We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. (Zhou, J.J., Trueman, L.J., Boorer, K.J., Theodoulou, F.L., Forde, B.G., Miller, A.J. 2000. A high-affinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894–9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H+-NO 3 − symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I NO 3 − . We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H++NO 3 − )o, the product concentration inside, [P] = [H+] i 2 · [NO 3 − i, may rise substantially near the plasma membrane, violating the condition [P] ≪ [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K 1/2 ≈ 20 µM for I NO 3 − at pHo from Zhou et al. is confirmed. According to our analysis, NrtA operates between about 0.2 and 0.6 of the electrical distance in the membrane (outside 0, inside 1). In absence of thermodynamic gradients, the predominant orientation of the binding site(s) is probably inwards. The activity of the enzyme is sensitive to the transmembrane voltage, V, with an apparent gating charge of +1.0 ± 0.5 for inactivation, and transition probabilities of 0.3–1.3 s−1 at V = 0. This gating mode impedes loss of cellular NO 3 − during depolarization.
The Journal of Membrane Biology – Springer Journals
Published: Oct 9, 2003
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