Role of Polyhydroxybutyrate in Mitochondrial Calcium Transport

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to polyesters class and ubiquitously present in all living organisms. It has been demonstrated that mammalian mitochondrial membranes contain PHB polymer consisting of up to 120 butyrate residues. We found before that mitochondrial PHB complexed with Ca2+ and inorganic polyphosphate forms channels with properties similar to mitochondrial permeability transition pore. It is also experimentally proven that PHB in its free form possesses ionophoretic properties and mediates Ca2+ transport through the model phospholipid membranes. We hypothesized that PHB might significantly contribute to the process of mitochondrial Ca2+ transport. To test this idea we investigated Ca2+ transport in mitochondria with decreased levels of PHB. Mitochondrial PHB was reduced enzymatically by targeted expression of specific bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. Experiments were performed using transiently transfected cultured cells: HepG2, U87 and HeLa. PHB deficiency induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential (measured by TMRM) in HepG2 but not in U87 and HeLa cells. Kinetics of mitochondrial Ca2+ transport was measured using mitochondrial Ca2+ sensitive fluorescent probe - x-Rhod-1 and confocal microscopy. Mitochondrial Ca2+ signal was stimulated either by addition of ATP or Histamine in experiments with intact cells or by addition of increased concentrations of calcium to the recording solution in digitonin permeabilized cells. We found that over-expression of mitochondrially targeted PHB depolymeraze in mammalian cultured cells dramatically inhibits their Ca2+ uptake. Ca2+ uptake in these cells was restored by addition of mimic of calcium uniporter with electrogenic Ca2+ ionophore properties - ferutinin. Our data suggest that PHB is previously unrecognized essential component of mitochondrial Ca2+ uptake system.