Structure-Function Relationships of ω-Conotoxin GVIA

Abstract

The structure-function relationships of the N-type calcium channel blocker, -conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1 H 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 of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg 17 , Tyr 22 and Lys 24 ) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly 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 affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys 2 and Tyr 13 .The fish-hunting marine cone snails produce a range of polypeptide toxins that rapidly immobilize their prey (1, 2). A number of such toxins targeted at ion channels have been isolated from the venoms of these cone shells, including several that selectively block N-and P-type calcium channels (3). The toxin studied here, -conotoxin GVIA (GVIA), 1 is a 27-residue polypeptide from Conus geographus (4) that selectively blocks N-type voltage-gated calcium channels (5, 6), an activity that may confer a number of useful therapeutic properties on GVIA, including antihypertensive, analgesic, and neuroprotective activities, as demonstrated for the closely related -conotoxin MVIIA (MVIIA) from Conus magus (7) .The amino acid sequence of GVIA (with the cystine bridges indicated by lines) is as show below.

Novel ω-Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes

“…Inhibition of neurotransmitter release was positively correlated with the displacement of 125 I-GVIA binding, consistent with N-type VSCCs being the major source of Ca 2ϩ influx contributing to neurotransmitter release in the rat vas deferens stimulated at low frequencies (37)(38)(39)(40). A correlation between inhibition of Ca 2ϩ influx and ligand displacement at the N-type VSCC was shown for a series of MVIIA and TVIA analogues (41) but not for a series of GVIA analogues (33). Potency estimates obtained using the vas deferens were lower than estimates obtained from radioligand displacement studies (Table II), consistent with previous observations (37,42).…”

“…This could stem from a long range interaction with loop 4, where the major differences in primary structure are located. Loop 2 has previously been the least defined region of -conotoxin structures, with residues of this loop characterized by relatively broad peaks in the 1 H NMR spectra, indicative of conformational exchange (30,33). This lack of structure definition has hindered attempts to understand the crucial role loop 2 plays in activity, function, and selectivity of -conotoxins, particularly that of the principal binding determinant Tyr 13 , as well as residues of secondary importance such as Leu 11 and Arg 10 in MVIIA (34).…”

Solution Structure and Alanine Scan of a Spider Toxin That Affects the Activation of Mammalian Voltage-gated Sodium Channels

“…All of the amino acid residues in Magi 5 except Gly, Cys, and Ala were substituted with Ala. Although Gly could have a functional role, it has a high propensity to form reverse turns in high molecular weight proteins (42) and is an essential component of reverse turns in low molecular weight proteins folded according to the ICK motif (43). Thus, glycines were excluded from the Ala scan.…”

Cardiovascular and autonomic effects of ω‐conotoxins MVIIA and CVID in conscious rabbits and isolated tissue assays

“…Only one animal out of ®ve in the CVID group showed behavioural eects with mild tremors at 2 h post-administration (at 24 ± 48 h no further tremors were noted). Previously published data from our laboratory for comparison (Lew et al, 1997). Table 1.…”

“…Table 1. GVIA values are reproduced from previously published data from our laboratory for comparison (Lew et al, 1997). Catheter placement was con®rmed at autopsy following completion of the 48 h experiment in all animals.…”