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.

1 The eects of a novel N-type voltage-operated calcium channel antagonist, o-conotoxin CVID, were compared with o-conotoxin MVIIA on sympathetic-evoked activation of right atria (RA), small mesenteric arteries (MA) and vasa deferentia (VD) isolated from the rat. Their eects were also compared on blood pressure and cardiovascular re¯exes in conscious rabbits. 2 The pIC 50 values for MVIIA and CVID, respectively, for inhibiting sympathetic-evoked responses were equivalent in RA (8.7 and 8.7) and VD (9.0 and 8.7); however, in MA the values were 8.4 and 7.7. The cardiac to vascular (RA/MA) potency ratios, antilog (plog RA ± plog MA), for MVIIA and CVID were 2 and 10. The oset rates for CVID and MVIIA were rapid, and peptide reapplication caused rapid onset of blockade, suggesting limited desensitization. 3 In the conscious rabbit, CVID and MVIIA (100 mg kg 71 i.v.) caused a similar fall in blood pressure and a tachycardia that rapidly reached maximum. Both peptides decreased the vagal-and sympathetic-mediated components of the barore¯ex, but had no eect on the vagal nasopharyngeal re¯ex. The orthostatic re¯ex to 908 tilt was blocked by MVIIA with sustained postural hypotension for 590 min after administration. In contrast, CVID caused postural hypotension at 30 min which recovered rapidly. 4 Neither CVID nor MVIIA (3 mg kg 71 i.t.) signi®cantly altered cardiovascular variables or autonomic re¯exes. 5 In conclusion, CVID appears to be relatively weak at inhibiting the re¯ex response to tilt consistent with its weaker inhibition of rat mesenteric artery constriction to perivascular nerve stimulation. This may point to subtype N-type calcium channel selectivity.

The effects of N-type calcium channel inhibition with omega-conotoxin GVIA (omega-CTX) on cardiovascular parameters and vagally mediated autonomic reflexes and the role of the renin-angiotensin system were assessed in conscious rabbits. Omega-CTX (10 microg/kg, i.v.) resulted in hypotension, tachycardia, and attenuation of the sympathetic and vagal components of the baroreceptor-heart rate reflex (baroreflex). In the control group (no pretreatment), the peak decrease in mean arterial pressure (MAP) of 13 +/- 3 mm Hg from 72 +/- 2 mm Hg occurred after 33 +/- 3 min, with a corresponding tachycardia of 80 +/- 20 beats/min (n = 6). The tachycardia was due to vagal withdrawal, as a similar increase in heart rate (84 +/- 8 beats/min) after omega-CTX was observed after pretreatment with the beta-adrenoceptor antagonist, propranolol (n = 6). Angiotensin-converting enzyme (ACE) inhibition with enalaprilat revealed a larger, more rapid decrease in MAP in response to omega-CTX (-19 +/- 4 mm Hg from 65 +/- 1 mm Hg after 18 +/- 2 min; n = 6) compared with the control group. Similar larger decreases in MAP were also observed in the presence of the AT1-receptor antagonist, losartan, or the bradykinin B2 receptor antagonist, HOE-140 (n = 5-6). Pretreatment with enalaprilat, losartan, or HOE-140 caused a 50% decrease in the reflex tachycardia after omega-CTX compared with that observed in the control group, and omega-CTX caused a greater attenuation of the vagal component of the baroreflex and a decrease in the bradycardia evoked by the Bezold-Jarisch-like reflex. Also, there was a significant decrease in the bradycardia induced by the nasopharyngeal reflex after omega-CTX in the presence of ACE inhibition and HOE-140. Thus in the conscious rabbit, angiotensin II and bradykinin have a role in attenuating and slowing the hypotensive effect of N-type calcium channel inhibition. Vagolytic effects of omega-CTX on the baroreflex are augmented, and on other vagal reflexes are unmasked, via inhibition of the renin-angiotensin system. The complexity and mechanism of the interaction between N-type calcium channels and the renin-angiotensin system remain to be elucidated.

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1. The effects of central administration of omega-conotoxin GVIA (omega-CTX), an N-type calcium channel blocker, were examined in conscious rabbits implanted with lateral intracerebroventricular (i.c.v.) cannulae. 2. Experiments were performed over 4 consecutive days. On day 1, the baroreceptor heart rate (induced by glyceryl trinitrate and phenylephrine) and Bezold-Jarisch like (elicited by serotonin) reflexes were measured before (0 h) and 2 h after central administration of omega-CTX (3 or 30 pmol/kg, i.c.v.) or vehicle. On days 2-4, resting parameters and reflexes were again monitored but no further omega-CTX was administered. 3. No change in heart rate (HR) was observed in any rabbit treatment group during the experimental period. In the vehicle (n = 6) and omega-CTX 3 pmol/kg (n = 6) groups, small falls in mean arterial pressure (MAP) of 6 +/- 2 and 10 +/- 3 mmHg, respectively, occurred between 0 and 24 h; MAP then remained stable. Baroreceptor-heart rate reflex curve parameters did not change in either of these groups during the 4 day period. 4. Following administration of omega-CTX 30 pmol/kg (n = 7), MAP decreased progressively and by 48 h had fallen by 19 +/- 4 mmHg. Also at 48 h, a 20% decrease in HR range of the baroreceptor-heart rate reflex curve was seen without any change in the lower HR plateau from the 0 h control. This indicates that there was an attenuation of the sympathetically mediated upper component of the curve while the vagally mediated component was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The selectivity of w-conotoxin GVIA (cu-CTX) for prejunctional N-type voltage-operated calcium channels (VOCC) was examined in rat isolated small mesenteric arteries mounted in a Mulvany-Halpern myograph. Contractile responses to perivascular nerve stimulation, noradrenaline (NA) and potassium (K*) were obtained before and after treatment with o-CTX. The effects of cvCTX were compared with those of felodipine, an L-type VOCC blocker.2. wCTX (3 nmol/L-10 pmol/L) inhibited contractions to electrical field stimulation by up to 94%, compared with the corresponding time control group. Felodipine (0.1 pmol/L) had little effect on the contractions to electrical stimulation compared with the vehicle-treated vessels.3. Concentration-response curves to exogenous NA (0.1-30 pmol/L) and contractions to a submaximal concentration of K* (50 mmol/L) were unaffected by o-CTX (3 nmoI/L-10 pmol/L). In contrast, the maximum contraction to N A in vessels exposed to felodipine (0.1 pmol/L) was reduced by 37%, and the contraction to K* (62mmol/L) was reduced by 84% compared with vehicle-treated arteries. 4.The results indicate that even at Concentrations up to 10 pmol/L (10 000-fold higher than required to inhibit prejunctional N-type VOCC), cu-CTX inhibits only neurotransmitter release. Its effects are clearly different to felodipine as o-CTX has no effect on post-junctional al-adrenoceptormediated vasoconstriction or direct smooth muscle depolarization considered to be mediated by L-type VOCC. Therefore, at least at the vascular neuroeffector junction, o-CTX appears to be highly selective for N-type VOCC with no effect on Ltype VOCC.