Mitochondria express α7 nicotinic acetylcholine receptors to regulate Ca2+ accumulation and cytochrome c release: study on isolated mitochondria

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 Ca(2+) accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl(2) or 0.5 mM H(2)O(2). α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca(2+) 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 Ca(2+) transport and mitochondrial permeability transition.

Figure 1. Electron microscopy images of mouse liver mitochondria.

Isolated mitochondria were stained with rabbit α7(1–208)-specific antibody followed by 10 nm colloidal gold-conjugated anti-rabbit IgG. A – secondary antibody only (control), B – α7(1–208)-specific antibody plus secondary antibody; arrows (1) indicate the sites of positive staining.

Figure 2. Identification of α7 nAChRs in isolated mitochondria by sandwich ELISA.

A and D – schemes of assays used; CTX-FITC – FITC-labeled α-cobratoxin, Bgt-Alexa – Alexa Fluor 555-labeled α-bungarotoxin, PO – horseradish peroxidase. B and E – the results of the CTX-FITC-developed (B, n = 6) or antibody-developed (E, n = 4) sandwich assays with the lysates of non-fractionated mitochondria from the wild-type (WT) or α7−/− mice and the outer (OM) and inner (IM) membranes of the wild-type mitochondria. *** – P<0.0005 compared to the data of the wild-type mitochondria and their outer membranes. C and F – the results of Bgt-Alexa-developed (C, n = 8) or antibody-developed (F, n = 5) sandwich assays with the outer membranes of mitochondria from the wild-type, α7−/− or β2−/− mice; non-specific binding detected in the presence of 200-fold molar excess of non-labeled α-cobratoxin (C) is subtracted. *** – P<0.0005 compared to the data of the outer membranes of the wild-type mitochondria.

Figure 3. The effects of α7-specific ligands and DIDS on Ca²⁺ accumulation in mitochondria studied by flow cytometry.

A – purified liver mitochondria gated by size (Forward scatter) and granularity (Side scatter) in flow cytometry. B – binding of 0.1 µM acridine orange 10-nonyl bromide (NAO) to the gated mitochondria population; Control – the non-stained mitochondria. C – Ca²⁺ accumulation in mitochondria loaded with Fluo 3-AM. D – Ca²⁺ accumulated in mitochondria during 2 min after 1 min pretreatment with DIDS, acetylcholine, choline, PNU-282987 or MLA; data are shown as normalized mean fluorescence values of 5 independent experiments for each ligand; * – P<0.05 compared to the fluorescence in the absence of α7 nAChR agonists or DIDS.

Figure 4. Connection of α7 nAChR and VDAC studied by sandwich assays.

The schemes (A, C) and results of direct (B, n = 3) and reverse (D, n = 3) sandwich assays demonstrating the connection of α7 nAChR and VDAC in the outer membranes (OM) of the wild-type (WT) mitochondria. Anti-TOM22 – antibody against mitochondrial outer membrane translocase.

Figure 5. Cytochrome c release into mitochondria supernatants.

Cyt c released from mitochondria in 2 min after addition of either 0.5 mM H₂O₂ or 90 µM CaCl₂ in the presence or absence of DIDS or α7 nAChR ligands. Each column corresponds to M±SE of three independent measurements. Mit – mitochondria without apoptogens; Contr – mitochondria treated with H₂O₂ or CaCl₂ only.