PATCH-CLAMP INVESTIGATION INTO THE PHOSPHATE CARRIER FROM SACCHAROMYCES-CEREVISIAE MITOCHONDRIA

After heterologous expression in E. coli, functionally active phosphat e carrier (PIC) from Saccharomyces cerevisiae mitochondria was purifie d and reconstituted into giant liposomes and used for patch clamp expe riments. Single channel currents across excised patches revealed an an ion channel function of the PIC protein. Besides the three transport m odes 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 chan nel activity was sensitive towards phosphate as its physiological subs trate. Phosphate (10 mM) blocked in a specific but reversible manner t he PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode. Furthermore, the current throug h the channel and its gating activity were affected by divalent cation s. In the presence of Ca2+ and Mg2+, the channel displayed a mean cond uctance of 25 +/- 5 pS whereas 40 +/- 10 pS was observed in the absenc e of divalent cations. Also, the dwell times in either the open or clo sed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+. The observed PIC channel characteristics are discuss ed with respect to previously reported electrophysiological in situ me asurements on anion channels of the inner mitochondrial membrane. Simi larities of the PIC channel to the inner mitochondrial anion channel ( IMAC) have been found. (C) 1997 Elsevier Science B.V.