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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Cloning and characterization of an electrogenic Na/HCO₃^– cotransporter from the squid giant fiber lobe

¹Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; and ²Department of Physiology, Emory University School of Medicine, Atlanta, Georgia

Submitted 23 October 2006 ; accepted in final form 31 January 2007

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na⁺-driven Cl-HCO₃^– exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO₃^–-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5' and 3' directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na⁺-coupled SLC4 transporters. However, when we measure pH_i and membrane potential—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO₃^– cotransporter, NBCe. That is, exposure to extracellular CO₂/HCO₃^– not only causes a fall in pH_i, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pH_i recovery and current require HCO₃^– and Na⁺, and are blocked by DIDS. Furthermore, neither K⁺ nor Li⁺ can fully replace Na⁺ in supporting the pH_i recovery. Extracellular Cl^– is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

intracellular pH; acid-base; axon; SLC4 family