Regulation of intracellular sodium concentrations in rat diaphragm muscle.

The concept is suggested that the sodium pump mechanism is influenced by the transmembrane electrical potential, and that the pump acts to maintain a constant electrochemical gradient for sodium. Evidence leading to this suggestion was obtained in rat diaphragm muscle by altering systematically the transmembrane chemical gradient for sodium ions and the transmembrane voltage. The voltage changes were produced by varying the extracellular and intracellular potassium ion concentrations. In each case the intracellular sodium concentration changed, presumably by activity of the sodium pump, so that the total electrochemical gradient for sodium was restored.

A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties.

The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.

Cardiac-specific external paths for lidocaine, defined by isoform-specific residues, accelerate recovery from use-dependent block.

Local anesthetic antiarrhythmic drugs block voltage-gated Na(+) channels from the cytoplasmic side. In addition, cardiac Na(+) channels can be also blocked by the membrane-impermeant local anesthetic QX via external paths not present in skeletal muscle or brain channels. Introduction of cardiac isoform-specific residues into wild-type skeletal muscle or brain channels creates access paths for external QX block. These paths should affect the characteristics of use-dependent block by influencing drug on- and off-rates. We investigated the effects of these external paths on drug kinetics of lidocaine, a lipophilic drug of clinical relevance, by studying use-dependent block using a two-electrode voltage clamp in Xenopus oocytes. Recovery from use-dependent block was slowed when cardiac isoform-specific residues important for external QX access were mutated to skeletal muscle or brain isoform-specific residues. As the fraction of charged lidocaine was decreased by raising external pH, differences in recovery kinetics diminished, indicating that these mutations mostly influenced block by charged lidocaine molecules. Data were fit into a model in which bound drug distributes into charged and neutral forms based on its pK(a) and external pH with separate dissociation paths and recovery-time constants. These isoform-specific mutations altered the recovery-time constants for the charged molecules with smaller effects on those for the neutral molecules. We conclude that the external egress paths created by isoform-specific residues influence the drug kinetics of lidocaine, and these residues define cardiac-specific external paths for local anesthetic drugs.

Effect of formaldehyde and glutaraldehyde on electrical properties of cardiac Purkinje fibers.

The effects of formaldehyde, glutaraldehyde, 1-fluoro-2,4-dinitrobenzene, and 1,5-difluoro-2,4-dinitrobenzene on the electrophysiological properties of cardiac Purkinje fibers were studied. At concentrations of 2.5 mM the aldehydes produced a transient hyperpolarization, lengthening of the plateau of the action potential, and an increase in action potential overshoot and upstroke velocity. If exposure to aldehyde was continued, the fiber failed to repolarize after an action potential and the membrane potential stabilized at about -30 mv. If exposure was terminated before this, recovery was usually complete. At the time the fibers were hyperpolarized the input resistance was increased without much change in length constant, leading to an increase in both calculated membrane resistance and calculated core resistance. Although it was anticipated that an effect of the aldehydes on the membrane was to increase fixed negative charge, it was difficult to explain all the electrophysiological changes on this basis. The major effects of the fluorobenzene compounds were not the same; they produced a shortening of the action potential and a rapid loss of excitability.

Activities of potassium and sodium ions in rabbit heart muscle.

Activities (a) of intracellular K and Na in rabbit ventricular papillary muslces were determined with cation-selectivve glass microelectrodes and concentrations (C) were estimated with flame photometry. The CK and aK of the muscles were 134.9 +/- 3.1 mM (mean value +/- SE) and 82.6 mM, respectively, at 25 degrees C. The corresponding CNa and aNa were 32.7 +/- 2.7 and 5.7, respectively. The apparent intracellular activity coefficients for K (gammaK) and Na (gammaNa) were 0.612 and 0.175, respectively. Similar results were obtained at 35 +/- 1 degree C. gammaK was substantially lower than the activity coefficient (0.745) of extracellular fluid (Tyrode's solution), which might be expected on the basis of a different intracellular ionic strength. gammaNa was much lower than that of extracellular fluid, and suggest that much of the Na was compartmentalized or sequestered. For external K concentrations greater than 5 mM, the resting membrane potentials agreed well with the potential differences calculated from the K activity gradients across the cell membrane as a potassium electrode. These results emphasize that potassium equilibrium potentials in heart muscle should be calculated by activities rather than concentrations.

Membrane response to current pulses in spheroidal aggregates of embryonic heart cells.

Hearts from chick embryos aged 4,7, or 14 days were dissociated into their component cells, and the cells allowed to reassociate in the form of smooth-surfaced spheroidal aggregates on a gyratory shaker. Records from intracellular electrodes inserted into two widely spaced cells in a spontaneously beating aggregate indicated that the action potentials occurred virtually simultaneously. In aggregates made quiescent with tetrodotoxin, the voltage response to a current pulse injected in one cell could be noted by recording with a second microelectrode at various distance from the current source. The magnitude of the response was found not to vary with distance. It is concluded that the component cells in an aggregate are normally tightly coupled electrically; the cell boundaries do not constitute an appreciable resistive barrier. Such ag-regates behave as virtually isopential systems, with properties similar to those of single spherical cells, as modeled by Eisenberg and Engel (1970. J. Gen. Physiol. 55:736-757). Passive membrane time constant ranged from 11 to 31 ms, with a mean value of 17 ms; this value did not vary with aggregate size. Input resistance (V/I) varied inversely with aggregate size, as predicted, but with much scatter in the measured values. Specific membrane resistance was calculated as either 13,000 or 800 ohm-cm2 depending on whether input resistance was attributed to the total cell surface membrane area or to the outer surface of the sphere alone. No systematic difference in passive electrical properties of aggregates composed of 4-, 7-, and 14-day cells was seen. It is concluded that these aggregates may be suitable for voltage clamp analysis of their excitable membrane properties.

Relationships between voltage and tension in sheep cardiac Purkinje fibers.

The two-microelectrode technique of voltage clamping sheep cardiac Purkinje fibers was used to examine the changes in contraction which occur during trains of voltage clamps. (A "train" is defined as a series of voltage clamps delivered at a particular rate, beginning after a rest long enough that the effects of previous stimulation have died away.) Contractions showed striking staircases, or progressive changes in peak isometric tension, during trains. Short clamps, clamps to voltages more negative than --20 or --30 mV, or holding potentials less negative than the resting potential favored negative staircases, while long clamps, clamps to positive voltages, and holding potentials near the resting potential each favored positive staircases. The staircase behavior appeared to be due to changes in the initial rate of recovery of the ability to contract. The changes in staircase behavior as a function of clamp voltage suggested that the relationship between peak tension and clamp voltage should depend on the experimental design. When the steady-state contraction was plotted as a function of clamp voltage, voltage-tension relations like those recently reported for working ventricle were obtained, with a threshold between --30 and --40 mV and a steep relation between tension and voltage. When the first contraction after a rest was plotted, the threshold voltage was more negative, the curve was flatter, and the peak tensions at inside positive voltages were reduced.

Slow inward current and contraction of sheep cardiac Purkinje fibers.

A "slow" inward current (Is) has been identified in ventricular muscle and Purkinje fibers of several mammalian species. The two-microelectrode voltage clamp technique is used to examine some of the relationships between Is and contraction of the sheep cardiac Purkinje fiber. "Tails" of inward current occurring on repolarization and extrapolation of Is recovery each show that the Is system may not inactivate completely during prolonged depolarization. The rate of recovery of Is after a depolarization is slow, and when a train of 300-ms clamps (frequency 1 s-1) is begun after a rest, Is is larger for the first clamp than it is for succeedings clamps. For the first clamp after a rest, the thresholds for Is and tension are the same and there is a direct correlation between peak tension and peak Is for clamp voltages between threshold and minus 40 mV. After a clamp, however, the ability to contract recovers much more slowly than does Is. Therefore, since Is may occur under certain conditions without tension, the realtionship between Is and tension must be indirect. Calcium entering the cell via this current may replenish or augment an intracellular calcium pool.

Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development.

Na+- and CA2+-sensitive microelectrodes were used to measure intracellular Na+ and Ca2+ activities (alpha iCa) of sheep ventricular muscle and Purkinje strands to study the interrelationship between Na+ and Ca2+ electrochemical gradients (delta muNa and delta muCa) under various conditions. In ventricular muscle, alpha iNa was 6.4 +/- 1.2 mM and alpha iCa was 87 +/- 20 nM ([Ca/+] = 272 nM). A graded decrease of external Na+ activity (alpha oNa) resulted in decrease of alpha iNa, and increase of alpha iCa. There was increase of twitch tension in low-alpha oNa solutions, and occasional increase of resting tension in 40% alpha oNa. Increase of external Ca2+ (alpha oCa) resulted in increase of alpha iCa and decrease of alpha iNa. Decrease of alpha oCa resulted in decrease of alpha iCa and increase of alpha iNa. The apparent resting Na-Ca energy ratio (delta muCa/delta muNa) was between 2.43 and 2.63. When the membrane potential (Vm) was depolarized by 50 mM K+ in ventricular muscle, Vm depolarized by 50 mV, alpha iNa decreased, and alpha iCa increased, with the development of a contracture. The apparent energy coupling ratio did not change with depolarization. 5 x 10(-6) M ouabain induced a large increase in alpha iNa ad alpha iCa, accompanied by an increase in twitch and resting tension. Under the conditions we have studied, delta muNa and delta muCa appeared to be coupled and n was nearly constant at 2.5, as would be expected if the Na-Ca exchange system was able to set the steady level of alpha iCa. Tension threshold was about 230 nM alpha iCa. The magnitude of twitch tension was directly related to alpha iCa.

High potassium and low sodium contractures in sheep cardiac muscle.

Contractures develop in sheep atrial trabeculae if Tyrode's solution is rapidly replaced by a solution containing elevated potassium, reduced sodium, or both. Two phases of the contracture can be identified on the basis of differences in physiological behavior: a rapid and transient phase that predominates during the first few seconds of the contracture, and a slowly developed phase that is responsible for the steady level of tension reached later in the contracture. The transient phase is particularly prominent if the muscle is stimulated rapidly before the contracture, and reduced or absent if the muscle is not stimulated or if calcium is not present before the contracture. Recovery of the transient phase after a contracture parallels the recovery of twitches. This transient phase appears to reflect the depolarization-induced release of activator (calcium) from an internal store, possibly the same store that is involved in the normal contraction. The slowly developed tension is dependent on the contracture solution used, and is decreased if the calcium concentration is reduced or if the sodium concentration is increased. It does not depend on conditions before the contracture and does not require time to recover. This phase of the contracture may be due to entry of calcium from the extracellular solution.

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers.

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers.

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.

Gating currents associated with Na channels in canine cardiac Purkinje cells.

Gating currents (Ig) were recorded in single canine cardiac Purkinje cells at 10-12 degrees C. Ig characteristics corresponded closely to macroscopic INa characteristics and appeared to exhibit little contamination from other voltage-gated channels. Charge density predicted by peak INa was 0.14-0.22 fC micron -2 and this compared well with the measured value of 0.19 +/- 0.10 fC micron -2 (SD; n = 28). The charge-voltage relationship rose over a voltage similar to the peak INa conductance curve. The midpoints of the two relationships were not significantly different although the conductance curve was 1.5 +/- 0.3 (SD; n = 9) times steeper. Consistent with this observation, which predicted that a large amount of the gating charge would be associated with transitions close to the open state, an analysis of activation from Hodgkin-Huxley fits to the macroscopic currents showed that tau m corresponded well with a prominent component of Ig. Ig relaxations fitted two exponentials better than one over the range of voltages in which Na channels were activated. When the holding potential was hyperpolarized, relaxation of Ig during step depolarizations to 0 mV was prolonged but there was no substantial increase in charge, further suggesting that early closed-state transitions are less in charge, further suggesting that early closed-state transitions are less voltage dependent. The single cardiac Purkinje cell appears to be a good candidate for combining Ig and single-channel measurements to obtain a kinetic description of the cardiac Na channel.

Kinetic analysis of single sodium channels from canine cardiac Purkinje cells.

Single sodium channel events were recorded from cell-attached patches on single canine cardiac Purkinje cells at 10-13 degrees C. Data from four patches containing two to four channels and one patch with one channel were selected for quantitative analysis. The channels showed prominent reopening behavior at voltages near threshold, and the number of reopenings declined steeply with depolarization. Mean channel open time was a biphasic function of voltage with the maximum value (1-1.5 ms) occurring between -50 and -40 mV and lower values at more and at less hyperpolarized levels. Inactivation without opening was also prominent near threshold, and this occurrence also declined with depolarization. The waiting time distributions and the probability of being open showed voltage and time dependence as expected from whole-cell current studies. The results were analyzed in terms of a five-state Markovian kinetic model using both histogram analysis and a maximum likelihood method to estimate kinetic parameters. The kinetic parameters of the model fits were similar to those of GH3 pituitary cells (Horn, R., and C. A. Vandenberg. 1984. Journal of General Physiology. 84:505-534) and N1E115 neuroblastoma cells (Aldrich, R. W., and C. F. Stevens. Journal of Neuroscience. 7:418-431). Both histogram and maximum likelihood analysis implied that much of the voltage dependence of cardiac Na current is in its activation behavior, with inactivation showing modest voltage dependence.

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)

mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains.

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

Electromechanical dissociation and its possible role in sudden cardiac death.

Electromechanical dissociation is a rare but important cause of death in some patients with cardiac ischemia. It is a well characterized syndrome involving loss of contractile function of the heart without arrhythmia. Several other clinical syndromes, including massive pulmonary embolism and cardiac rupture, can mimic electromechanical dissociation, so its incidence is difficult to estimate. Experimental studies of early ischemic contractile failure show preservation of adenosine triphosphate levels, and of the calcium ion release mechanism. Intracellular acidosis is temporarily correlated, but direct causality cannot be assigned to the acidosis because of the lack of necessary direct studies. The role of the autonomic nervous system in triggering the sequence of events leading to electromechanical dissociation is unclear and is a promising area for further study.

Cellular mechanism of action of cardiac glycosides.

It has long been known that cardiac glycosides can inhibit the membrane sodium-potassium (Na+-K+) pump, raising intracellular Na+. However, at clinical concentrations of cardiac glycosides, a change in intracellular Na+ that correlates with a change in cardiac contraction has been very difficult to demonstrate. The recent use of Na+-sensitive microelectrodes in the experimental laboratory has made intracellular Na+ measurements possible. A doubling of contraction strength in vitro is associated with a change of only approximately 1 mM intracellular Na+. Another membrane transport system, the Na+-Ca2+ exchange system, exchanges extracellular Na+ for intracellular Ca2+. If this system is responsible for regulating intracellular Ca2+, then it would be very sensitive to the transmembrane Na+ concentration gradient. This influence of intracellular Na+ on Na+-Ca2+ exchange is though to be the cellular basis of the positive inotropic action of digitalis. However, a number of issues remain unresolved, such as the extent of Na+-K+ pump inhibition by the level of cardiac glycoside achieved clinically.

Sodium currents in single cardiac Purkinje cells.

The sodium (Na) channel is the fundamental unit of excitability in heart muscle. This channel has been very difficult to study in detail, because the major experimental tool, the voltage clamp, has been difficult to use in multicellular tissue. In the absence of more direct studies in the heart, it has been assumed that the sodium channel in the heart was the same as that in nerve tissue, where it could be studied quantitatively. However, the sodium channel is not likely to be the same as in nerve, because it responds differently to local anesthetics and to other drugs such as tetrodotoxin. It is essential to learn the details of the cardiac sodium channel, because it is the membrane process that underlies many lethal cardiac arrhythmias, and it is the molecular site of action of the most effective antiarrhythmic drugs. Single cardiac Purkinje cells were dialyzed at room temperature through a large bore pipette, and their Na+ currents were studied under voltage clamp control. The peak currents were 0.5 to 1.0 mA/cm2, assuming a 1 mu farad/cm2 membrane. Peak currents near 0 mV were achieved in less than 1 ms. The decay of the Na+ current did not correspond to a single exponential process. This result and the observation that recovery from inactivation occurred with a latency are inconsistent with the original Hodgkin-Huxley model, but they qualitatively fit a model with two sequential inactivated states or a model with two kinetically different types of Na+ channels. The steady state inactivation curve shifted in the negative direction after initiation of intracellular dialysis, stabilizing with a half-availability voltage of -115 mV.

Mechanism of local anesthetic drug action on voltage-gated sodium channels.

Local anesthetic drugs interfere with excitation and conduction by action potentials in the nervous system and in the heart by blockade of the voltage-gated Na channel. Drug affinity varies with gating state of the channel. The drugs show low affinity at slow excitation rates, but high affinity when the channels are opened and inactivated during action potentials at high frequency, as they are during pain or during a cardiac arrhythmia. The drugs are thought to access their binding site in the inner pore by passage through the membrane and entry through the inner pore vestibule. There have been three major developments in the last decade that greatly increase our understanding of their mechanism of action. Firstly, amino acid residues critical to drug binding have been located by mutagenesis, and it is possible to develop a molecular model of the drug binding site. Secondly, a path for drug access directly from the outside has been characterized in the cardiac isoform of the channel. Thirdly, the hypothesis that high affinity binding stabilizes the fast inactivated conformation of the channel has been challenged. Rather, the drug may stabilize a slow inactivated state and immobilize the voltage sensor in domain III in its activated outward position. The combination of mutational study of the cloned Na channels and patch clamp offers the opportunity to understand the detailed molecular mechanism of drug action and to resolve drug structure-function.