α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6ß4 and α6ß2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release

Abstract

α6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of α6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. α-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of α-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC(50) by 2800-fold at α6/α3β2β3 but only by 6-fold at α6/α3β4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in α6-null mutant mice. α-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in β4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain α6β2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related α6* nAChRs; α6β4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

Figure 1.

Sequences of native α-CTx BuIA and analogs. For each analog, the amino acid that is substituted is bold and underscored. Disulfide bond connectivity is also shown. Pound sign (#) indicates an amidated C terminus.

Figure 2.

Concentration-response curves for blocking of nAChR subtypes α6/α3β2β3 (A, B) and α6/α3β4 (C, D) by α-CTx BuIA analogs. Data are means ± se from ≥3 separate oocytes. IC₅₀ and Hill slope values are given in Table 1.

Figure 3.

Concentration-response curves for blocking of nAChR subtypes by α-CTx BuIA[T5A;P6O]. A) α-CTx BuIA blocks rat α6/α3β4 with nanomolar potency, whereas it displays micromolar potency in blocking α3β4 and has little to no effect on the other nAChR subtypes. B) α-CTx BuIA blocks the mouse α6/α3β4 subtype with nanomolar potency, whereas it displays micromolar potency in blocking α3β4 and little effect on the other subtypes. Data are means ± se from ≥3 separate oocytes. IC₅₀ and Hill slope values are given in Table 2.

Figure 4.

Differential binding and kinetics of α-CTx BuIA and α-CTx BuIA[T5A;P6O] on α6/α3β2β3 andα6/α3β4 nAChRs. Representative responses in a single oocyte expressing each nAChR subtype are shown. A) Wild-type α-CTx BuIA blocks both α6/α3β4 and α6/α3β2β3 nAChRs. Block is pseudoirreversible on α6/α3β4 nAChRs, whereas block reverses in minutes after washout from α6/α3β2β3 nAChRs. nAChRs were expressed in Xenopus oocytes. After a control response to 100 μM ACh, toxin was perfusion applied at the indicated concentrations. Toxin was then washed out, and responses to ACh were again measured at 1-min intervals. B) α-CTx BuIA[T5A;P6O] blocks α6/α3β4 but not α6/α3β2β3 nAChRs. Blocking of α6/α3β4 by α-CTx BuIA[T5A;P6O] reverses on a minute time frame. Broken bar indicates perfusion application of toxin; solid bar indicates static bath application for 5 min. C) Individual peak currents for α-CTx BuIA[T5A;P6O] blocking of each subtype. Lack of effect of 10 μM α-CTx BuIA[T5A;P6O] on α6/α3β2β3 nAChR is shown for comparison.

Figure 5.

Deletion of the α6 subunit abolishes nicotine-stimulated [³H]NE release. Data are shown as percentage release over baseline. Values are means ± se from ≥3 separate experiments. ***P < 0.001 vs. wild-type release; Student's unpaired t test.

Figure 6.

Effect of α-CTxs on nicotine-stimulated NE release. A) α-CTx BuIA[T5A;P6O] blocks nicotine-stimulated [³H]NE release from mouse hippocampal synaptosomes by 32 ± 5.3%. Release is blocked by 90 ± 7.8% by α-CTx MII. ***P < 0.001, F(2,51) = 79.35, Dunnett's post hoc test. B) α-CTx MII[E11A] potently blocks nicotine-stimulated [³H]NE release. ***P < 0.001; F(4,9) = 20.77; Dunnett's post hoc test. C) α-CTx BuIA[T5A;P6O] does not block nicotine-stimulated [³H]NE release in hippocampal synatosomes from β4^−/− mice.

Figure 7.

Effect of α-CTx BuIA[T5A;P6O] on nicotine-stimulated DA release. α-CTx BuIA[T5A;P6O] does not inhibit nicotine-stimulated [³H]DA release from mouse striatal synaptosomes at concentrations as high as 1 μM. α-CTx MII blocks 41 ± 6.7% of [³H]DA release. Data are means ± se from ≥3 separate experiments. ***P < 0.001; F(4,35) = 6.85; Dunnett's post hoc test analysis.

Figure 8.

Structure of α-CTx BuIA, as determined by NMR (45). Arrows indicate residues that are important for discrimination between α6/α3β2β3 and α6/α3β4 (Thr5 and Pro6). Two views; panel B shows 180° rotation along the axis of view in panel A.

Figure 9.

Different nAChR subtypes modulate nicotine-stimulated DA vs. NE release. A) Principal nAChR subtypes that mediate DA release in mouse striatum. These receptors lack a β4 subunit, and only a subpopulation contains the α6 subunit (25). B) Principal AChR subtypes that mediate NE release in mouse hippocampus (31). Each subtype contains the α6 subunit. Only a subpopulation contains an α6/β4 subunit interface that is sensitive to blocking by α-CTx BuIA[T5A;P6O]. Arrow indicates the highest-affinity target of α-CTx. Arrow with label in parentheses indicates the lower-affinity site for α-CTx MII. Note that the majority of subtypes are sensitive to α-CTx MII, whereas only α6β4* nAChR is sensitive to α-CTx BuIA[T5A;P6O].