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 alpha 7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional alpha 7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early proapoptotic events like cytochrome c release. The binding of alpha 7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and beta 2-/- but not alpha 7-/- mice bound alpha 7 nAChR-specific antibody and toxins: FITC-labeled alpha-cobratoxin or Alexa 555-labeled alpha-bungarotoxin. alpha 7 nAChR agonists (1 mu M acetylcholine, 10 mu M choline or 30 nM PNU-282987) impaired intramitochondrial Ca2+ accumulation and significantly decreased cytochrome c release stimulated with either 90 mu M CaCl2 or 0.5 mM H2O2. alpha 7-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 mu M) decreased cytochrome c release stimulated with apoptogens similarly to alpha 7 nAChR agonists, and VDAC was co-captured with the alpha 7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that alpha 7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca2+ transport and mitochondrial permeability transition.