Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity.
Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.
D000165:Acridine Orange / Q000378:metabolism; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D002523:Edible Grain / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D012430:Ruthenium Red / Q000494:pharmacology; D013379:Substrate Specificity; D013997:Time Factors
D012430:Ruthenium Red; D004024:Dicyclohexylcarbodiimide; D006859:Hydrogen; D000165:Acridine Orange; D002118:Calcium