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

Galyna Gergalova,Olena Lykhmus,Elena Kryukova,Maryna Skok,Lyudmyla Koval,Sergiy Komisarenko,Olena Kalashnyk,Victor Tsetlin,Volodymyr Chernyshov,Joseph Najbauer

doi:10.1371/journal.pone.0031361

Galyna Gergalova, Olena Lykhmus + Show 8 more

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https://doi.org/10.1371/journal.pone.0031361

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Abstract

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.

Highlights

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that were initially explored in muscle and autonomic ganglia and shown to mediate fast synaptic transmission [1]
We posed a question: whether functional a7 Nicotinic acetylcholine receptors (nAChRs) can be found in intracellular organelles, in in mitochondria, which are involved in inducing intracellular apoptotic pathway? Here we show that a7 nAChRs are expressed in the outer mitochondria membrane to regulate Ca2+ accumulation and cytochrome c release stimulated with apoptogens like high Ca2+ dose or H2O2
In other studies, the decrease of mitochondria membrane potential caused by ethanol was prevented with specific a7 nAChR agonist 2,4-dimethoxibenziliden anabasein and this effect was blocked with MLA [23]

Summary

Introduction

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that were initially explored in muscle and autonomic ganglia and shown to mediate fast synaptic transmission [1]. In the brain, they regulate glutamate-, GABAand dopamine-mediated transmission and are involved in the establishment of nicotine dependence [2]. The nAChRs are composed of combinations of ten alpha (a1 to a10) and four beta (b1 to b4) subunits, muscle-type receptors contain c, d or e subunits. They can be assembled as heteropentamers (eg. (a1)2b1cd (a3)2(b4), (a3)2a5(b4), (a4)2(b2), etc.) or as homopentamers (eg. (a7)5) and, correspondingly, differ in their cation selectivity, kinetics of the ion channel opening and desensitization [1]

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