The voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa flagellar membrane vesicles studied with an entrapped pH probe.
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.
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D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D005407:Flagella / Q000502:physiology* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials; D008854:Microscopy, Electron; D012617:Sea Urchins; D017923:Sodium-Hydrogen Exchangers; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers