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J. Biol. Chem., Vol. 276, Issue 28, 25919-25928, July 13, 2001
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From the Departments of We have recently described a novel
K+-dependent
Na+/Ca2+ exchanger, NCKX2, that is abundantly
expressed in brain neurons (Tsoi, M., Rhee, K.-H., Bungard, D., Li,
X.-F., Lee, S.-L., Auer, R. N., and Lytton, J. (1998)
J. Biol. Chem. 273, 4115-4162). The precise role for
NCKX2 in neuronal Ca2+ homeostasis is not yet clearly
understood but will depend upon the functional properties of the
molecule. Here, we have performed whole-cell patch clamp analysis to
characterize cation dependences and ion stoichiometry for rat brain
NCKX2, heterologously expressed in HEK293 cells. Outward currents
generated by reverse NCKX2 exchange depended on external
Ca2+ with a K1/2 of 1.4 or 101 µM without or with 1 mM Mg2+, and
on external K+ with a K1/2 of about 12 or 36 mM with choline or Li+ as counter ion,
respectively. Na+ inhibited outward currents with a
K1/2 of about 60 mM. Inward currents
generated by forward NCKX2 exchange depended upon external
Na+ with a K1/2 of 30 mM
and a Hill coefficient of 2.8. K+ inhibited the inward
currents by a maximum of 40%, with a K1/2 of 2 mM or less, depending upon the conditions. The transport stoichiometry of NCKX2 was determined by observing the change in
reversal potential as individual ion gradients were altered. Our data
support a stoichiometry for rat brain NCKX2 of 4 Na+:(1
Ca2+ + 1 K+). These findings provide the first
electrophysiological characterization of rat brain NCKX2, and the first
evidence that a single recombinantly expressed NCKX polypeptide encodes
a K+-transporting Na+/Ca2+
exchanger with a transport stoichiometry of 4 Na+:(1
Ca2+ + 1 K+).
Electrophysiological Characterization and Ionic Stoichiometry
of the Rat Brain K+-dependent
Na+/Ca2+ Exchanger, NCKX2*
§¶,
,§
Biochemistry and Molecular
Biology and § Physiology and Biophysics, University of
Calgary, Calgary, Alberta T2N 4N1, Canada
*
This work was supported in part by Canadian Institutes of
Health Research grants (to J. L. and to R. J. F.).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 Pharmacology, University of Alberta,
Edmonton, Alberta T6G 2H7, Canada.
**
Scientist of the Alberta Heritage Foundation for Medical Research
and a Distinguished Scientist of the Canadian Institutes of Health Research.
Senior Scholar of the Alberta Heritage Foundation for Medical
Research and an Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Calgary
Health Sciences Center, 3330 Hospital Dr. NW, Calgary, Alberta
T2N 4N1, Canada. Tel.: 403-220-2893; Fax: 403-283-4841; E-mail:
jlytton@ucalgary.ca.
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