The sequences of polyclonal antibodies against the α2, α3, α4, α5, α6, β2, β3, and β4 peptides were raised and characterized as previously described.
11 We used antibodies directed only against peptides located in the cytoplasmic loop between M3 and M4 (CYT) of the α2, α3, α4, α5, α6, β2, β3, and β4 subunits. The specificity and immunoprecipitation capacity of most of the antibodies have been previously described.
11,12
The eyes and visual cortex were dissected, immediately frozen in liquid nitrogen, and then stored at −80°C for later use. In each experiment, the eyes or visual cortex were separately homogenized in an excess of 50 mM Na phosphate (pH 7.4), 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonylfluoride (PMSF) for 2 minutes in a commercial homogenizer (Ultra Turrax; IKA Works, Wilmington, NC), after which the homogenates were diluted and centrifuged for 1.5 hours at 60,000g. The whole eyes and visual cortex were homogenized, diluted, and centrifuged twice, after which the pellets were collected, rapidly rinsed with 50 mM Tris-HCl (pH 7), 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 2 mM PMSF, and then resuspended in the same buffer containing a mixture of 10 μg/mL of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Triton X-100 at a final concentration of 2% was added to the washed membranes, which were extracted for 2 hours at 4°C. The tissue extracts were then centrifuged for 1.5 hours at 60,000g, recovered, and an aliquot of the resultant supernatants was collected for protein measurement using the BCA protein assay (Pierce Protein Research Products, Thermo Fisher Scientific, Rockford, IL) with bovine serum albumin as the standard.
The specific activity of the [
125I]α-Bgtx (PerkinElmer, Boston, MA) was 150 Ci/mmol. Binding to the visual cortex membranes was performed by means of overnight incubation with a saturating concentration of 5 nM [
125I]α-Bgtx at room temperature. Nonspecific binding was determined in parallel by means of incubation in the presence of 1 μM unlabeled α-Bgtx. After incubation, the samples were filtered and the bound radioactivity was directly counted in a γ counter. To ensure that the α7 subtype did not contribute to
3H-epibatidine (
3H-Epi) binding, the binding to the Triton X-100 extracts and the immunoprecipitation experiments were performed in the presence of 1 μM αBgtx, which specifically binds to the α7 subtype and prevents Epi from binding to the subtypes containing this subunit. The binding to the tissue extracts was performed by incubating the extracts with 2 nM
3H-Epi in the presence and absence of 100 nM cold Epi using an ion-exchange resin (DE52; Whatman, Maidstone, UK) as previously described.
11,12
For the immunoprecipitation experiments, the extracts obtained from tissues preincubated with 1 μM αBgtx were labeled with 2 nM 3H-Epi and then incubated overnight with a saturating concentration of affinity-purified anti–subunit-specific IgG (20 μg). The immunoprecipitation was recovered by incubating samples with beads containing bound anti-rabbit goat IgG (Technogenetics, Milan, Italy). The level of antibody immunoprecipitation was expressed as fmol of immunoprecipitated receptors/mg of protein.