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-butylamin oethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable ary l azido moiety, which is known to form a covalent bond to adjacent mol ecules 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. Bioc himica et Biophysica Acta. 559:39-69.). We studied the effects of DNB- AB on the sodium current (I-Na) under whole-cell voltage clamp in clon al mammalian GH(3) cells and on (3)[H]-BTX-B binding to sheep brain sy naptoneurosomes. In the absence of UV illumination, DNB-AB behaved sim ilarly to known LAs, producing both reversible block of peak I-Na (IC5 0 26 mu M, 20 degrees C) and reversible inhibition of (3)[H]-BTX-B (50 nM in the presence of 0.12 mu g/liter Leiurus quinquestriatus scorpio n venom) binding (IC50 = 3.3 mu M, 37 degrees C), implying a noncovale nt association between DNB-AB and its receptor(s). After exposure to U V light, both block of I-Na and inhibition of (3)[H]-BTX-B binding wer e only partially reversible (I-Na = 42% of control; (3)[H]-BTX-B bindi ng = 23% of control) showing evidence of a light-dependent, covalent a ssociation between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on I-Na (post exposure I-Na = 96% of control ) or on (3)[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of I-Na was partially protected by coincubatio n of DNB-AB with 1 mM bupivacaine (IC50 = 45 mu M, for I-Na inhibition at 20 degrees C, Wang, G. K., and S. Y. Wang. 1992. Journal of Genera l Physiology. 100:1003-1020.), (post exposure I-Na = 73% of control). The irreversible inhibition of (3)[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 mu M, 37 degrees C) ( post exposure binding = 51% of control), suggesting that the site of i rreversible inhibition of both I-Na and (3)[H]-BTX-B binding is shared with the clinical LA bupivacaine.