying either multiple receptor sites or allosteric modulation of receptor affinity. Alternatively, the lidocaine receptor may be guarded by the channel gates. To test these distinct hypotheses, inhibition of Na⁺channels by lidocaine was studied by voltage-clamp methods in both native and heterologous expression systems. Native Na⁺channels were studied in guinea pig ventricular myocytes, and recombinant human heart Na⁺channels were expressed inXenopus laevisoocytes. Fast inactivation was eliminated by mutating three amino acids (isoleucine, phenylalanine, and methionine) in the III-IV interdomain to glutamines or by enzymatic digestion with α-chymotrypsin. In channels with intact fast inactivation, lidocaine block developed with a time constant of 589±42 ms (n=7) at membrane potentials between −50 and +20 mV, as measured by use of twin pulse protocols. The IC₅₀was 36±1.8 μmol/L. Control channels inactivated within 20 ms, and slow inactivation developed much later (time constant of slow inactivation, 6.2±0.36 s). The major component of block developed long after activated and open channels were no longer available for drug binding. Control channels recovered fully from inactivation in <50 ms at −120 mV (time constant, 11±0.5 ms; n=50). In 30 μmol/L lidocaine, 49±3% of the current recovered with a second larger time constant of 398±46 ms (n=5). After removal of fast inactivation, either enzymatically or by mutagenesis, the channels were no longer blocked by 50 μmol/L lidocaine (n=8). Much higher concentrations of lidocaine produced a tonic block (IC₅₀, 0.4±0.07 mmol/L; n=13) and time dependence suggestive of open-channel block. The results indicate the importance of inactivated state block by therapeutic concentrations of lidocaine and suggest that the molecular site of action is the structural region of the channel that is responsible for inactivation.

Background Zatebradine is a bradycardic agent that inhibits the hyperpolarization-activated current (I _f ) in the rabbit sinoatrial node. It also prolongs action potential duration in papillary muscles in guinea pigs and in Purkinje fibers in rabbits. The underlying mechanism by which zatebradine induces this effect has not been explored, but it is likely to involve K ⁺ channel block. Methods and Results Cloned human cardiac K ⁺ delayed rectifier currents (hKv1.5) were recorded in Ltk ⁻ cells transfected with their coding sequence. Zatebradine 10 μmol/L did not modify the initial activation time course of the current but induced a subsequent decline to a lower steady-state current level with a time constant of 109±16 ms. Zatebradine inhibited hKv1.5 with an apparent K _D of 1.86±0.14 μmol/L. Block was voltage dependent (electrical distance δ=0.177±0.003) and accumulated in a use-dependent manner during 0.5- and 1-Hz pulse trains because of slower recovery kinetics in the presence of the drug. Zatebradine reduced the tail current amplitude, recorded at −30 mV, and slowed the deactivation time course, which resulted in a “crossover” phenomenon when control and zatebradine tail currents were superimposed. Conclusions These results indicate that (1) zatebradine is an open-channel blocker of hKv1.5, (2) binding occurs in the internal mouth of the ion pore, (3) unbinding is required before the channel can close, and (4) zatebradine-induced block is use dependent because of slower recovery kinetics in the presence of the drug. These effects may explain the prolongation of the cardiac action potential and could be clinically relevant.

Voltage-dependent potassium (K_v) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K_v channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K⁺ currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K_v1.3/K_v1.5 channels, which generate the main K_v in macrophages. An increase in K⁺ current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K_v1.3 subunits in the K_v1.3/K_v1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K_v1.3 expression. Neither of these treatments apparently altered the expression of K_v1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K_v1.3/K_v1.5 hybrid channels by altering the subunit stoichiometry.