Abstract
Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.
Highlights
EXPERIMENTAL PROCEDURESConstructs, Oocytes, and Injection of cRNA and Carbonic Anhydrase—The human CAII cDNA (CAII-WT) and the mutant CAII-HEX with mutations of the amino acids at positions H3P, H4Q, L9A, H10K, H15Q, and H17S was kindly provided by Dr Reinhart Reithmeier
We investigated a possible interaction between NBCe1 and CAII by expressing the transporter in Xenopus oocytes either injected with CAII or coexpressing CAII-WT and CAII mutants
To check for possible interaction between the two proteins, we measured the changes in membrane current and cytosolic Naϩ concentration, two parameters that are directly and exclusively related to the NBCe1 transport activity [25, 32]. Both parameters did not change in native oocytes or in oocytes only injected with CAII, showing that they are both attributable to the expression of NBCe1
Summary
Constructs, Oocytes, and Injection of cRNA and Carbonic Anhydrase—The human CAII cDNA (CAII-WT) and the mutant CAII-HEX with mutations of the amino acids at positions H3P, H4Q, L9A, H10K, H15Q, and H17S was kindly provided by Dr Reinhart Reithmeier. Oocytes of stages V and VI were selected and injected with 14 ng of NBCe1/cRNA dissolved in DEPC/H2O using glass micropipettes and a microinjection device (Nanoliter 2000, World Precision Instruments, Berlin, Germany). Voltage Clamp Recording—A borosilicate glass capillary, 1.5 mm in diameter, was pulled to a micropipette and backfilled with 3 M KCl. The resistance of the electrodes measured in oocyte saline was around 1 M⍀. To obtain current-voltage relationships, the CAII activity of the sample, the rate of 18O degradation was the membrane potential was changed stepwise in 20-mV incre- obtained from the linear slope of the log (enrichment) over the ments between Ϫ120 and ϩ20 mV. The cuvette was filled with 10 ml of oocyte saline with a pH of 7.35 according to the mean intracellular pH of CAII-injected oocytes at 25 °C. A significance level of p Յ 0.05 is marked with a single asterisk, p Յ 0.01 a double asterisk, and p Յ 0.001 with triple asterisks
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