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J. Biol. Chem., Vol. 275, Issue 51, 39894-39899, December 22, 2000
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From the Biochemistry and Physiology Department, Integrated
Approach to Crop Research (IACR)-Rothamsted, Harpenden, Herts, AL5 2JQ,
United Kingdom
We have expressed the CRNA high affinity nitrate
transporter from Emericella (Aspergillus)
nidulans in Xenopus oocytes and used
electrophysiology to study its properties. This method was used because
there are no convenient radiolabeled substrates for the transporter.
Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the
gene was originally identified by chlorate selection there was no
evidence for transport of this anion. The gene selection is explained
by the high affinity of the transporter for chlorite, and the fact that
this ion contaminates solutions of chlorate. The pH-dependence of the
anion-elicited currents was consistent with H+-coupled
mechanism of transport. At any given voltage, currents showed
hyperbolic kinetics with respect to extracellular H+, and
these data could be fitted with a Michaelis-Menten relationship. But
this equation did not adequately describe transport of the anion
substrates. At higher concentrations of the anion substrates and more
negative membrane voltages, the currents were decreased, but this
effect was independent of changes in external pH. These more
complicated kinetics could be fit by an equation containing two
Michaelis-Menten terms. The substrate inhibition of the currents could
be explained by a transport reaction cycle that included two routes for
the transfer of nitrate across the membrane, one on the empty carrier
and the other proton coupled. The model predicts that the substrate
inhibition of transporter current depends on the cytosolic nitrate
concentration. This is the first time a high affinity nitrate transport
activity has been characterized in a heterologous system and the
measurements show how the properties of the CRNA transporter are
modified by changes in the membrane potential, external pH, and nitrate
concentration. The physiological significance of these observations is discussed.
A High Affinity Fungal Nitrate Carrier with Two Transport
Mechanisms*
,
*
This work was supported by Grant PG206/0549 from the
Biotechnology and Biological Sciences Research Council (BBSRC) and by Grants BIO2-CT93-0400 and BIO4-CT97-2231 from the EU BIOTECH Program. Work at IACR-Rothamsted received grant-aided support from the BBSRC of
the United Kingdom.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Plant Genetics and Biotechnology,
Horticulture Research International, Wellesbourne, Warwick CV35 9EF,
United Kingdom.
§
Present address: Dept. of Biological Sciences, University of
Lancaster, Lancaster LA1 4YQ, United Kingdom.
¶
To whom correspondence should be addressed. Tel.:
44-1582-763133; Fax: 44-1582-763010; E-mail:
tony.miller@bbsrc.ac.uk.
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