Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Recently we have cloned electrogenic Na/HCO3 cotransporters (NBC) isoforms from several spe-cies and tissues (for review see Romero & Boron, Ann. Rev. Physiol. 61, 1999). Here we report the molecular identification and expression of a Na/HCO3 cotransporter from the round worm, Caeno-rhabditis elegans (ceNBC). A C. elegans genomic clone (F52B5.1) was identified when the Ambys-toma kidney NBC (aNBC) was used in a BLAST search against GenBank (Romero et al, Nature 387:409, 1997). We used the predicted protein in another BLAST search against the expressed se-quence tag (EST) database and identified several clones corresponding to the predicted protein. We obtained these clones as a generous gift of Dr. Yuji Kohara (Gene Network Lab, Structural Biology Center, National Institute of Genetics, Mishima 411, Japan). EST sequences implied that all clones were likely part of the same cDNA, so we chose the largest clone for sequencing (yk41h3 or ceNBC). Sequencing revealed that ceNBC has a start Met and a complete open reading frame (ORF). The ORF of ceNBC (GenBank AF004926) is 1119 amino acids and predicts a protein of ~125 kDa. The encoded protein is ~40% identical to the vertebrate NBC clones, indicating that along with NBCs and the anion exchangers (AE1-3), ceNBC belongs to the Bicarbonate Transporter Superfamily. Using hydropathy analysis and comparison to NBCs and the anion exchangers, we propose a simplified model in which both N- and C-termini, a large central extracellular loop, and with 12 transmembrane spans. Sequence and model comparison predict 3 potential, N-linked glycosylation sites and several putative, phospho-rylation sites (9 protein kinase C, 9 casein kinase II, and 3 protein kinase A). We subcloned this cDNA into a Xenopus expression vector, transcribed cRNA, and injected the RNA into oocytes for expression. Using microelectrodes we monitored membrane voltage (Vm) and intracellular pH (pHi) of oocytes ex-pressing ceNBC. Addition of even 5% CO2 / 33 mM HCO3 (pH 7.5) to ceNBC oocytes, did not elicit a membrane hyperpolarization, typical of our vertebrate NBC clones. However, in the continued pres-ence of CO2/HCO3 there is a detectable pHi recovery not observed in water-injected control oocytes. Removal of CO2/HCO3 caused pHi to overshoot the pre-HCO3- steady-state pHi. If CO2/HCO3 is added to the bath in the absence of bath Na+ (choline replacement), there is no recovery in CO2 and no over-shoot upon CO2 removal. Bath Cl- removal in the presence of CO2/HCO3, does not seem to effect pHi. Thus, our data indicate that ceNBC is a new member of the bicarbonate transporter superfamily. Functionally, ceNBC is an electroneutral, Na(+)-coupled HCO3 transporter, most likely an electroneutral Na/HCO3 cotransporter.

Back to the top.
Presentation Number SAromero0784
Keywords: acid-base, intracellular pH, bicarbonate, cloning, Xenopus oocyteRec