High-affinity NO3--H+ cotransport in the fungus Neurospora: Induction and control by pH and membrane voltage

M R Blatt, L Maurousset, A A Meharg

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using Electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3--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 NO3--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 mu M NO3-. 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 NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 mu M NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3--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 NO3- uptake using NO3--selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K-m and J(max) values around 25 mu M and 75 pmol NO3- cm(-2)sec(-1), respectively, and combined measurements of pH(o) and [NO3-](o) showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- 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 [NO3-](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 NO3-. 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 NO3- anion transported across the membrane, and implicate a carrier cycle in which NO3- 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 NO3- transport system in Arabidopsis, and under line the importance of voltage as a kinetic factor controlling NO3- transport.

, finally, they distinguish metabolite repression of NO3- 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.

Original languageEnglish
Pages (from-to)59-76
Number of pages18
JournalJournal of Membrane Biology
Volume160
Issue number1
Publication statusPublished - 1 Nov 1997

Keywords

  • plasma membrane NO3--H+ cotransport
  • Neurospora crassa
  • NH4+ repression
  • metabolic blockade
  • voltage clamp
  • reaction kinetic model
  • STOMATAL GUARD-CELLS
  • NITRATE-REDUCTASE-ACTIVITY
  • POTASSIUM-PROTON SYMPORT
  • ION-SPECIFIC ELECTRODES
  • ARABIDOPSIS-THALIANA
  • PLASMA-MEMBRANE
  • SELECTIVE MICROELECTRODES
  • POTENTIAL CHANGES
  • BARLEY PLANTS
  • NO3 EFFLUX

Cite this

High-affinity NO3--H+ cotransport in the fungus Neurospora: Induction and control by pH and membrane voltage. / Blatt, M R ; Maurousset, L ; Meharg, A A .

In: Journal of Membrane Biology, Vol. 160, No. 1, 01.11.1997, p. 59-76.

Research output: Contribution to journalArticle

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T1 - High-affinity NO3--H+ cotransport in the fungus Neurospora: Induction and control by pH and membrane voltage

AU - Blatt, M R

AU - Maurousset, L

AU - Meharg, A A

PY - 1997/11/1

Y1 - 1997/11/1

N2 - High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using Electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3--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 NO3--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 mu M NO3-. 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 NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 mu M NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3--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 NO3- uptake using NO3--selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K-m and J(max) values around 25 mu M and 75 pmol NO3- cm(-2)sec(-1), respectively, and combined measurements of pH(o) and [NO3-](o) showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- 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 [NO3-](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 NO3-. 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 NO3- anion transported across the membrane, and implicate a carrier cycle in which NO3- 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 NO3- transport system in Arabidopsis, and under line the importance of voltage as a kinetic factor controlling NO3- transport., finally, they distinguish metabolite repression of NO3- 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.

AB - High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using Electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3--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 NO3--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 mu M NO3-. 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 NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 mu M NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3--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 NO3- uptake using NO3--selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K-m and J(max) values around 25 mu M and 75 pmol NO3- cm(-2)sec(-1), respectively, and combined measurements of pH(o) and [NO3-](o) showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- 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 [NO3-](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 NO3-. 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 NO3- anion transported across the membrane, and implicate a carrier cycle in which NO3- 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 NO3- transport system in Arabidopsis, and under line the importance of voltage as a kinetic factor controlling NO3- transport., finally, they distinguish metabolite repression of NO3- 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.

KW - plasma membrane NO3--H+ cotransport

KW - Neurospora crassa

KW - NH4+ repression

KW - metabolic blockade

KW - voltage clamp

KW - reaction kinetic model

KW - STOMATAL GUARD-CELLS

KW - NITRATE-REDUCTASE-ACTIVITY

KW - POTASSIUM-PROTON SYMPORT

KW - ION-SPECIFIC ELECTRODES

KW - ARABIDOPSIS-THALIANA

KW - PLASMA-MEMBRANE

KW - SELECTIVE MICROELECTRODES

KW - POTENTIAL CHANGES

KW - BARLEY PLANTS

KW - NO3 EFFLUX

M3 - Article

VL - 160

SP - 59

EP - 76

JO - Journal of Membrane Biology

JF - Journal of Membrane Biology

SN - 0022-2631

IS - 1

ER -