Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle.

Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.     

Kinetic analysis of phasic inhibition of neuronal sodium currents by lidocaine and bupivacaine.

Phasic ("use-dependent") inhibition of sodium currents by the tertiary amine local anesthetics, lidocaine and bupivacaine, was observed in voltage-clamped node of Ranvier of the toad, Bufo marinus. Local anesthetics were assumed to inhibit sodium channels through occupation of a binding site with 1:1 stoichiometry. A three-parameter empirical model for state-dependent anesthetic binding to the Na channel is presented: this model includes two discrete parameters that represent the time integrals of binding and unbinding reactions during a depolarizing pulse, and one continuous parameter that represents the rate of unbinding of drug between pulses. The change in magnitude of peak sodium current during a train of depolarizing pulses to 0 mV was used as an assay of the extent of anesthetic binding at discrete intervals; estimates of model parameters were made by applying a nonlinear least-squares algorithm to the inhibition of currents obtained at two or more depolarizing pulse rates. Increasing the concentration of drug increased the rate of binding but had little or no effect on unbinding, as expected for a simple bimolecular reaction. The dependence of the model parameters on pulse duration was assessed for both drugs: as the duration of depolarizing pulses was increased, the fraction of channels binding drug during each pulse became significantly larger, whereas the fraction of occupied channels unbinding drug remained relatively constant. The rate of recovery from block between pulses was unaffected by pulse duration or magnitude. The separate contributions of open (O) and inactivated (I) channel binding of drug to the net increase in block per pulse were assessed at 0 mV: for lidocaine, the forward binding rate ko was 1.3 x 10(5) M-1 s-1, kl was 2.4 x 10(4) M-1 s-1; for bupivacaine, ko was 2.5 x 10(5) M-1 s-1, kl was 4.4 x 10(4) M-1 s-1. These binding rates were similar to those derived from time-dependent block of maintained Na currents in nodes where inactivation was incomplete due to treatment with chloramine-T. The dependence of model parameters on the potential between pulses (holding potential) was examined. All three parameters were found to be nearly independent of holding potential from -70 to -100 mV. These results are discussed with respect to established models of dynamic local anesthetic-Na channel interactions.     

Tonic and phasic block of neuronal sodium currents by 5-hydroxyhexano- 2'',6''-xylide, a neutral lidocaine homologue

The effects of a neutral lidocaine homologue, 5-hydroxyhexano-2',6'-xylidide (5-HHX), on the kinetics and amplitude of sodium currents in voltage-clamped amphibian nerve fibers are described. 5-HHX produced two types of sodium current inhibition: (a) tonic block, in resting fibers (IC50 approximately 2 mM), and (b) phasic block, an additional, incremental inhibition, in repetitively depolarized fibers (frequency greater than 1 Hz). The kinetics of phasic block were characterized by a single-receptor, switched-affinity model, in which binding increases during a depolarizing pulse and decreases between pulses. In the presence of 4 mM 5-HHX, binding increased during pulses from -80 to 0 mV, with an apparent rate constant of 6.4 +/- 1.4 s-1. Binding decreased between pulses with an apparent rate constant of 1.1 +/- 0.3 s-1. There was little effect of extracellular pH on the kinetics of phasic block. These findings demonstrate that neither the presence of a terminal amine nor a net charge on a local anesthetic is required for phasic block of sodium channels.     

The effects of external pH on calcium channel currents in bullfrog sympathetic neurons.

We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance.     

Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, approximately 50% neutral at pH 7.4) have similar structures but markedly different effects on Na(+) channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na(+) channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na(+) channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na(+) channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na(+) channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.     

Determination of rate constants for nucleotide dissociation from Na,K-ATPase.

A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.     

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.     

Multiple open channel states revealed by lidocaine and QX-314 on rat brain voltage-dependent sodium channels

We have recently reported that brain sodium channels display periods with high (low-Kd) and low (high-Kd) levels of lidocaine-induced open channel block (Salazar, B.C., D.O. Flash, J.L. Walewski, and E. Recio- Pinto. 1995. Brain Res. 699:305-314). In the present study, we further characterize this phenomenon by studying the effects of the permanently charged lidocaine analogue, QX-314. We found that the detection of high- and low-Kd periods does not require the presence of the uncharged form of lidocaine. The level of block, for either period, at various QX-314 concentrations indicated the presence of a single local anesthetic binding site. Increasing the concentration of QX-314 decreased the lifetime of the high-Kd periods while it increased the lifetime of the low-Kd periods. These results could be best fitted to a model with two open channel conformations that display different local anesthetic Kd values (low and high Kd), and in which the channel area defining the local anesthetic Kd consists of multiple interacting regions. Amplitude distribution analysis showed that changes in the Kd values reflected changes in the kon rates, without changes in the koff rates. Both lidocaine and QX-314 were found to be incapable of blocking small- channel subconductance states (5-6 pS). Changes in the local anesthetic kon rates for blocking the fully open state and the lack of local anesthetic block of the small subconductance state are consistent with the presence of channel conformational changes involving the intracellular permeation pathway leading to the local anesthetic binding site.     

Antagonism by Local Anesthetics of Sodium Channel Activators in the Presence of Scorpion Toxins: Two Mechanisms for Competitive Inhibition

1. The interaction of veratridine (VTD), a Na+ channel activator, scorpion alpha-toxin (LQ), an open state Na+ channel stabilizer, and the local anesthetic, lidocaine (LID), a channel inhibitor, at the neuronal sodium channel was assessed by measuring VTD-dependent slow depolarizations of frog sciatic nerve using the sucrose-gap method. 2. The slow depolarizing action of veratridine was potentiated more than 10-fold by the peptide LQ toxin, whereas its competitive inhibition by lidocaine was unchanged by LQ. 3. We conclude that the antagonism between VTD and a LID molecule during slow depolarization is allosteric, involving a trapping of the Na+ channel by LID in the inactivated state that has a very low affinity for VTD. 4. The binding of VTD to the open state of the channel, which is stabilized by LQ, may be inhibited by orthosteric competition at overlapping sites since both LID and VTD bind avidly and rapidly to open channels.     

pH-dependent binding of local anesthetics in single batrachotoxin-activated Na+ channels. Cocaine vs. quaternary compounds.

The effects of internal and external pH on the binding kinetics of local anesthetics (LAs) were studied in single batrachotoxin-activated Na+ channels incorporated into planar bilayers. With internal quaternary QX-314 and RAC421-II drugs, the binding interactions were little affected by either external or internal pH. With tertiary cocaine, the binding kinetics were drastically altered by pH. A decrease in the internal pH from 9.3 to 6.2 decreased the apparent equilibrium dissociation constant (Kd) of internal cocaine by more than 100-fold. This increase in the binding affinity was mostly accounted for by an increase in the apparent cocaine on-rate constant (kon) of approximately 80-fold. The cocaine off-rate constant (koff) was little changed (between 3-4 s-1). These results demonstrate quantitatively that the charged form of cocaine is the active form for BTX-activated Na+ channels. Surprisingly, the apparent pKa of cocaine near its binding site was estimated to be 1.4 units lower than that in bulk solution (7.1 vs. 8.5), indicating that the LA drug encounters a relatively hydrophobic environment. Opposite to the internal pH effect, a decrease of external pH from 8.4 to 6.2 increased the Kd value of internally and externally applied cocaine by approximately 8- and approximately 25-fold, respectively. External pH effect was primarily mediated by modulation of kon; koff was again relatively unaffected. Our findings support a model in which neutral cocaine can readily cross the membrane barrier, but needs to be protonated internally to bind to its binding site.     

Local anesthetic inhibition of a bacterial sodium channel

Recent structural breakthroughs with the voltage-gated sodium channel from Arcobacter butzleri suggest that such bacterial channels may provide a structural platform to advance the understanding of eukaryotic sodium channel gating and pharmacology. We therefore set out to determine whether compounds known to interact with eukaryotic Na(V)s could also inhibit the bacterial channel from Bacillus halodurans and NaChBac and whether they did so through similar mechanisms as in their eukaryotic homologues. The data show that the archetypal local anesthetic (LA) lidocaine inhibits resting NaChBac channels with a dissociation constant (K(d)) of 260 μM, and channels displayed a left-shifted steady-state inactivation gating relationship in the presence of the drug. Extracellular application of QX-314 to expressed NaChBac channels had no effect on sodium current, whereas internal exposure via injection of a bolus of the quaternary derivative rapidly reduced sodium conductance, consistent with a hydrophilic cytoplasmic access pathway to an internal binding site. However, the neutral derivative benzocaine applied externally inhibited NaChBac channels, suggesting that hydrophobic pathways can also provide drug access to inhibit channels. Alternatively, ranolazine, a putative preopen state blocker of eukaryotic Na(V)s, displayed a K(d) of 60 μM and left-shifted the NaChBac activation-voltage relationship. In each case, block enhanced entry into the inactivated state of the channel, an effect that is well described by a simple kinetic scheme. The data suggest that although significant differences exist, LA block of eukaryotic Na(V)s also occurs in bacterial sodium channels and that NaChBac shares pharmacological homology to the resting state of vertebrate Na(V) homologues.     

Distinct local anesthetic affinities in Na+ channel subtypes.

Lidocaine is a widely used local anesthetic and antiarrhythmic drug that is believed to exert its clinically important action by blocking voltage-gated Na+ channels. Studies of Na+ channels from different species and tissues and the complexity of the drug-channel interaction create difficulty in understanding whether there are Na+ channel isoform specific differences in the affinity for lidocaine. Clinical usage suggests that lidocaine selectively targets cardiac Na+ channels because it is effective for the treatment of arrhythmias with few side effects on muscle or neuronal channels except at higher concentrations. One possibility for this selectivity is an intrinsically higher drug-binding affinity of the cardiac isoform. Alternatively, lidocaine may appear cardioselective because of preferential interactions with the inactivated state of the Na+ channel, which is occupied much longer in cardiac cells. Recombinant skeletal muscle (hSkM1) and cardiac sodium channels (hH1) were studied under identical conditions, with a whole-cell voltage clamp used to distinguish the mechanisms of lidocaine block. Tonic block at high concentrations of lidocaine (0.1 mM) was greater in hH1 than in hSkM1. This was also true for use-dependent block, for which 25-microM lidocaine produced an inhibition in hH1 equivalent to 0.1 mM in the skeletal muscle isoform. Pulse protocols optimized to explore inactivated-state block revealed that hSkM1 was five to eight times less sensitive to block by lidocaine than was hH1. The results also indicate that relatively more open-state block occurs in hSkM1. Thus, the cardiac sodium channel is intrinsically more sensitive to inhibition by lidocaine.     

Interactions between H+ and Ca2+ near cardiac L-type calcium channels: evidence for independent channel-associated binding sites.

Monovalent and divalent ions are known to affect voltage-gated ion channels by the screening of, and/or binding to, negative charges located on the surface of cell membranes within the vicinity of the channel protein. In this investigation, we studied gating shifts of cardiac L-type calcium channels induced by extracellular H+ and Ca2+ to determine whether these cations interact at independent or competitive binding sites. At constant pHo (7.4), Cao-induced gating shifts begin to approach a maximum value (approximately equal to 17 mV) at concentrations of extracellular calcium of > or = 40 mM. A fraction of the calcium-dependent gating shift could be titrated with an effective pKa = 6.9 indicating common and competitive access to H+ and Ca2+ ions for at least one binding site. However, if pHo is lowered when Cao is > or = 40 mM, additional shifts in gating are measured, suggesting a subpopulation of sites to which Ca2+ and H+ bind independently. The interdependence of L-channel gating shifts and Cao and pHo was well described by the predictions of surface potential theory in which two sets of binding sites are postulated; site 1 (pKa = 5.5) is accessible only to H+ ions and site 2 (pKa = 6.9) is accessible to both Ca2+ and H+ ions. Theoretical computations generated with this model are consistent with previously determined data, in which interactions between these two cations were not studied, in addition to the present experiments in which interactions were systematically probed.     

Guanine nucleotide regulation of B2 kinin receptors. Time-dependent formation of a guanine nucleotide-sensitive receptor state from which [3H]bradykinin dissociates slowly

We have examined the binding of [3H]bradykinin to bovine myometrial membranes and assessed its sensitivity to guanine nucleotides. Total binding displayed a typical B2 kinin receptor specificity. However, saturation binding isotherms were resolved into at least two components with KD values of 8 pM (45%) and 378 pM (55%). Low affinity binding exhibited relatively rapid rates of association (kobs = 1.40 x 10(-2) s-1) and dissociation (k-1 = 3.82 x 10(-3) s-1), while high affinity binding exhibited considerably slower rates (kobs = 9.52 x 10(-4) s-1 and k-1 = 4.43 x 10(-5) s-1). Pre-equilibrium dissociation kinetics revealed that formation of high affinity binding was characterized as a time-dependent accumulation of the slow dissociation rate at the expense of at least one other more rapid dissociation rate. In the presence of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), at least two binding components were resolved with KD values of 37 pM (12%) and 444 pM (88%). Gpp(NH)p apparently specifically perturbed high affinity binding by completely preventing the accumulation of the slow dissociation phase. Instead, two more rapid dissociation rates (k-1 = 8.53 x 10(-3) s-1 and 4.43 x 10(-4) s-1) were observed. These results suggest that [3H]bradykinin interacts with at least two B2 kinin receptor-like binding sites in bovine myometrial membranes. A three-state model for the guanine nucleotide-sensitive agonist interaction with the high affinity binding sites is proposed.     

Kinetic effects of quaternary lidocaine block of cardiac sodium channels: a gating current study

The interaction of antiarrhythmic drugs with ion channels is often described within the context of the modulated receptor hypothesis, which explains the action of drugs by proposing that the binding site has a variable affinity for drugs, depending upon whether the channel is closed, open, or inactivated. Lack of direct evidence for altered gating of cardiac Na channels allowed for the suggestion of an alternative model for drug interaction with cardiac channels, which postulated a fixed affinity receptor with access limited by the conformation of the channel (guarded receptor hypothesis). We report measurement of the gating currents of Na channels in canine cardiac Purkinje cells in the absence and presence of QX-222, a quaternary derivative of lidocaine, applied intracellularly, and benzocaine, a neutral local anesthetic. These data demonstrate that the cardiac Na channel behaves as a modulated rather than a guarded receptor in that drug-bound channels gate with altered kinetics. In addition, the results suggest a new interpretation of the modulated receptor hypothesis whereby drug occupancy reduces the overall voltage- dependence of gating, preventing full movement of the voltage sensor.     

Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.     

Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.     

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.     

Post-repolarization block of cloned sodium channels by saxitoxin: the contribution of pore-region amino acids.

Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat IIa co-expressed with beta 1 subunit, Br2a + beta 1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX. This block/unblock process for Br2a + beta 1 was 10-fold slower than that for RHI. A model accounting for these results predicts a faster toxin dissociation rate and a slower association rate for the cardiac isoform, and it also predicts a shorter dwell time in a putative high STX affinity conformation for the cardiac isoform. The RHI mutation (Cys374-->Phe), which was previously shown to be neuronal-like with respect to high affinity tonic toxin block, was also neuronal-like with respect to the kinetics of post-repolarization block, suggesting that this single amino acid is important for conferring isoform-specific transition rates determining post-repolarization block. Because the same mutation determines both sensitivity for tonic STX block and the kinetics of phasic STX block, the mechanisms accounting for tonic block and phasic block share the same toxin binding site. We conclude that the residue at position 374, in the putative pore-forming region, confers isoform-specific channel kinetics that underlie phasic toxin block.     

Mutant bacterial sodium channels as models for local anesthetic block of eukaryotic proteins

Voltage gated sodium channels are the target of a range of local anesthetic, anti-epileptic and anti-arrhythmic compounds. But, gaining a molecular level understanding of their mode of action is difficult as we only have atomic resolution structures of bacterial sodium channels not their eukaryotic counterparts. In this study we used molecular dynamics simulations to demonstrate that the binding sites of both the local anesthetic benzocaine and the anti-epileptic phenytoin to the bacterial sodium channel NavAb can be altered significantly by the introduction of point mutations. Free energy techniques were applied to show that increased aromaticity in the pore of the channel, used to emulate the aromatic residues observed in eukaryotic Nav1.2, led to changes in the location of binding and dissociation constants of each drug relative to wild type NavAb. Further, binding locations and dissociation constants obtained for both benzocaine (660 μM) and phenytoin (1 μ M) in the mutant channels were within the range expected from experimental values obtained from drug binding to eukaryotic sodium channels, indicating that these mutant NavAb may be a better model for drug binding to eukaryotic channels than the wild type.