INHIBITION OF BINDING OF [BATRACHOTOXININ-H-3 A 20-ALPHA-BENZOATE TO SODIUM-CHANNELS BY LOCAL-ANESTHETICS

  • 1 January 1984
    • journal article
    • research article
    • Vol. 25  (2) , 219-227
Abstract
The effects of several local anesthetics on the binding of ligands to receptors associated with voltage-sensitive Na channels in rat brain synaptosomes were examined. In the presence of 0.3 .mu.M scorpion toxin, the local anesthetics [tetracaine, etidocaine, bupivacaine, prilocaine, propanolol, mepivacaine, procaine, tocainide, W36017, benzocaine] tested inhibited the specific binding of [3H]batrachotoxinin A 20.alpha.-benzoate ([3H]BTX-B), a ligand which binds to a receptor site responsible for the activation of Na channel ion flux, in a dose-dependent fashion, with Kd values ranging from 1.2 .mu.M for tetracaine to 1.58 mM for benzocaine. A plot of log Kd from these binding experiments against log K0.5 for inhibition of Na currents by local anesthetics from electrophysiological experiments yielded a regression line with a slope of 0.84 and a correlation coefficient, r, of 0.86, demonstrating that the inhibition of [3H]BTX-B binding by local anesthetics occurs within a concentration range of physiological relevance. Tetracaine had little effect on basal 125I-labeled scorpion toxin binding to synaptosomes in the absence of batrachotoxin. However, in the presence of batrachotoxin, tetracaine inhibited the batrachotoxin-dependent increase in scorpion toxin binding (Kd = 2.0 .mu.M) in a dose-dependent manner, suggesting that inhibition of [3H]BTX-B binding by local anesthetics does not occur through binding at the scorpion toxin binding site. The inhibition of [3H]BTX-B binding by lidocaine was reversible within 30 min when samples were diluted from 10-3 M to 10-4 M lidocaine. Scatchard analysis of [3H]BTX-B binding to synaptosomes showed that bupivacaine and tetracaine reduced receptor affinity without decreasing maximal binding capacity. This reduction in receptor affinity in the presence of local anesthetics appears to be due, at least in part, to an increased rate of ligand dissociation from the receptor-ligand complex, suggesting an indirect allosteric mechanism for the inhibition of [3H] BTX-B binding by local anesthetics. Analysis of the effects of local anesthetics in terms of an allosteric model of drug action showed that they bind to inactive states of Na channels with at least a 10-fold higher affinity than active states. A 7-fold difference in Kd for inhibition of [3H]BTX-B binding between the local anesthetic stereoisomers RAC 109 I and RAC 109 II was observed. Similarly, the dissociation rate constant for the [3H]BTX-B/receptor complex was increased 9.3-fold in the presence of RAC 109 II and 4.3-fold in the presence of a comparable concentration of RAC 109 I. Taken together, the results suggest that the action of local anesthetics involves interaction with a specific receptor site(s) involved in the activation of voltage-sensitive Na channels. Since this interaction is stereospecific, this putative receptor site(s) probably includes protein components of the Na channel. Occupancy of the local anesthetic receptor site blocks batrachotoxin binding by an indirect allosteric mechanism.