Functional consequences of lidocaine binding to slow-inactivated sodium channels

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use- dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to - 20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow- inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow- inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.     

Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use- dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1- beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.     

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.     

Differences in steady-state inactivation between Na channel isoforms affect local anesthetic binding affinity.

Cocaine and lidocaine are local anesthetics (LAs) that block Na currents in excitable tissues. Cocaine is also a cardiotoxic agent and can induce cardiac arrhythmia and ventricular fibrillation. Lidocaine is commonly used as a postinfarction antiarrhythmic agent. These LAs exert clinically relevant effects at concentrations that do not obviously affect the normal function of either nerve or skeletal muscle. We compared the cocaine and lidocaine affinities of human cardiac (hH1) and rat skeletal (mu 1) muscle Na channels that were transiently expressed in HEK 293t cells. The affinities of resting mu 1 and hH1 channels were similar for cocaine (269 and 235 microM, respectively) and for lidocaine (491 and 440 microM, respectively). In addition, the affinities of inactivated mu 1 and hH1 channels were also similar for cocaine (12 and 10 microM, respectively) and for lidocaine (19 and 12 microM, respectively). In contrast to previous studies, our results indicate that the greater sensitivity of cardiac tissue to cocaine or lidocaine is not due to a higher affinity of the LA receptor in cardiac Na channels, but that at physiological resting potentials (-100 to -90 mV), a greater percentage of hH1 channels than mu 1 channels are in the inactivated (i.e., high-affinity) state.     

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.     

Time-dependent Outward Currents through the Inward Rectifier Potassium Channel IRK1 : The Role of Weak Blocking Molecules

Outward currents through the inward rectifier K⁺ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg²⁺ (0.44–1.1 mM) or putrescine (∼0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K⁺. Without internal Mg²⁺, small outward currents flowed only at potentials between −80 (the reversal potential) and ∼−40 mV during voltage steps applied from −110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg²⁺, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between −40 and 0 mV were concurrent with the contribution of Mg²⁺ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to −50 mV after short depolarizing steps (>0 mV), a transient increase appeared in outward currents at −50 mV. Since the peak amplitude depended on the fraction of Mg²⁺-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg²⁺ block, followed by a re-block of channels by spermine. Shift in the holding potential (−110 to −80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg²⁺-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg²⁺. When both spermine (1 μM, an estimated free spermine level during whole-cell recordings) and putrescine (300 μM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg²⁺ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.     

Voltage-dependent block by tetrodotoxin of the sodium channel in rabbit cardiac Purkinje fibers.

The two-microelectrode, voltage-clamp technique was applied to rabbit cardiac Purkinje fibers to study the interaction of tetrodotoxin (TTX) with the slowly inactivating Na current. Binding of TTX to rested, inactivated, and activated channels was estimated by measuring the relative decrease of current at the beginning (rested and inactivated channels) and the end (activated channels) of a 1 s depolarizing clamp to -45 mV. The accelerated decline of the Na current in the presence of a submaximal dose of TTX was interpreted as an increase in blocking efficiency upon depolarization. The experiments show that activated as well as inactivated channels are more sensitive to TTX than are rested channels. The dissociation equilibrium constants for the three states are 3.5 X 10(-6) M for the rested, 0.94 X 10(-6) M for the activated, and 0.75 X 10(-6) M for the inactivated channels. The time course of activation block was dependent on TTX concentration. Rate constants for association and dissociation of the activated state are 1.3 X 10(6) M-1 X s-1 and 1.5 s-1, respectively.     

Effect of lidocaine on the slow Na+ channels of Xenopus oocytes

The membrane of immature Xenopus oocytes is known to possess a peculiar type of sodium channels, which are not activatable unless the membrane has been depolarized for some time. Once induced by a long-lasting depolarization, the channels behave like voltage-dependent channels, but they slowly activate and apparently do not inactivate. In addition, these channels were shown to be insensitive to the toxins classically used to inhibit the voltage-dependent Na+ channels. The effects of lidocaine on these slow Na+ channels were investigated using current-and voltage-clamped oocytes. Lidocaine reversibly blocked the channels when they were in their open configuration, but not when the channels were in their closed state. The concentration of lidocaine required for half-inhibition of the slow inward current was 270 +/- 67 micromol/l. The current/voltage relationships indicated that lidocaine blocked the sodium current (inward as well as outward) for all the potentials investigated. At a concentration of 0.3 mmol/l, lidocaine caused a shift of 5 +/- 1 mV of the activation curve. This suggests that the gating properties of the channels were alterated. The effect of lidocaine was found to be non-selective since at least two other channels were affected by the drug, namely the voltage-dependent calcium channels and the monovalent non-selective channels.     

The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na+ Channels

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na⁺ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na⁺ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel α subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Inactivated sodium channels are blocked by the anti-arrhythmic cordaron

The blocking action of the antiarrhythmic cordaron (amiodaron hydrochloride) on Na currents in the frog node of Ranvier was studied by the voltage-clamp technique. It was established that the degree of the cordaron-induced block can be reversibly increased by long-term (1 s and more) membrane depolarization. The time constants of both the development and recovery from the block caused by membrane depolarization are of the order of hundreds of seconds ("induced slow sodium inactivation"). The fraction of blocked channels rises to some limiting level with an increase in the magnitude of the depolarizing step, the region of strong voltage dependency being between -130 and -70 mv. The inhibition of outward potassium current by 7.10(-4) M cordaron does not exceed 15%. The time- and voltage-dependent block of Na channels is supposed to result from interaction of the antiarrhythmic with inactivated channels.     

Lidocaine alters the input resistance and evokes neural activity in crayfish sensory neurons

Lidocaine, a use-dependent Na(+) channel blocker, paradoxically evokes neural activation in the slowly adapting stretch receptor organ of crayfish at 5-10 mmol/l concentration. For elucidating the underlying mechanisms of this paradoxical effect, a series of conventional electrophysiological experiments were performed in the stretch receptor neurons of crayfish. In the presence of tetrodotoxin, lidocaine did not evoke impulse activity, however, a slowly developing and dose-dependent depolarization occurred in both the rapidly and slowly adapting stretch receptors. Similar effects were observed by perfusion of equivalent concentrations of benzocaine but not of procaine or prilocaine. Lidocaine did not evoke neural activity in the rapidly adapting neuron which fires action potential(s) in response to rapid changes in membrane potential. Slowly developing mode of the depolarization indicated the reason why only depolarization but not action potential responses were observed in the rapidly adapting neuron. The depolarizing effect of lidocaine was independent from any ionic channel or exchanger system. However, lidocaine and benzocaine but not procaine and prilocaine evoked a dose-dependent alteration in the input resistance of the neuron. It was proposed that the principal mechanism of the effect could stem from a change in the physical properties of the neuronal membrane.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT_1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT_1A autoreceptor-activated inwardly rectifying K⁺ conductance in rat dorsal raphe 5-HT neurons. 5-HT_1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba²⁺ (EC₅₀ = 9.4 μM, full block with 100 μM) and by SCH23390 (EC₅₀ = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT_1A autoreceptor-activated GIRK conductance with high potency (EC₅₀ = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC₅₀s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K⁺ channels coupled to 5-HT_1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.     

Mechanisms of atrial fibrillation termination by rapidly unbinding Na+ channel blockers: insights from mathematical models and experimental correlates

Atrial fibrillation (AF) is the most common sustained clinical arrhythmia and is a problem of growing proportions. Recent studies have increased interest in fast-unbinding Na(+) channel blockers like vernakalant (RSD1235) and ranolazine for AF therapy, but the mechanism of efficacy is poorly understood. To study how fast-unbinding I(Na) blockers affect AF, we developed realistic mathematical models of state-dependent Na(+) channel block, using a lidocaine model as a prototype, and studied the effects on simulated cholinergic AF in two- and three-dimensional atrial substrates. We then compared the results with in vivo effects of lidocaine on vagotonic AF in dogs. Lidocaine action was modeled with the Hondeghem-Katzung modulated-receptor theory and maximum affinity for activated Na(+) channels. Lidocaine produced frequency-dependent Na(+) channel blocking and conduction slowing effects and terminated AF in both two- and three-dimensional models with concentration-dependent efficacy (maximum approximately 89% at 60 microM). AF termination was not related to increases in wavelength, which tended to decrease with the drug, but rather to decreased source Na(+) current in the face of large ACh-sensitive K(+) current-related sinks, leading to the destabilization of primary generator rotors and a great reduction in wavebreak, which caused primary rotor annihilations in the absence of secondary rotors to resume generator activity. Lidocaine also reduced the variability and maximum values of the dominant frequency distribution during AF. Qualitatively similar results were obtained in vivo for lidocaine effects on vagal AF in dogs, with an efficacy of 86% at 2 mg/kg iv, as well as with simulations using the guarded-receptor model of lidocaine action. These results provide new insights into the mechanisms by which rapidly unbinding class I antiarrhythmic agents, a class including several novel compounds of considerable promise, terminate AF.     

Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.     

Late Sodium Current in Human Atrial Cardiomyocytes from Patients in Sinus Rhythm and Atrial Fibrillation

Slowly inactivating Na⁺ channels conducting “late” Na⁺ current (I_Na,late) contribute to ventricular arrhythmogenesis under pathological conditions. I_Na,late was also reported to play a role in chronic atrial fibrillation (AF). The objective of this study was to investigate I_Na,late in human right atrial cardiomyocytes as a putative drug target for treatment of AF. To activate Na⁺ channels, cardiomyocytes from transgenic mice which exhibit I_Na,late (ΔKPQ), and right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF were voltage clamped at room temperature by 250-ms long test pulses to -30 mV from a holding potential of -80 mV with a 100-ms pre-pulse to -110 mV (protocol I). I_Na,late at -30 mV was not discernible as deviation from the extrapolated straight line IV-curve between -110 mV and -80 mV in human atrial cells. Therefore, tetrodotoxin (TTX, 10 μM) was used to define persistent inward current after 250 ms at -30 mV as I_Na,late. TTX-sensitive current was 0.27±0.06 pA/pF in ventricular cardiomyocytes from ΔKPQ mice, and amounted to 0.04±0.01 pA/pF and 0.09±0.02 pA/pF in SR and AF human atrial cardiomyocytes, respectively. With protocol II (holding potential -120 mV, pre-pulse to -80 mV) TTX-sensitive I_Na,late was always larger than with protocol I. Ranolazine (30 μM) reduced I_Na,late by 0.02±0.02 pA/pF in SR and 0.09±0.02 pA/pF in AF cells. At physiological temperature (37°C), however, I_Na,late became insignificant. Plateau phase and upstroke velocity of action potentials (APs) recorded with sharp microelectrodes in intact human trabeculae were more sensitive to ranolazine in AF than in SR preparations. Sodium channel subunits expression measured with qPCR was high for SCN5A with no difference between SR and AF. Expression of SCN8A and SCN10A was low in general, and lower in AF than in SR. In conclusion, We confirm for the first time a TTX-sensitive current (I_Na,late) in right atrial cardiomyocytes from SR and AF patients at room temperature, but not at physiological temperature. While our study provides evidence for the presence of INa,late in human atria, the potential of such current as a target for the treatment of AF remains to be demonstrated.     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.