Mitochondria Express α7 Nicotinic Acetylcholine Receptors to Regulate Ca2+ Accumulation and Cytochrome c Release: Study on Isolated Mitochondria

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate synaptic transmission in the muscle and autonomic ganglia and regulate transmitter release in the brain. The nAChRs composed of α7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional α7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early pro-apoptotic events like cytochrome c release. The binding of α7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and β2−/− but not α7−/− mice bound α7 nAChR-specific antibody and toxins: FITC-labeled α-cobratoxin or Alexa 555-labeled α-bungarotoxin. α7 nAChR agonists (1 µM acetylcholine, 10 µM choline or 30 nM PNU-282987) impaired intramitochondrial Ca2+ accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl2 or 0.5 mM H2O2. α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca2+ accumulation in mitochondria but attenuated the effects of agonists on cytochrome c release. Inhibitor of voltage-dependent anion channel (VDAC) 4,4′-diisothio-cyano-2,2′-stilbene disulfonic acid (0.5 µM) decreased cytochrome c release stimulated with apoptogens similarly to α7 nAChR agonists, and VDAC was co-captured with the α7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that α7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca2+ transport and mitochondrial permeability transition

Synthesis and pharmacological characterization of novel analogues of the nicotinic acetylcholine receptor agonist (±)-UB-165

(+/-)-UB-165 (1) is a potent neuronal nicotinic acetylcholine receptor (nAChR) ligand, which displays functional selectivity between nAChR subtypes. Using UB-165 as a lead structure, two classes of racemic ligands were synthesized and assessed in binding assays for three major nAChR subtypes (alpha4beta2*, alpha3beta4, and alpha7). The first class of compounds comprises the three pyridine isomers 4-6, corresponding to the 3-, 2-, and 4-substituted pyridine isomers, respectively. Deschloro UB-165 (4) displayed a 2-3-fold decrease in affinity at alpha4beta2* and alpha3beta4 nAChR subtypes,. as compared with (+/-)-UB- 165, while at the alpha7 subtype a 31-fold increase in affinity was observed. At each of the nAChR subtypes, high affinity binding was dependent on the presence of a 3-substituted pyridine, and the other isomers, 5 and 6, resulted in marked decreases in binding affinities. The second class of compounds is based on replacing the pyridyl unit of 1 with a diazine moiety, giving pyridazine (7), pyrimidine (8), and pyrazine (9), which retain the "3-pyridyl" substructure. Modest reductions in binding affinity were observed for all of the diazine ligands at all nAChR subtypes, with the exception of 7, which retained potency comparable to that of 4 in binding to alpha7 nAChR. In functional assays at the alpha3beta4 nAChR, all analogues 4-9 were less potent, as compared with 1, and the rank order of functional potencies correlated with that of binding potencies. Computational studies indicate that the 3-substituted pyridine 4 and 2-substituted pyridine 5, as well as the diazine analogues 7-9, all conform to a distance-based pharmacophore model recently proposed for the alpha4beta2* receptor. However, the nicotinic potencies of these ligands vary considerably and because 5 lacks appreciable nicotinic activity, it is clear that further refinements of this model are necessary in order to describe adequately the structural and electronic demands associated with this nAChR subtype. This rational series of compounds based on UB-165 presents a systematic approach to defining subtype specific pharmacophores

UB-165: A Novel Nicotinic Agonist with Subtype Selectivity Implicates the α4β2 Subtype in the Modulation of Dopamine Release from Rat Striatal Synaptosomes

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the α3β2-selective α-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this α-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the α-conotoxin-MII-insensitive subtype is composed of α4 and β2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at α4β2* and α3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over α7-type and muscle binding sites. (±)-Epibatidine, (±)-anatoxin-a, and (±)-UB-165 stimulated [3H]-dopamine release from striatal synaptosomes with EC50values of 2.4, 134, and 88 nm, and relative efficacies of 1:0.4:0.2, respectively. α-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (±)-UB-165 is a very poor agonist at the α-conotoxin-MII-insensitive nAChR subtype. In assays of86Rb+efflux from thalamic synaptosomes, a model of an α4β2* nAChR response, (±)-UB-165 was a very weak partial agonist; the low efficacy of (±)-UB-165 at α4β2 nAChR was confirmed inXenopusoocytes expressing various combinations of human nAChR subunits. In contrast, (±)-UB-165 and (±)-anatoxin-a were similarly efficacious and similarly sensitive to α-conotoxin-MII in increasing intracellular Ca2+in SH-SY5Y cells, a functional assay for native α3-containing nAChR. These data support the involvement of α4β2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.</jats:p