We have recently found that the mitochondrial ATP synthase forms a large voltage-gated channel with the biophysical characteristics of mitochondrial permeability transition pore (mPTP). We have shown that minimal unit required for the channel activity is the ATP synthase monomer with its membrane embedded c-subunit ring.
1. We have optimized the large-scale purification procedure of ATP synthase from porcine heart mitochondria for cryo-EM structural studies and single-channel recordings. We confirmed the monomeric state of ATP synthase by cryo-electron microscopy of ATP synthase reconstituted proteoliposomes. We then used this same monomeric ATP synthase reconstituted proteoliposome for patch-clamp recordings, which revealed that the ATP synthase monomers form large conductance, Ca2+-sensitive and voltage-gated channels with the biophysical characteristics of mPTP.
2. We have purified human ATP synthase c-subunit from HEK 293 cells. We have also heterologously overexpressed and purified human c-subunit from E. coli plasma membranes. Human c-subunit purified from both HEK 293 cells and E.coli forms a large conductance channels, which can be gated by purified F1 portion of ATP synthase.
3. We observed that disassembly/dissociation of ATP synthase F1 from FO occurs when we expose primary hippocampal neurons to glutamate toxicity (elevated intracellular calcium conditions), suggesting that the non-reversible dissociation of F1 from FO occurs in pathology and confirming that the F1 ATPase is forming the gate of the c-subunit channel.
4. We have used CRISPR-Cas9 technology to knocked out five/six alleles of the ATP5G1/G2/G3 genes encoding ATP synthase c-subunit in mouse embryonic stem cells, which resulted in 85% KD of c-subunit in mitochondrial membranes. Patch-clamp recordings of c-subunit KD mitoplasts demonstrate low conductance activity that is not sensitive to calcium and CsA and lack the high-conductance (~1.5 nS) mPTP-like activity.
5. We have successfully made and purified the low-conductance mutant of c-subunit from HEK 293 cells. We are now generating CRISPR/Cas9-edited mice, which will have the low probability of c-subunit channel (mPTP) opening. We will introduce these mutations in transgenic Alzheimer's disease (AD) mice to study if they will protect the mice from the onset of AD-like features. We hypothesize that this mouse will be protected from degenerative diseases of brain and heart.
These findings confirm that the ATP synthase monomer and more specifically its membrane-embedded c-subunit ring forms the high conductance channel of mitochondrial permeability transition.
Alzheimer Disease; Mitochondria; Synaptic Transmission; Apoptosis; Neurodegenerative Diseases; ATP Synthetase Complexes; alpha7 Nicotinic Acetylcholine Receptor