Inhibition of wild-type and mutant neuronal nicotinic acetylcholine receptors by local anesthetics

Inhibition of neuronal nicotinic receptors can be regulated by the presence of specific amino acids in the subunit second transmembrane domain (TM2) d omain. We show that the incorporation of a mutant beta4 subunit, which cont ains sequence from the muscle beta subunit at the TM2 6' and 10' positions of the neuronal beta4 subunit, greatly reduces the sensitivity of receptors to the local anesthetic [2-(triethylaminc)-N-(2,6-dImethylphenyl)acetamide ] (QX-314). Although differing in potency, the inhibition of both wild-type alpha3 beta4 receptors and alpha3 beta4(6'F10'T) receptors by QX-314 is vo lage-dependent and noncompetitive. Interestingly, the potency of the local anesthetic tetracaine for the inhibition of alpha3 beta4 and alpha3 beta4(6 'F10'T) receptors seems unchanged when measured at -50 mV. However, whereas the onset of inhibition of wild-type alpha3 beta4 receptors is voltage-dep endent and noncompetitive, the onset of inhibition of alpha3 beta4(6'F10`T) receptors by tetracaine is unaffected by membrane voltage, and at concentr ations less than or equal to 30 muM seems to be competitive with acetylchol ine. This may be due to either direct effects of tetracaine at the acetylch oline binding site or preferential block of closed rather than open channel s in the mutant receptors. Further analysis of receptors containing the 6' mutation alone suggests that although the 6' mutation is adequate to alter the voltage dependence of tetracaine inhibition, both point mutations are r equired to produce the apparent competitive effects.