Citrate transport in the human prostate epithelial PNT2-C2 cell line: electrophysiological analyses

摘要

Although prostate synthesizes and releases large amounts of citrate, the mechanism of the release is not well understood. Most known citrate transporters mediate uptake of citrate from extracellular space and, consequently, are driven by the transmembrane Na⁺ gradient, which would not be appropriate for prostatic function. In the present study, we investigated citrate transport in a normal human prostate cell line, PNT2-C2, using mainly electrophysiological methods. Intracellular application of citrate through the patch pipette in the whole-cell recording mode induced an outward current whilst in response to extracellular citrate an inward current was recorded. Membrane currents induced by citrate were bigger than those elicited by other (equimolar) Krebs cycle intermediates. Both inward and outward citrate-induced currents had the same ionic dependence, inhibitor profile and reversal potential. In particular, the currents were strongly dependent on the transmembrane K⁺ gradient. Uptake and release of citrate and their K⁺ dependence were confirmed by spectrophotometric enzyme analyses. Citrate-induced membrane currents were also sensitive to pH, consistent with the transporter preferring the trivalent form. Application of intracellular Zn²⁺ generated an outward current which had the same quantitative K⁺ dependence as the citrate-induced currents. Extracellular application of a membrane-permeant Zn²⁺ chelator generated an inward current. These experiments suggested that m-aconitase was tonically active in PNT2-C2 cells. Determination of ‘forward’ and ‘reverse’ K⁺ stoichiometry both suggested a citrate: K⁺ ratio of 1 : 4. We conclude that normal prostatic epithelial cells possess an electrogenic citrate transporter which mediates the cotransfer of 1 trivalent citrate anion alongside 4 K⁺ out of cells and thus generates a net outward current.