High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO− 3 challenge and to quantify transport activity. The NO− 3-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO− 3-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μm NO− 3. In the latter, induction showed a latency of 40–80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO− 3; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO− 3 additions which, after induction, resulted in reversible membrane depolarizations of (+)54–85 mV in the presence of 50 μm NO− 3; and it was suppressed when NH4 + was present during the first, inductive exposure to NO− 3. Voltage clamp measurements carried out immediately before and following NO− 3 additions showed that the NO− 3-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (−400 to +100 mV). Measurements of NO− 3 uptake using NO− 3-selective macroelectrodes indicated a charge stoichiometry for NO− 3 transport of 1(+):1(NO− 3) with common K m and J max values around 25 μm and 75 pmol NO− 3 cm−2sec−1, respectively, and combined measurements of pH o and [NO− 3] o showed a net uptake of approx. 1 H+ with each NO− 3 anion. Analysis of the NO− 3 current demonstrated a pronounced voltage sensitivity within the normal physiological range between −300 and −100 mV as well as interactions between the kinetic parameters of membrane voltage, pH o and [NO− 3] o . Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH o of 6.1, driving the membrane voltage from −350 to −150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO− 3. By contrast, the same depolarization effected an approx. 20% fall in the K m for transport as a function in [H+] o . These, and additional results are consistent with a charge-coupling stoichiometry of 2(H+) per NO− 3 anion transported across the membrane, and implicate a carrier cycle in which NO− 3 binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO− 3 transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO− 3 transport; finally, they distinguish metabolite repression of NO− 3 transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.
The Journal of Membrane Biology – Springer Journals
Published: Nov 1, 1997
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