Ion channel properties of the reconstituted chloroplast triose phosphate/phosphate translocator.
The chloroplast triose phosphate/phosphate translocator (cTPT) was isolated from envelope membranes or from transformed yeast cells and reconstituted into artificial membranes. Ionic currents mediated by the cTPT across these membranes were investigated by flux measurements and by the patch-clamp technique. The results of the flux measurements indicate that inorganic phosphate (Pi) at saturating concentrations on both sides of the membrane and chloride (Cl-) at all applied concentrations are transported by the cTPT at rates about 20-fold higher than those measured in intact chloroplasts. After reconstitution of the protein into giant liposomes, single channel currents mediated by the cTPT were resolved with the patch-clamp technique. The protein was shown to be a voltage-dependent anion channel with complex gating revealing sublevels with conductances of 12, 54, 96, and 138 pS for Cl- and 6 pS and 18 pS for Pi, respectively. Recordings from patches compromising multiple channels show a synchronously appearing non-linear current voltage (I/V) relationship in symmetrical buffers, and a different gating at positive and negative membrane potentials. This suggests that the cTPT is incorporated into the membrane in a unidirectional orientation. 3-Phosphoglycerate, a high affinity substrate of the transporter protein, induced a reversible flickering of open channel, and the channel open probability was decreased 60%. It is concluded that, besides its normal counter-exchange mode, the cTPT can also work as a voltage-dependent anion selective channel.
Schwarz
M
M
Fachbereich Biologie/Chemie, Universität Osnabrück, Federal Republic of Germany.
Gross
A
A
Steinkamp
T
T
Flügge
U I
UI
Wagner
R
R
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001692:Biological Transport; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000502:physiology*; D005988:Glyceric Acids / Q000494:pharmacology; D015640:Ion Channel Gating; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008081:Liposomes; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D026901:Membrane Transport Proteins; D010940:Plant Proteins / Q000187:drug effects / Q000502:physiology*; D018724:Spinacia oleracea
D060365:Chloroplast Proteins; D005988:Glyceric Acids; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; C005156:3-phosphoglycerate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid