| Abstract: | Flagellar plasma membrane vesicles were isolated from sea urchin sperm using osmotic lysis. A membrane impermeant fluorescence pH indicator, pyranine, was incorporated into the vesicles as they resealed after lysis and was used to measure the intravesicular pH (pHi). Addition of Na+ rapidly alkalinized the pHi of vesicles prepared with an internal acidic pH gradient. The pHi increase showed ionic selectivity in the order of Na+ greater than Li+ much greater than K+ approximately equal to Cs+ approximately equal to O. Complete removal of monovalent anions such as Cl- and HCO3- did not affect the exchange, thus ruling out the participation of an anion carrier in the process. The optimal operation of the exchanger, however, required the presence of a transmembrane potential, which could be generated by the diffusion potential of either K+, a naturally permeant ion, or Cs+ which was artificially made permeant by the ionophore valinomycin. Depolarization inhibited the exchange in both the forward and the reverse directions, which is consistent with the voltage-gated electroneutral exchange mechanism proposed previously for this exchanger (Lee, H. C. J. Biol. Chem. (1984) 259, 15315-15319). The voltage sensitivity of the Na+/H+ exchanger was found to be modulated by the presence of Mg2+. A model involving the screening of the internal surface potential was proposed to account for the Mg2+ effect. The vesicle preparation used in this study allows complete control of the internal contents and represents a major simplification of the system as compared with the intact sperm and the isolated flagella used previously. |
