STRUCTURE-FUNCTION-RELATIONSHIPS OF OMEGA-CONOTOXIN GVIA - SYNTHESIS,STRUCTURE, CALCIUM-CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGS

The structure-function relationships of the N-type calcium channel blo cker, omega-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substi tuted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence, In most cases the structure o f the analogues in aqueous solution was shown to be native-like by H-1 NMR spectroscopy, Minor conformational changes observed in some cases were characterized by two-dimensional NMR, Replacement of Lys(2) and Tyr(13) with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation o f rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay, Replacement of several other residu es with Ala (particularly Arg(17), Tyr(22) and Lys(24)) resulted in si gnificant reductions in potency (< 100-fold) in the functional assays, but not the binding assay, The potencies of the analogues were strong ly correlated between the different functional assays but not between the functional assays and the binding assay, Thus, the physiologically relevant assays employed in this study have shown that the high affin ity of GVIA for the N-type calcium channel is the result of interactio ns between the channel binding site and the toxin at more sites than t he previously identified Lys(2) and Tyr(13).