Michael F. Sheets

The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.

Molecular Action of Lidocaine on the Voltage Sensors of Sodium Channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Qmax) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel–gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Qmax of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaines most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near −100 mV. These findings suggest that lidocaines actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.

Extracellular divalent and trivalent cation effects on sodium current kinetics in single canine cardiac Purkinje cells

1. The effects of the extracellular divalent cations barium, calcium, cadmium, cobalt, magnesium, manganese, nickel and zinc and the trivalent cation lanthanum on macroscopic sodium current (INa) were characterized in enzymatically isolated single canine cardiac Purkinje cells under voltage clamp at 9‐14 degrees C. 2. All di(tri)valent cations produced depolarizing shifts in the conductance‐voltage relationship. The order of efficacy, taken as the concentration required to produce a 5 mV shift in the mid‐point of peak INa conductance, from least to most effective was (mM): Ca2+ (2.97) approximately Mg2+ (2.67) approximately Ba2+ (1.93) > CO2+ (1.02) approximately Mn2+ (0.88) > Ni2+ (0.54) > La3+ (0.095) approximately Cd2+ (0.083) approximately Zn2+ (0.076). 3. Addition of di(tri)valent cations also produced depolarizing shifts in voltage‐dependent availability. The order of efficacy from the least to most effective was (mM): Cd2+ (7.70) approximately Mg2+ (6.86) approximately Ba2+ (4.50) > Ca2+ (2.47) approximately CO2+ (1.87) approximately Mn2+ (1.24) approximately Ni2+ (1.20) > Zn2+ (0.300) > La3+ (0.060). 4. The Gouy‐Chapman‐Stern equations were used to evaluate di(tri)valent cation efficacy in binding to surface charges. Surface charge density was estimated as 0.72 sites nm‐2, and it was assumed that Mg2+, the divalent cation that produced the smallest shift, screened but did not bind to surface charges. Based on voltage‐dependent availability, KD from lowest to highest affinity were (mM): Ba2+ (2500) > CO2+ (1670) approximately Mn2+ (1430) approximately Ca2+ = Cd2+ = Ni2+ (1200) > Zn2+ (250) > La3+ (30). 5. All di(tri)valent cations also produced a concentration‐dependent acceleration of INa tail current relaxation. The addition of Ca2+ and La3+ produced acceleration of tail current relaxations that could be accounted for by the surface charge effects predicted from the shift in voltage‐dependent availability. Cd2+, which produced almost no change in voltage‐dependent availability, dramatically accelerated tail current relaxation. Zn2+, Ni2+, Mn2+ and CO2+ also produced greater acceleration of tail current relaxation than could be accounted for by surface charge effects. 6. Di(tri)valent cations delayed time to peak INa in a concentration‐dependent manner. The time to peak INa‐voltage relationship was well described by an exponential plus a constant, and di(tri)valent cations did not affect the slope factor or constant but shifted the relationship in the depolarizing direction. Similar to their effect on tail currents, addition of some di(tri)valent cations produced larger effects on time to peak INa than expected from the shift of voltage‐dependent availability.(ABSTRACT TRUNCATED AT 400 WORDS)

Gating of skeletal and cardiac muscle sodium channels in mammalian cells

1 Sodium channel ionic current (INa) and gating current (Ig) were compared for rat skeletal (rSkM1) and human heart Na+ channels (hH1a) heterologously expressed in cultured mammalian cells at ∼13 °C before and after modification by site‐3 toxins (Anthopleurin A and Anthopleurin B). 2 For hH1a Na+ channels there was a concordance between the half‐points (V½) of the peak conductance‐voltage (G–V) relationship and the gating charge‐voltage (Q–V) relationship with no significant difference in half‐points. In contrast, the half‐point of the Q–V relationship for rSkM1 Na+ channels was shifted to more negative potentials compared with its G–V relationship with a significant difference in the half‐points of −8 mV. 3 Site‐3 toxins slowed the decay of INa in response to step depolarizations for both rSkM1 and hH1a Na+ channels. The half‐point of the G–V relationship in rSkM1 Na+ channels was shifted by −8.0 mV while toxin modification of hH1a Na+ channels produced a smaller hyperpolarizing shift of the V½ by −3.7 mV. 4 Site‐3 toxins reduced maximal gating charge (Qmax) by 33% in rSkM1 and by 31% in hH1a, but produced only minor changes in the half‐points and slope factors of their Q–V relationships. In contrast to measurements in control solutions, after modification by site‐3 toxin the half‐points of the G–V and the Q–V relationships for rSkM1 Na+ channels demonstrated a concordance similar to that for hH1a. 5 Q max vs. G max for rSkM1 and hH1a Na+ channels exhibited linear relationships with almost identical slopes, as would be expected if the number of electronic charges (e−) per channel was comparable. 6 We conclude that the faster kinetics in rSkM1 channels compared with hH1a channels may arise from inherently faster rate transitions in skeletal muscle Na+ channels, and not from major differences in the voltage dependence of the channel transitions.

Using lidocaine and benzocaine to link sodium channel molecular conformations to state-dependent antiarrhythmic drug affinity.

Rationale: Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in NaV1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block. Objective: Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes. Methods and Results: The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed. Conclusions: These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.

Outward stabilization of the S4 segments in domains III and IV enhances lidocaine block of sodium channels

The anti‐arrhythmic drug lidocaine has been shown to have a lower affinity for block of voltage‐gated sodium channels at hyperpolarized potentials compared to depolarized potentials. Concomitantly, lidocaine reduces maximum gating charge (Qmax) by 40% resulting from the complete stabilization of the S4 in domain III in an outward, depolarized position and partial stabilization of the S4 in domain IV in wild‐type Na+ channels (NaV1.5). To investigate whether the pre‐positioning of the S4 segments in these two domains in a depolarized conformation increases affinity for lidocaine block, a cysteine residue was substituted for the 3rd outermost charged residue in the S4 of domain III (R3C‐DIII) and for the 2nd outermost Arg in S4 of domain IV (R2C‐DIV) in NaV1.5. After biotinylation by exposure to extracellular MTSEA‐biotin the mutated S4s became stabilized in an outward, depolarized position. For Na+ channels containing both mutations (R3C‐DIII + R2C‐DIV) the IC50 for rested‐state lidocaine block decreased from 194 ± 15 μm in control to 28 ± 2 μm after MTSEA‐biotin modification. To determine whether an intact inactivation gate (formed by the linker between domains III and IV) was required for local anaesthetic drugs to modify Na+ channel gating currents, a Cys was substituted for the Phe in the IFM motif of the inactivation gate (ICM) and then modified by intracellular MTSET (WT‐ICMMTSET) before exposure to intracellular QX‐222, a quarternary amine. Although WT‐ICMMTSET required higher concentrations of drug to block INa compared to WT, Qmax decreased by 35% and the V1/2 shifted leftward as previously demonstrated for WT. The effect of stabilization of the S4s in domains III and IV in the absence of an intact inactivation gate on lidocaine block was determined for R3C‐DIII + ICM, R2C‐DIV + ICM and R3C‐DIII + R2C‐DIV + ICM, and compared to WT‐ICM. IC50 values were 1360 ± 430 μm, 890 ± 70 μm, 670 ± 30 μm and 1920 ± 60 μm, respectively. Thermodynamic mutant‐cycle analysis was consistent with additive (i.e. independent) contributions from stabilization of the individual S4s in R3C‐DIII + ICM and R2C‐DIV + ICM. We conclude that the positions of the S4s in domains III and IV are major determinants of the voltage dependence of lidocaine affinity.

Sodium channel molecular conformations and antiarrhythmic drug affinity.

Class I cardiac antiarrhythmic drugs, for example, lidocaine, mexiletine, flecainide, quinidine, and procainamide, continue to play an important role in the therapy for cardiac arrhythmias because of the presence of use-dependent block. Lidocaine, as well as related drugs such as mepivacaine, bupivacaine, and cocaine, also belong to the class of medications referred to as local anesthetics. In this review, we will consider lidocaine as the prototypical antiarrhythmic drug because it continues to be widely used both as an antiarrhythmic drug (first used as an antiarrhythmic drug in 1950) as well as a local anesthetic agent. Both of these clinical uses depend upon block of sodium current (I(Na)), but it is the presence of use-dependent I(Na) block, that is, an increasing amount of block at faster heart rates, which enables a local anesthetic agent to be a useful antiarrhythmic drug. Although many early studies investigated the action of antiarrhythmic drugs on Na currents, the availability of site-directed mutant Na channels has enabled for major advances in understanding their mechanisms of action based upon molecular conformations of the Na channel.

Isolation and characterization of single canine cardiac purkinje cells.

Single cardiac Purkinje cells should permit improved control of membrane potential during voltage clamp studies. We have developed a method for isolation of single canine Purkinje cells and studied their basic elecrrophysiological properties using conventional single and double microelectrode techniques. The single Purkinje cells appeared free of connective tissue, had regular striations, excluded trypan blue vital stain, and remained quiescent in solutions containing 1.8 mM calcium. Elecrrophysiological studies at 22°C showed normal resting membrane potentials, and action potentials could be elicited by extracellular or intracellular stimulation. Plot of the upstroke velocity of the action potential (Vn,) vs. the holding potential showed a sigmoid curve with the peak mean V, of 167 V/sec, and voltage corresponding to half-maximal V−* was about −70 mV. Plot of the overshoot of the action potential vs. the holding potential was similar, with maximal values of about +30 mV. The mean membrane input resistance was 21 Mil and the mean membrane capacitance was 360 pF. These experiments demonstrate that single Purkinje cells have electrical properties similar to intact Purkinje fibers and that they should be useful for more detailed elecrrophysiological experiments.

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.