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

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

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.     

Saxitoxin and tetrodotoxin. Electrostatic effects on sodium channel gating current in crayfish axons.

The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (IgON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block greater than 99% of sodium channels. A smaller suppression of IgON was observed when 1 nM STX was used (KD = 1-2 nM STX). The suppression of IgON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, -140 to -120 mV. The maximal effect of these toxins on IgON was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of IgON induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of 4.9 +/- 1.7 mV and 10.0 +/- 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus.     

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl- aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX- B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX- B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.     

Aluminum enhances the voltage activated sodium currents in the neurons of the pond snail Lymnaea stagnalis L

1. The effects of aluminum on voltage activated sodium currents (VASCs) were investigated by using the conventional two-electrode voltage clamp technique in Lymnaea stagnalis L. neurons. The peak amplitude, kinetics, and voltage-dependence of activation and inactivation of the sodium currents were studied in the presence of 5-500 microM AlCl3, at pH = 7.7. 2. There was a significant concentration-dependent increase in the peak amplitude of sodium currents after Al treatment, ED50 = 67 microM. The threshold concentration of the enhancement was 50 microM. The maximal peak increase of 143% was caused by a 500 microM aluminum. The action of aluminum on VASCs developed slowly, and it is not recovered by washing within 20 min. 3. There was little alteration of the voltage-dependence of the current. It was not a significant effect on the activation- and inactivation time constants of INa, but the steady-state inactivation curve shifted to negative direction on the voltage axis in the presence of Al. 4. The leak currents were not influenced by aluminum up to the highest concentration applied.     

Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration-dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations < 50 microM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 microM, and the blocking site is located 41 +/- 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 microM) and TPeA (20 microM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology. 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.     

Kinetic study of N-type calcium current modulation by delta-opioid receptor activation in the mammalian cell line NG108-15

The voltage-dependent inhibition of N-type Ca2+ channel current by the delta-opioid agonist [D-pen2, D-pen5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 microM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased omega-conotoxin GVIA-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with 1 microM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from -20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at -20 mV and of reinhibition at -40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca2+ channel activation at the same potentials measured during application of DPDPE (approximately 7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel.     

Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

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.     

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.     

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.     

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.     

State-independent block of BK channels by an intracellular quaternary ammonium

Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca(2+)- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43-57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between -200 and -100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in P(o), in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.     

Fast and slow inactivation of sodium channels: effects of photodynamic modification by methylene blue.

Illumination of crayfish giant axons, during internal perfusion with 0.5 mM methylene blue (MB), produces photodynamic effects that include (i) reduction in total sodium conductance, (ii) shifting of the steady-state inactivation curve to the right along the voltage axis, (iii) reduction in the effective valence of steady-state inactivation and, (iv) potentially complete removal of fast inactivation. Additionally, the two kinetic components of fast inactivation in crayfish axons are differentially affected by MB+light. The intercept of the faster component (tau h1) is selectively reduced at shorter MB+light exposure times. Neither tau h1 nor the slower (tau h2) process was protected from MB+light by prior steady-state inactivation of sodium channels. However, carotenoids provide differing degrees of protection against each of the photodynamic actions listed above, suggesting that the four major effects of MB+light are mediated by changes occurring within different regions of the sodium channel molecule.     

Fast and slow steps in the activation of sodium channels

Kinetic features of sodium conductance (gNa) and associated gating current (Ig) were studied in voltage-clamped, internally perfused squid axons. Following a step depolarization Ig ON has several kinetic components: (a) a rapid, early phase largely preceding gNa turn-on; (b) a delayed intermediate component developing as gNa increases; and (c) a slow component continuing after gNa is fully activated. With small depolarizations the early phase shows a quick rise (less than 40 mus) and smooth decay; the slow component is not detectable. During large pulses all three components are present, and the earliest shows a rising phase or initial plateau lasting approximately 80 mus. Steady-state and kinetic features of Ig are minimally influenced by control pulse currents, provided controls are restricted to a sufficiently negative voltage range. Ig OFF following a strong brief pulse also shows a rising phase. A depolarizing prepulse producing gNa inactivation and Ig immobilization eliminates the rising phase of Ig OFF. gNa, the immobilized portion of Ig ON, and the rising phase reappear with similar time-courses when tested with a second depolarizing pulse after varying periods of repolarization. 30 mM external ZnCl2 delays and slows gNa activation, prolongs the rising phase, and slows the subsequent decay of Ig ON. Zn does not affect the kinetics of gNa tails or Ig OFF as channels close, however. We present a sequential kinetic model of Na channel activation, which adequately describes the observations. The rapid early phase of IgON is generated by a series of several fast steps, while the intermediate component reflects a subsequent step. The slow component is too slow to be clearly associated with gNa activation.     

Interaction of lidocaine with the cardiac sodium channel: effects of low extracellular pH are consistent with an external blocking site

Brief extracellular application of millimolar concentrations of lidocaine affected sodium currents recorded in isolated rat ventricular myocytes in two ways: 1) a reduction of the maximum current consistent with a channel blocking action, and 2) a negative shift in the voltage dependence of inactivation consistent with an interaction with the inactivated state of the channel. Both effects occurred very rapidly (< 1 s). Decreasing extracellular pH to 6.4 increased the potency for channel block (EC50 1.8 +/- 0.2 mM at pH 7.4 and 0.8 +/- 0.1 mM at pH 6.5) and decreased the potency to shift inactivation (V(1/2) shift -42 mV by 1 mM lidocaine at pH 7.4 and -12.6 mV at pH 6.5). Channel block was slightly less at +90 mV compared to -40 mV at either pH (not statistically significant). The increase in potency for block at decreased extracellular pH, while intracellular pH is buffered, and the lack of voltage dependence of block, suggest that the charged form of lidocaine can block the channel by interacting with a site near the extracellular mouth, although alternative explanations are discussed.     

Molecular and structural basis of resting and use-dependent block of sodium current defined using disopyramide analogues.

The effects of disopyramide (Norpace) and 14 closely related structural analogues on the Na current of voltage clamped squid axons were examined to determine which physico-chemical properties and which changes in the structure of the Norpace molecule can alter the nature of its sodium channel blocking actions. Conventional voltage clamp technique for internally perfused giant axons was used. Axons were exposed to 100 microM concentrations via the internal perfusion solution, and the actions of the 15 analogues to produce resting and use-dependent block of Na current were assessed. The roles of Na ions and the activation and inactivation processes in the development of and recovery from use-dependent block of Na current induced by the Norpace analogues were also examined. The results indicate that for both mono-tertiary and bis-tertiary amines the potency to produce use-dependent block was proportional to molecular weight, whereas the correlation between potency to produce resting block and molecular weight was significant only for bis-tertiary amines. The mono- were more potent than the bis-compounds. However, comparisons between compounds having similar molecular weights and/or pKa values indicate that other factors also can influence blocking potency. For compounds within each homologous mono- or bis-tertiary amine series, hydrophobicity as estimated from log P values (P = octanol/water partition coefficient) was found to influence the potency to produce use dependent block of Na current. Use-dependent block was extant in axons internally exposed to pronase to remove the inactivation process, which indicates that inactivation is not an obligate condition for development of use-dependent block of Na current. An important role for the activation process in the development of use-dependent block of Na current is suggested by the finding that, in general, the voltage dependence of Na current activation paralleled that of use-dependent block. However, the potential dependence of use-dependent block produced by less hydrophobic but not by more hydrophobic compounds was shifted in the hyperpolarizing direction by removing Na+ from the external solution. Compounds with intermediate hydrophobicities altered the time course of Na current during its activating and inactivating phases. This finding can be explained by the kinetics of association and dissociation of drug molecules with channel receptor sites during the development and relaxation of use-dependent block rather than by postulating any major effect of drug to alter channel gating kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)