BETA-SUBUNIT COEXPRESSION AND THE ALPHA(1), SUBUNIT DOMAIN I-II LINKER AFFECT PIPERIDINE BLOCK OF NEURONAL CALCIUM CHANNELS

The effects of local anesthetics were examined on a family of transien tly expressed neuronal calcium channels. Fomocaine, a local anesthetic containing a morpholine ring, preferentially blocked alpha(1E) channe ls (K-i = 100 mu M), and had a lower affinity (3- to 15-fold) for alph a(1A), alpha(1B), and alpha(1C), channels. Block was incompletely reve rsible, followed 1:1 kinetics, and did not affect steady-state inactiv ation properties. Fomocaine block was sensitive to the concentration o f permeant ion and enhanced in the presence of external pore blockers, suggesting a site of action in the conducting pathway. Flecainide, wh ich carries a piperidine ring, and the diphenylbutylpiperidine antipsy chotic, penfluridol, caused qualitatively similar block, suggesting th at morpholine rings are compatible with the piperidine receptor site. In contrast, procaine, which contains an alkyl chain, caused reversibl e low affinity block of the different calcium channels (K-d values bet ween 2 and 5 mM) and was least effective on alpha(1E) and did not comp ete with fomocaine, suggesting that local anesthetics interact with at least two distinct receptor sites. Compared to coexpression with the Ca channel beta(1b) subunit, block at the piperidine receptor site was significantly weakened with the beta(2a) subunit suggesting that the nature of the beta subunit contributes to drug binding. Amino acid cha nges in the cytoplasmic linker between domains I and II resulted in de creased fomocaine and penfluridol blocking affinity. Furthermore, the blocking affinity observed with alpha(1B) was conferred on alpha(1A) b y substitution of the domain I-II linker of alpha(1B) into alpha(1A). Taken together, the data suggest that beta subunit binding and the dom ain I-II linker contribute to the piperidine receptor site on neuronal calcium channels.