Abstract
It is generally agreed that solute transport across the Chara plasma membrane is energized by a proton electrochemical gradient maintained by an H(+)-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H(+)-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the true I-V profile for the pump when treatments alter the thermodynamic pressure on transport. We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cell I-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl3, so that steady-state currents could be recorded under voltage clamp between -400 and +100 mV. Exposures to 1 mM NaCN (CN) and 0.4 mM salicylhydroxamic acid (SHAM) depolarized (positive-going) Chara membrane potentials by 44-112 mV with a mean half time of 5.4 +/- 0.8 sec (n = 13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3 +/- 0.9 sec [( ATP]t = 0, 0.74 +/- 0.3 mM; [ATP]t = infinity, 0.23 +/- 0.02 mM). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses of dI-V curves (+/- CN + SHAM) as well as of families of I-V curves taken at times during CN + SHAM exposures indicated a stoichiometry for the pump of one charge (H+) transported per ATP hydrolyzed and an equilibrium potential near -420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60-75% of the total membrane conductance near Vm. Complementary results were obtained also in fitting previously published I-V data gathered over the external pH range 4.5-7.5. Kinetic features deduced for the pump were dominated by a slow step preceding H+ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium.