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.

Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at ~ 12~ Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Q~ax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-oi. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O---~I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O--~I transition determined. The O--*Itransition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O---)I transition at 0 mV could also be estimated (0.53 ms, ~ 12~ Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O---)Cand O---)Irate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.

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 lidocaine's 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 lidocaine's 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.

The site 3 toxin, Anthopleurin-A (Ap-A), was used to modify inactivation of sodium channels in voltage-clamped single canine cardiac Purkinje cells at ~12~Although Ap-A toxin markedly prolonged decay of sodium current (Iya) in response to step depolarizations, there was only a minor hyperpolarizing shift by 2.5 z 1.7 mV (n = 13) of the half-point of the peak conductance-voltage relationship with a slight steepening of the relationship from -8.2 --0.8 mV to -7.2 + 0.8 mV (n = 13). Increases in Gm~, were dependent on the choice of cation used as a Na substitute intracellularly and ranged between 26 -+ 15% (Cs, n = 5) to 77 -19% (TMA, n = 8). Associated with Ap-A toxin modification time to peak INa occurred later, but analysis of the time course INa at multiple potentials showed that the largest effects were on inactivation with only a small effect on activation. Consistent with little change in Na channel activation by Ap-A toxin, INa tail current relaxations at very negative potentials, where the dominant process of current relaxation is deactivation, were similar in control and after toxin modification. The time course of the development of inactivation after Ap-A toxin modification was dramatically prolonged at positive potentials where Na channels open. However, it was not prolonged after Ap-A toxin at negative potentials, where channels predominately inactivate directly from closed states. Steady state voltage-dependent availability (h= or steady state inactivation), which predominately reflects the voltage dependence of closed-closed transitions equilibrating with closed-inactivated transitions was shifted in the depolarizing direction by only 1.9 _+ 0.8 mV (n = 8) after toxin modification. The slope factor changed from 7.2 -+ 0.8 to 9.9 -+ 0.9 mV (n = 8), consistent with a prolongation of inactivation from the open state of Ap-A toxin modified channels at more depolarized potentials. We conclude that Ap-A selectively modifies Na channel inactivation from

Na channels are the source of excitatory currents for the nervous system and muscle. They are the target for a class of drugs called local anesthetics (LA), which have been used for local and regional anesthesia and for excitatory problems such as epilepsy and cardiac arrhythmia. These drugs are prototypes for new analgesic drugs. The drug-binding site has been localized to the inner pore of the channel, where drugs interact mainly with a phenylalanine in domain IV S6. Drug affinity is both voltage- and use-dependent. Voltage-dependency is the result of changes in the conformation of the inner pore during channel activation and opening, allowing high energy interaction of drugs with the phenylalanine. LA drugs also reduce the gating current of Na channels, which represents the movement of charged residues in the voltage sensors. Specifically, drug binding to phenylalanine locks the domain III S4 in its outward (activated) position, and slows recovery of the domain IV S4. Although strongly affecting gating, LA drugs almost certainly also block by steric occlusion of the pore. Molecular definition of the binding and blocking interactions may help in new drug development.