Cloning and characterization of an electrogenic Na/HCO3- cotransporter from the squid giant fiber lobe.
The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na(+)-driven Cl-HCO(3)(-) exchanger, sqNDCBE--related to the SLC4 superfamily and cloned from giant fiber lobe cDNA--is the only HCO(3)(-)-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/HCO(3)(-) cotransporter, NBCe. That is, exposure to extracellular CO(2)/HCO(3)(-) 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 HCO(3)(-) 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.
Piermarini
Peter M
PM
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA. pmp26@cornell.edu
Choi
Inyeong
I
Boron
Walter F
WF
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D049832:Decapodiformes / Q000378:metabolism*; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014981:Xenopus
D018076:DNA, Complementary; D012333:RNA, Messenger; D027982:Sodium-Bicarbonate Symporters