Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria.
After heterologous expression in E. coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments. Single channel currents across excised patches revealed an anion channel function of the PIC protein. Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC. The PIC channel activity was sensitive towards phosphate as its physiological substrate. Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode. Furthermore, the current through the channel and its gating activity were affected by divalent cations. In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25 +/- 5 pS whereas 40 +/- 10 pS was observed in the absence of divalent cations. Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+. The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane. Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found.
Herick
K
K
Institut für Biotechnologie I, Forschungszentrum Jülich GmbH, Jülich, Germany.
Krämer
R
R
Lühring
H
H
D002352:Carrier Proteins / Q000502:physiology*; D002413:Cations, Divalent; D003902:Detergents; D004594:Electrophysiology; D005656:Fungal Proteins / Q000737:chemistry; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D010085:Oxidative Phosphorylation; D018408:Patch-Clamp Techniques; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D011510:Proteolipids; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000502:physiology
D002352:Carrier Proteins; D002413:Cations, Divalent; D003902:Detergents; D005656:Fungal Proteins; D007473:Ion Channels; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes