Coronaridine congeners modulate mitochondrial a3b4* nicotinic acetylcholine receptors with different potency and through distinct intra-mitochondrial pathways Hugo R. Arias a, * , Olena Lykhmus b , Kateryna Uspenska b , Maryna Skok b a Department of Basic Sciences, California Northstate University College of Medicine, Elk Grove, CA, USA b Department of Molecular Immunology, Palladin Institute of Biochemistry NAS of Ukraine, 9, Leontovycha str., 01030 Kyiv, Ukraine article info Article history: Received 7 November 2017 Received in revised form 8 December 2017 Accepted 21 December 2017 Available online 23 December 2017 Keywords: Coronaridine congeners (þ)-Catharanthine (±)-18-Methoxycoronaridine Mitochondrial nicotinic acetylcholine receptors Mitochondria-driven apoptosis abstract In contrast to plasma membrane-expressed nicotinic acetylcholine receptors (nAChRs), mitochondrial nAChRs function in an ion-independent manner by triggering intra-mitochondrial kinases that regulate the release of cytochrome c (Cyt c), an important step in cellular apoptosis. The aim of this study is to determine the structural requirements for mitochondrial a3b4* nAChR activation by measuring the modulatory effects of two noncompetitive antagonists of these receptors, (þ)-catharanthine and (±)-18- methoxycoronaridine [(±)-18-MC], on Cyt c release from wild-type and a7/ mice mitochondria. The sandwich ELISA results indicated that a3b4* nAChRs are present in liver mitochondria in higher amounts compared to that in brain mitochondria and that these receptors are up-regulated in a7/ mice. Correspondingly, (±)-18-MC decreased Cyt c release from liver mitochondria of wild-type mice and from brain and liver mitochondria of a7/ mice. The effect in wild-type mice mitochondria was mediated mainly by the Src-dependent pathway, regulating the apoptogenic activity of reactive oxygen species, while in a7/ mice mitochondria, (±)-18-MC strongly affected the calcium-calmodulin kinase II- dependent pathway. In contrast, (þ)-catharanthine was much less potent than (±)-18-MC and trig- gered several signaling pathways, suggesting the involvement of multiple nAChR subtypes. These results show for the first time that noncompetitive antagonists can induce mitochondrial a3b4* nAChR signaling, giving a more comprehensive understanding on the function of intracellular nAChR subtypes. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating fast synaptic transmission in nerve and muscle cells and regulating cell proliferation, survival or cytokine pro- duction in many, if not all, non-excitable tissues (Kalamida et al., 2007; Kawashima and Fujii, 2008). Previous studies reported that a3b2, a4b2, a7b2 and a9 nAChR subtypes are expressed in the outer membrane of mitochondria, where they regulate the internal pathway of apoptosis in an ion channel-independent manner (Gergalova et al., 2012, 2014; Lykhmus et al., 2014; reviewed in Skok et al., 2016). Both nicotinic agonists and antagonists as well as positive allosteric modulators (PAMs) with selectivity for a7-or b2- containing nAChRs attenuated cytochrome c (Cyt c) release from isolated mitochondria (Gergalova et al., 2014; Uspenska et al., 2017), an important step in the process of cellular apoptosis. Our laboratory has hypothesized that the observed decrease is triggered by ligand-induced conformational changes in the receptor (reviewed in Skok et al., 2016). As an attempt to understand such novel molecular mechanisms, we contrasted the previous results using selective agonists and PAMs with that gathered in this work on noncompetitive antagonists (NCAs) such as (±)-18- methoxycoronaridine [(±)-18-MC] and (þ)-catharanthine (see Fig. 1 for the molecular structures). Pharmacologically, (±)-18-MC and (þ)-catharanthine behave as NCAs of several neuronal nAChR subtypes with preferential Abbreviations: a3b4* nicotinic acetylcholine receptors, a3b4-containing nAChRs; NCA, noncompetitive antagonist; WT, wild-type; RT, room temperature; (±)-18-MC, (±)-18-methoxycoronaridine; (þ)-catharanthine, (þ)-3,4- didehydrocoronaridine; Cyt c, cytochrome c; PAM, positive allosteric modulator; CaKMII, Src; MHb, medial habenula; apparent IC 50 , ligand concentration that pro- duces half-maximal inhibition. * Corresponding author. Department of Basic Sciences, California Northstate University College of Medicine, 9700 W. Taron Dr., Elk Grove, CA, USA. E-mail address: hugo.arias@cnsu.edu (H.R. Arias). Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci https://doi.org/10.1016/j.neuint.2017.12.008 0197-0186/© 2017 Elsevier Ltd. All rights reserved. Neurochemistry International 114 (2018) 26e32