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.

No abstract

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10 -7 to 10 -5 M Са 2+ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Са 2+ , Sr 2+ , and Ва 2+ , were compared. ATP induced membrane fusion both in the presence and in the absence of Са 2+ , and the effects of ATP and Са 2+ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Са 2+ but also ATP. The intensity of Са 2+ -dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Са 2+ -regulated membrane fusion is possible without the involvement of the entire SNARE complex.

No abstract

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10 -7 to 10 -5 M Са 2+ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Са 2+ , Sr 2+ , and Ва 2+ , were compared. ATP induced membrane fusion both in the presence and in the absence of Са 2+ , and the effects of ATP and Са 2+ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Са 2+ but also ATP. The intensity of Са 2+ -dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Са 2+ -regulated membrane fusion is possible without the involvement of the entire SNARE complex.