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J. Biol. Chem., Vol. 282, Issue 6, 3720-3729, February 9, 2007

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Na⁺-dependent Inactivation of the Retinal Cone/Brain Na⁺/Ca²⁺-K⁺ Exchanger NCKX2^*

From the Department of Physiology and Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada

The SLC24 gene family Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are bidirectional plasma membrane transporters whose main function is the extrusion of Ca²⁺ from the cytosol. In this study, we used human embryonic kidney 293 cells expressing human retinal cone/brain NCKX2 to examine its Na⁺ affinity and kinetic parameters of Ca²⁺ transport. With the use of the ionophore gramicidin to control alkali cation concentrations across the plasma membrane, application of high intracellular Na⁺ promoted large NCKX2-mediated increases in intracellular free Ca²⁺ in the 15–20 µM range; this also resulted in inactivation of NCKX2 transport, the first description of this novel kinetic state. The affinity of NCKX2 for internal Na⁺ was found to be sigmoidal, with a Hill coefficient of 2.6 and K_d = 50 mM. The time-dependent (t 40s) inactivation of NCKX2 required high intracellular Na⁺ levels (K_d > 50 mM) as well as high occupancy of the extracellular Ca²⁺-binding site. Also reported are two residues whose substitution resulted in an increase in internal Na⁺ affinity to values of 19 mM; these mutants also displayed enhanced inactivation, suggesting that inactivation requires binding of Na⁺ to its intracellular transport sites. These findings are the first report of a regulatory kinetic state of Ca²⁺ transport via NCKX2 Na⁺/Ca²⁺-K⁺ exchangers that may play a prominent role in regulation of Ca²⁺ extrusion in cellular environments such as neuronal synapses that experience frequent and dynamic Ca²⁺ fluxes.

Received for publication, October 2, 2006 , and in revised form, December 8, 2006.

^* This work was supported in part by an operating grant from the Canadian Institutes of Health Research (to P. P. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a studentship from the Foundation Fighting Blindness-Canada.

² To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-5448; Fax: 403-283-8731; E-mail: pschnetk{at}ucalgary.ca.

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