Exposure to Hypoxia Rapidly Induces Mitochondrial Channel Activity within a Living Synapse

Abstract

One of the earliest effects of hypoxia on neuronal function is to produce a run-down of synaptic transmission, and more prolonged hypoxia results in neuronal death. An increase in the permeability of the outer mitochondrial membrane, controlled by BCL-2 family proteins, occurs in response to stimuli that trigger cell death. By patch clamping mitochondrial membranes inside the presynaptic terminal of a squid giant synapse, we have now found that several minutes of hypoxia trigger the opening of large multiconductance channels. The channel activity is induced concurrently with the attenuation of synaptic responses that occurs under hypoxic conditions. Hypoxia-induced channels are inhibited by NADH, an agent that inhibits large conductance channels produced by a pro-apoptotic fragment of BCL-xL in these synaptic mitochondria. The appearance of hypoxia-induced channels was also prevented by the caspase/cysteine protease inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk), which inhibits proteolysis of BCL-xL during hypoxia. Both NADH and Z-VAD-fmk reduced significantly the rate of decline of synaptic responses during hypoxia. Our results indicate that an increase in outer mitochondrial channel activity is a very early event in the response of neurons to hypoxia and suggest that this increase in activity may contribute to the decline in synaptic function during hypoxia.

Bcl-xL Inhibitor ABT-737 Reveals a Dual Role for Bcl-xL in Synaptic Transmission

“…Even in the absence of cell death, it is possible that such increases in permeability influence synaptic transmission (Jonas et al 2005a). In the squid synapse, the effects of hypoxia have served as a model for the effects of a cell death stimulus on synaptic activity (Jonas et al 2005b). In particular, the presynaptic terminal is sensitive to hypoxia, which attenuates synaptic transmission over 10-30 min (Jonas et al 2005b).…”

“…Previous studies have determined, using a Clark type oxygen electrode (Hansatech, Norfolk, UK), that levels of oxygen in the medium surrounding the ganglia fall by 48.1 ± 1.1% in 20 min (Jonas et al 2005b) after cessation of flow.…”

“…ΔN BCLxL produces large conductance ion channel activity in the outer mitochondrial membrane and may induce cell death in part by contributing to the increase in conductance of this membrane and the release of cytochrome c from mitochondria after a cell death stimulus (Clem et al 1998;Jonas et al 2004). In addition to these death-promoting activities, the pro-apoptotic form of BCL-xL, when injected into a presynaptic terminal, causes a decline in synaptic responses that mimics that recorded during acute hypoxic insults to the synapse (Jonas et al , 2005b.…”

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

“…62 These channels have conductances and other properties similar to mitochondrial channels detected in squid giant synapses during early stages of hypoxia-mediated apoptosis, when Bcl-x L is cleaved by caspases. 63 In particular, conductances were detected of up to 3.8 nS in media containing 570 mM KCl in the micropipette. 63 For purposes of comparison, this peak conductance should correspond to about 1 nS in symmetrical 150 mM KCl.…”

“…63 In particular, conductances were detected of up to 3.8 nS in media containing 570 mM KCl in the micropipette. 63 For purposes of comparison, this peak conductance should correspond to about 1 nS in symmetrical 150 mM KCl. Hence, the pore size is expected to be too small to allow for cytochrome c transport through the outer membrane and therefore is unlikely to have the same role as MAC during early steps of the intrinsic apoptotic pathway.…”