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J. Biol. Chem., Vol. 281, Issue 28, 19241-19250, July 14, 2006

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Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO₃ Cotransporter NBCe1-A in Xenopus Oocytes^*

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO₃ cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I_NBC) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I_NBC or the initial rate at which the intracellular pH fell (dpH_i/dt) upon applying 5% CO₂ (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I_NBC or dpH_i/dt (Day 4). Because dpH_i/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I_NBC slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I_NBC or dpH_i/dt (days 3-4), exposed oocytes to EZA, and measured I_NBC or dpH_i/dt (Day 3-4). dpH_i/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.

Received for publication, March 8, 2006 , and in revised form, April 24, 2006.

^* This work was supported in part by National Institutes of Health Grant DK-30344. 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.

² Supported by Yale Liver Center NIDDK National Institutes of Health Grant P30-34989.

¹ To whom correspondence may be addressed. Tel.: 203-785-4483; Fax: 203-785-4951; E-mail: Jing.Lu{at}Yale.edu.

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