A mutation in the local anaesthetic binding site abolishes toluene effects in sodium channels

Toluene is a solvent of abuse that inhibits cardiac sodium channels in a manner that resembles the action of local anaesthetics. The purpose of this work was to analyze toluene effects on skeletal muscle sodium channels with and without beta1 subunit (Nav1.4+beta1 and Nav1.4-beta1, respectively) expressed in Xenopus laevis oocytes and to compare them with those produced in the F1579A mutant channel lacking a local anaesthetic binding site. Toluene inhibited Nav1.4 sodium currents (IC50=2.7 mM in Nav1.4+beta1 and 2.2 mM in Nav1.4-beta1 in a concentration dependent way. Toluene (3 mM) blocked sodium currents in Nav1.4 channels proportionally throughout the entire current-voltage relationship producing inactivation at more negative potentials. Minimal inhibition was produced by 3 mM toluene in F1579A mutant channels. Recovery from inactivation was slower both in Nav1.4 and F1579A channels in the presence of 3 mM toluene. The solvent blocked sodium currents in a use-dependent and frequency-dependent manner in Nav1.4 channels. A single mutation in the local anaesthetic binding site of Nav1.4 channels almost abolished toluene effects. These results suggest that this site is important for toluene action.     

State-dependent mibefradil block of Na+ channels

Mibefradil is a T-type Ca2+ channel antagonist with reported cross-reactivity with other classes of ion channels, including K+, Cl-, and Na+ channels. Using whole-cell voltage clamp, we examined mibefradil block of four Na+ channel isoforms expressed in human embryonic kidney cells: Nav1.5 (cardiac), Nav1.4 (skeletal muscle), Nav1.2 (brain), and Nav1.7 (peripheral nerve). Mibefradil blocked Nav1.5 in a use/frequency-dependent manner, indicating preferential binding to states visited during depolarization. Mibefradil blocked currents of all Na+ channel isoforms with similar affinity and a dependence on holding potential, and drug off-rate was slowed at depolarized potentials (k(off) was 0.024/s at -130 mV and 0.007/s at -100 mV for Nav1.5). We further probed the interaction of mibefradil with inactivated Nav1.5 channels. Neither the degree nor the time course of block was dependent on the stimulus duration, which dramatically changed the residency time of channels in the fast-inactivated state. In addition, inhibiting the binding of the fast inactivation lid (Nav1.5 ICM + MTSET) did not alter mibefradil block, confirming that the drug does not preferentially interact with the fast-inactivated state. We also tested whether mibefradil interacted with slow-inactivated state(s). When selectively applied to channels after inducing slow inactivation with a 60-s pulse to -10 mV, mibefradil (1 microM) produced 45% fractional block in Nav1.5 and greater block (88%) in an isoform (Nav1.4) that slow-inactivates more completely. Our results suggest that mibefradil blocks Na+ channels in a state-dependent manner that does not depend on fast inactivation but probably involves interaction with one or more slow-inactivated state(s).     

Functional and pharmacological properties of human and rat NaV1.8 channels

The aim of this work is to characterise the functional properties of human and rat Na_V1.8 channels and to investigate the action of anti-nociceptive agents. Na_V1.8 α-subunits were expressed in mammalian sensory neuron-derived ND7/23 cells, and sodium currents were recorded using whole-cell patch clamp. The current-voltage curves for activation were similar for human and rat Na_V1.8 channels. However, for inactivation, human Na_V1.8 showed more hyperpolarised voltage-dependence than for the rat channel, faster development of inactivation, slower recovery from the fast component of inactivation, and faster recovery from the slow component. Thus, this would imply that the human channel is more inactivated at normal resting potentials. Compounds 227c89, A-803467, V102862, ralfinamide and tetracaine all showed greater affinity for the inactivated state than for the resting state. Compounds A-803467 and V102862 were the most potent, and A-803467 showed greater inactivated state affinity for human than for rat channels. Surprisingly, during recovery from inactivation, an increase in current was observed for V102862 and A-803467, probably due to disinhibition of resting block. Rather than the use-dependent inhibition normally seen with inactivated state blockers, for A-803467 this disinhibition led to an increase in current during repetitive stimulation, while V102862 showed less inhibition than otherwise expected at lower frequencies. Thus the data supports the suggestion that, while both V102862 and A-803467 are potent inhibitors of Na_V1.8, the compound V102862, rather than A-803467, may be useful as an analgesic where physiological firing frequencies are higher.     

State- and use-dependent block of muscle Nav1.4 and neuronal Nav1.7 voltage-gated Na+ channel isoforms by ranolazine

Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na(+) channels. However, ranolazine block of muscle and neuronal Na(+) channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na(+) currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na(+) channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC(50)) >/= 60 microM. Use-dependent block of Na(+) channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 microM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na(+) currents by ranolazine with estimated IC(50) values of 2.4, 6.2, and 1.7 microM, respectively. On- and off-rates of ranolazine were 8.2 microM(-1) s(-1) and 22 s(-1), respectively, for Nav1.4 open channels and 7.1 microM(-1) s(-1) and 14 s(-1), respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na(+) channels reduced the potency of ranolazine approximately 17-fold. We conclude that: 1) both muscle and neuronal Na(+) channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na(+) channels; and 3) ranolazine block of open Na(+) channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.     

Point mutations at L1280 in Nav1.4 channel D3-S6 modulate binding affinity and stereoselectivity of bupivacaine enantiomers

Local anesthetics (LAs) block voltage-gated sodium channels. Parts of the LA binding site are located in the pore-lining transmembrane segments 6 of domains 1, 3, and 4 (D1-S6, D3-S6, D4-S6). We suggested previously that residue N434 in D1-S6 interacts directly with bupivacaine enantiomers in inactivated channels because side-chain properties of different residues substituted at N434 correlated with changes in blocking potencies of bupivacaine enantiomers. Furthermore, mutation N434R exhibited significant stereoselectivity for block of inactivated channels that resulted from a selective decrease in block by S(-)-bupivacaine. In the present study, we analyzed the role of residue L1280 in D3-S6 of the rat skeletal muscle Nav1.4 channel in interactions with the enantiomers of bupivacaine. We substituted native leucine at L1280 with amino acids of different physicochemical properties. Wild-type and mutant channels were expressed transiently in human embryonic kidney 293t cells and were investigated under whole-cell voltage clamp. Block of resting mutant channels by bupivacaine enantiomers revealed little difference compared with wild-type channels. Block of inactivated channels was increased in a mutation containing an aromatic group (L1280W) and decreased in mutations containing a positive charge (L1280K, L1280R). Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels. More surprisingly, stereoselectivity resulted from a selective decrease in block by R(+)-bupivacaine, in contrast to mutation N434R in D1-S6. We propose that in inactivated channels, residues L1280 in D3-S6 and N434 in D1-S6 interact directly with LAs and thereby face each other in the ion-conducting pore.     

V102862 (Co 102862): a potent, broad-spectrum state-dependent blocker of mammalian voltage-gated sodium channels

1. 4-(4-Fluorophenoxy)benzaldehyde semicarbazone (V102862) was initially described as an orally active anticonvulsant with robust activity in a variety of rodent models of epilepsy. The mechanism of action was not known. We used whole-cell patch-clamp techniques to study the effects of V102862 on native and recombinant mammalian voltage-gated Na+ channels. 2. V102862 blocked Na+ currents (I(Na)) in acutely dissociated cultured rat hippocampal neurons. Potency increased with membrane depolarization, suggesting a state-dependent mechanism of inhibition. There was no significant effect on the voltage dependence of activation of I(Na). 3. The dissociation constant for the inactivated state (K(I)) was approximately 0.6 microM, whereas the dissociation constant for the resting state (K(R)) was >15 microM. 4. The binding to inactivated channels was slow, requiring a few seconds to reach steady state at -80 mV. 5. The mechanism of inhibition was characterized in more detail using human embryonic kidney-293 cells stably expressing rat brain type IIA Na+ (rNa(v)1.2) channels, a major Na+ channel alpha subunit in rat hippocampal neurons. Similar to hippocampal neurons, V102862 was a potent state-dependent blocker of rNa(v)1.2 channels with a K(I) of approximately 0.4 microM and K(R) approximately 30 microM. V102862 binding to inactivated channels was relatively slow (k(+) approximately = 1.7 microM(-1) s(-1)). V102862 shifted the steady-state availability curve in the hyperpolarizing direction and significantly retarded recovery of Na+ channels from inactivation. 6. These results suggest that inhibition of voltage-gated Na+ channels is a major mechanism underlying the anticonvulsant properties of V102862. Moreover, understanding the biophysics of the interaction may prove to be useful in designing a new generation of potent Na+ channel blocker therapeutics.     

Differential interaction of R-mexiletine with the local anesthetic receptor site on brain and heart sodium channel alpha-subunits

Mexiletine is a class I antiarrhythmic drug with neuroprotective effects in models of brain ischemia attributable to inhibition of brain sodium channels. We compared effects of R-mexiletine on wild-type and mutant rat brain (rbIIA) and heart (rh1) sodium channel alpha-subunits transiently expressed in tsA-201 cells. R-mexiletine induced tonic and frequency-dependent block and bound with a 26-fold (brain) or 35-fold (heart) higher affinity to inactivated sodium channels. Affinities of both resting and inactivated channels for R-mexiletine block were approximately 2-fold higher for heart than for brain channels. Mutations in transmembrane segment IVS6 of heart (rhF1762A) and brain (rbF1764A and rbY1771A) channels, which reduce block by other local anesthetics, reduced high-affinity block of inactivated channels and frequency-dependent block of open channels by R-mexiletine and abolished the difference in affinity between brain and heart sodium channels. Unlike previous local anesthetics studied, the strongest effect was observed for mutation rbY1771A. Comparison of mutations of the homologous phenylalanine residue in brain and heart channels showed striking differences in the effects of the mutations. rbF1764A reduced drug block by slowing R-mexiletine binding to inactivated channels, whereas rhF1762A reduced block by increasing the rate of dissociation from inactivated and resting channels. Thus, rbF1764/rhF1762 is a critical determinant of affinity and tissue-specific differences in mexiletine block of brain and heart sodium channels, but its role in drug interaction differs in these two channel isoforms.     

State-dependent verapamil block of the cloned human Ca(v)3.1 T-type Ca(2+) channel

Verapamil is a potent phenylalkylamine antihypertensive believed to exert its therapeutic effect primarily by blocking high-voltage-activated L-type calcium channels. It was the first clinically used calcium channel blocker and remains in clinical use, although it has been eclipsed by other calcium channel blockers because of its short half-life and interactions with other channels. In addition to blocking L-type channels, it has been reported to block T-type (low-voltage activated) calcium channels. This type of cross-reactivity is likely to be beneficial in the effective control of blood pressure. Although the interactions of T channels with a number of drugs have been described, the mechanisms by which these agents modulate channel activity are largely unknown. Most calcium channel blockers exhibit state-dependence (i.e., preferential binding to certain channel conformations), but little is known about state-dependent verapamil block of T channels. We stably expressed human Ca(v)3.1 T-type channels in human embryonic kidney 293 cells and studied the state-dependence of the drug with macroscopic and gating currents. Verapamil blocked currents at micromolar concentrations at polarized potentials similar to those reported for L-type channels, although unlike for L-type currents, it did not affect current time course. The drug exhibited use-dependence and significantly slowed the apparent recovery from inactivation. Current inhibition was dependent on potential. This dependence was restricted to negative potentials, although all data were consistent with verapamil binding in the pore. Gating currents were unaffected by verapamil. We propose that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel.     

The opioid methadone induces a local anaestheticlike inhibition of the cardiac Na+ channel,Nav1.5

Background and Purpose: Treatment with methadone is associated with severe cardiac arrhythmias, a side effect that seems to result from an inhibition of cardiac hERG K⁺ channels. However, several other opioids are inhibitors of voltage-gated Na⁺ channels. Considering the common assumption that an inhibition of the cardiac Na⁺ channel Na_v1.5, is the primary mechanism for local anaesthetic (LA)-induced cardiotoxicity, we hypothesized that methadone has LA-like properties leading to a modulation of Na_v1.5 channels.Experimental Approach: The whole-cell patch clamp technique was applied to investigate the effects of methadone on wild-type and mutant human Na_v1.5 channels expressed in HEK293 cells. A homology model of human Na_v1.5 channels was used to perform automated ligand-docking studies.Key Results: Methadone inhibited Na_v1.5 channels in a state-dependent manner, that is, tonic block was stronger with inactivated channels than with resting channels and a use-dependent block at 10 Hz. Methadone induced a concentration-dependent shift of the voltage dependency of both fast and slow inactivation towards more hyperpolarized potentials, and impaired recovery from fast and slow inactivation. The LA-insensitive mutants N406K and F1760A exhibited reduced tonic and use-dependent block by methadone, and docking predictions positioned methadone in a cavity that was delimited by the residue F1760. Dextromethadone and levomethadone induced discrete stereo-selective effects on Na_v1.5 channels.Conclusions and Implications: Methadone interacted with the LA-binding site to inhibit Na_v1.5 channels. Our data suggest that these channels are a hitherto unrecognized molecular component contributing to cardiac arrhythmias induced by methadone.     

Inhibition of cardiac sodium currents by toluene exposure

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration-dependent manner, with an IC50 of 274 microm (confidence limits: 141-407 microm). The inhibition was complete, voltage-independent, and slowly reversible. Toluene had no effect on: (i). the shape of the I-V curves; (ii). the reversal potential of Na+; and (iii). the steady-state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use- and frequency-dependent. In rat cardiac myocytes, 300 microm toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use- and frequency-dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect.     

T-type calcium channels are inhibited by fluoxetine and its metabolite norfluoxetine

Fluoxetine, a widely used antidepressant that primarily acts as a selective serotonin reuptake inhibitor, also inhibits various neuronal ion channels. Using the whole-cell patch-clamp technique, we have examined the effects of fluoxetine and norfluoxetine, its major active metabolite, on cloned low-voltage-activated T-type calcium channels (T channels) expressed in tsA 201 cells. Fluoxetine inhibited the three T channels Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 in a concentration-dependent manner (IC(50) = 14, 16, and 30 microM, respectively). Norfluoxetine was a more potent inhibitor than fluoxetine, especially on the Ca(V)3.3 T current (IC(50) = 5 microM). The fluoxetine block of T channels was voltage-dependent because it was significantly enhanced for T channels in the inactivated state. Fluoxetine caused a hyperpolarizing shift in steady-state inactivation, with a slower rate of recovery from the inactivated state. These results indicated a tighter binding of fluoxetine to the inactivated state than to the resting state of T channels, suggesting a more potent inhibition of T channels at physiological resting membrane potential. Indeed, fluoxetine and norfluoxetine at 1 microM strongly inhibited cloned T currents (approximately 50 and approximately 75%, respectively) in action potential clamp experiments performed with firing activities of thalamocortical relay neurons. Altogether, these data demonstrate that clinically relevant concentrations of fluoxetine exert a voltage-dependent block of T channels that may contribute to this antidepressant's pharmacological effects.     

Preferred mexiletine block of human sodium channels with IVS4 mutations and its pH-dependence

The effects of extracellular pH (6.2, 7.4 and 8.2) and 0.1 mM mexiletine, a channel blocker of the lidocaine type, are studied on two mutations of the fourth voltage sensor of the Nav1.4 sodium channel, R1448H/C. The fast inactivated channel state to which mexiletine preferentially binds is destabilized by the mutations. By contrast to the expected low response of R1448H/C carriers, mexiletine is particularly effective in preventing exercise-induced stiffness and paralysis from which these patients suffer. Our measurements performed in the whole-cell mode on stably transfected HEK cells show for the first time that the mutations strikingly accelerate closed-state inactivation and, as steady-state fast inactivation is shifted to more negative potentials, stabilize the fast inactivated channel state in the potential range around the resting potential. At pH 7.4 and 8.2, the phasic mexiletine block is larger for R1448C (55%) and R1448H (47%) than for wild-type channels (31%) due to slowed recovery from block (tau is approximately 520 ms for R1448C versus 270 ms for wild-type at pH 7.4) although the recovery from inactivation is slightly faster for the mutants (tau is approximately 1.9 ms for R1448C versus 3.8 ms for wild-type at pH 7.4). At pH 6.2, recovery from block is relatively fast (tau is approximately 35 ms for R1448H/C and 14 ms for wild-type) and thus shows no use-dependence. We conclude that enhanced closed-state inactivation expands the concept of a mutation-induced uncoupling of channel inactivation from activation to a new potential range and that the higher mexiletine efficacy in R1448H/C carriers compared to other myotonic patients offers a pharmacogenetic strategy for mutation-specific treatment.     

Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels

Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.     

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.     

Prenylamine block of Nav1.5 channel is mediated via a receptor distinct from that of local anesthetics

We have shown previously that prenylamine, a calcium channel blocker, has potent local anesthetic activity in vivo and in vitro. We now characterize the tonic and use-dependent block of prenylamine on wild-type human cardiac voltage-gated sodium channels (hNav1.5) transiently expressed in human embryonic kidney 293t cells under whole-cell voltage-clamp condition. We also determine whether prenylamine and local anesthetics interact with a common binding site on the Nav1.5 channel by analyzing prenylamine block on mutant hNav1.5 channels that have substitution mutations in amino acids at the putative local anesthetic binding sites. Prenylamine exhibits tonic block at both hyperpolarizing and depolarizing potentials on hNav1.5 channels with 50% inhibitory concentrations of 9.67 +/- 0.25 microM and 0.72 +/- 0.02 microM, respectively. Substitutions of the amino acids at the putative local anesthetic binding site (i.e., F1760, N1765, Y1767, and N406) with lysine had much lesser effects on prenylamine block of the mutant hNav1.5 channels compared with local anesthetic block. The affinity of prenylamine was reduced at most by 5.8-fold, whereas that of bupivacaine, a known local anesthetic, was reduced by as much as 68-fold compared with wild-type by the mutations at the local anesthetic receptor site. Furthermore, equilibrium results between prenylamine-bupivacaine mixtures suggest two independent receptors. Thus, the data demonstrate that prenylamine has both tonic and use-dependent block of hNav1.5 channels similar to that of local anesthetics, but the location of the prenylamine binding site on hNav1.5 differs from that of the local anesthetic binding site.     

A phenylalanine residue at segment D3-S6 in Nav1.4 voltage-gated Na(+) channels is critical for pyrethroid action

Mammalian voltage-gated Na(+) channels were less sensitive to pyrethroids than their insect counterparts by 2 to 3 orders of magnitude. Deltamethrin at 10 microM elicited weak gating changes in rat skeletal muscle alpha-subunit Na(+) channels (Nav1.4) after > 30 min of perfusion. About 10% of the peak current was maintained during the 8-ms, +50-mV pulse and, upon repolarization to -140 mV, the amplitude of the slow tail current corresponded to less than 3% of total Na(+) channels modified by deltamethrin. A background mutation, Nav1.4-I687M (within D2:S4-S5 cytoplasmic linker), enhanced the deltamethrin-induced maintained current by approximately 2.5-fold, whereas Nav1.4-I687T, a homologous superkdr mutation, reduced it by approximately 2-fold. Repetitive pulses at 2 Hz further augmented the effects of deltamethrin on Nav1.4-I687M mutant channels so that approximately 75% of peak currents were maintained. A second mutation, Nav1.4-I687M/F1278I at the middle of D3-S6, rendered the channel greatly resistant to deltamethrin. This double mutant channel remained sensitive to batrachotoxin (BTX), even though nearby residues S1276 and L1280 were critical for BTX action. We hypothesize that the deltamethrin receptor and the BTX receptor are situated at the middle but opposite surface of the D3-S6 alpha-helical structure. Another mutant, Nav1.4-I687M/N784K, exhibited a partial deltamethrin-resistant phenotype but was completely resistant to BTX. Consistent with the BTX-resistant phenotype of N784K and the known adjacent kdr mutation at position L785F, deltamethrin and BTX were probably situated next to each other upon binding at D2-S6. Evidently, distinct residues from multiple S6 segments were critical for deltamethrin and BTX actions.     

Serotonin involvement in cocaine sensitization: Clues from studies with cocaine analogs

While the behavioral effects of cocaine are generally ascribed to its ability to inhibit the uptake of dopamine, there is evidence to indicate that some of the other pharmacological properties of cocaine may play a significant role in its actions. Behavioral, biochemical, and electrophysiological data suggest that the potent inhibition of serotonin uptake elicited by cocaine is a mechanism that may contribute to its overall effects in vivo. Cocaethylene is the ethyl ester of benzoylecognine which is formed in vivo during concurrent ingestion of cocaine and ethanol. Cocaethylene is equipotent with cocaine as an inhibitor of dopamine uptake, but less potent as an inhibitor of serotonin uptake. We have compared the effects of acute and chronic cocaine and cocaethylene on rat locomotor activity in an attempt to determine the serotonin component in this behavior. Acute dose-response studies revealed that at higher doses (20 mg/kg ip) cocaethylene produced less stimulation of locomotor activity than cocaine. Prior exposure to cocaine resulted in an augmented response to a subsequent challenge dose of either cocaine (sensitization) or cocaethylene (cross-sensitization). However, previous treatment with cocaethylene did not significantly affect the locomotor activity produced by challenges with cocaethylene or cocaine. The involvement of serotonergic systems in the development of cocaine-induced sensitization is one intriguing possible explanation of these data. Biochemical studies have shown that other cocaine analogs possess even more selectivity for the dopamine uptake site than cocaethylene. In terms of comparative potency as inhibitors of dopamine, serotonin, and norepinephrine uptake, isopropylcocaine (isopropyl ester of benzoylecognine) is more selective than even cocaethylene. A metabolically resistant analog of cocaine, β-CIT (2β-carbomethoxy-3β-(4-iodophenyl)tropane), was shown to be extraordinarily long-acting, stimulating locomotor activity for 10 h following a dose of 0.1 mg/kg ip. Furthermore, β-CIT was extremely potent, inhibiting dopamine or serotonin uptake in vitro at a concentration 100 times lower than is required for cocaine. Thus, isopropyl or other substitutions at the carbomethoxy position of the cocaine or β-CIT structures may provide useful tools for the analysis of the serotonin or norepinephrine components in cocaine's actions and as selective probes of central dopamine systems in imaging studies. © 1993 wiley-Liss, Inc.     

Molecular determinants for activation of human ether-à-go-go-related gene 1 potassium channels by 3-nitro-n-(4-phenoxyphenyl) benzamide

Human ether-à-go-go-related gene 1 (hERG1) channels mediate repolarization of cardiac action potentials. Inherited long QT syndrome (LQTS) caused by loss-of-function mutations, or unintended blockade of hERG1 channels by many drugs, can lead to severe arrhythmia and sudden death. Drugs that activate hERG1 are a novel pharmacological approach to treat LQTS. 3-Nitro-n-(4-phenoxyphenyl) benzamide [ICA-105574 (ICA)] has been discovered to activate hERG1 by strong attenuation of pore-type inactivation. Here, we used scanning mutagenesis of hERG1 to identify the molecular determinants of ICA action. Three mutations abolished the activator effects of 30 μM ICA, including L622C in the pore helix, F557L in the S5 segment, and Y652A in the S6 segment. One mutation in S6 (A653M) switched the activity of ICA from an activator to an inhibitor, revealing its partial agonist activity. This was confirmed by showing that the noninactivating mutant hERG1 channel (G628C/S631C) was inhibited by ICA and that the addition of the F557L mutation rendered the channel drug-insensitive. Simulated molecular docking of ICA to homology models of hERG1 corroborated the scanning mutagenesis findings. Together, our findings indicate that ICA is a mixed agonist of hERG1 channels. Activation or inhibition of currents is mediated by the same or overlapping binding site located in the pore module between two adjacent subunits of the homotetrameric channel.     

Differential modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by the local anesthetic lidocaine

1 Voltage-gated Na+ channels are transmembrane proteins that are essential for the propagation of action potentials in excitable cells. Nav1.7 and Nav1.8 dorsal root ganglion Na+ channels exhibit different kinetics and sensitivities to tetrodotoxin (TTX). We investigated the properties of both channels in the presence of lidocaine, a local anesthetic (LA) and class I anti-arrhythmic drug. 2 Nav1.7 and Nav1.8 Na+ channels were coexpressed with the beta1-subunit in Xenopus oocytes. Na+ currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dose-response curves for both channels had different EC50 (dose producing 50% maximum current inhibition) (450 microm for Nav1.7 and 104 microm for Nav1.8). Lidocaine enhanced current decrease in a frequency-dependent manner. Steady-state inactivation of both channels was also affected by lidocaine, Nav1.7 being the most sensitive. Only the steady-state activation of Nav1.8 was affected while the entry of both channels into slow inactivation was affected by lidocaine, Nav1.8 being affected to a larger degree. 4 Although the channels share homology at DIV S6, the LA binding site, they differ in their sensitivity to lidocaine. Recent studies suggest that other residues on DI and DII known to influence lidocaine binding may explain the differences in affinities between Nav1.7 and Nav1.8 Na+ channels. 5 Understanding the properties of these channels and their pharmacology is of critical importance to developing drugs and finding effective therapies to treat chronic pain.