Invited Symposium: Na-H Exchangers and Intracellular pH Regulation
NOTE to the reader: We apologize for the apparent lack of details in this presentation. However, since our abstract represents new and previously unpublished results, providing those results in this Internet forum would constitute publication. Presenting these results in this forum with current copyright practices would prohibit us from publishing in peer review journals in which we would otherwise publish. Consequently, we will only provide text descriptions and general conclusions of our work.
Recently we have cloned electrogenic Na/HCO3 cotransporters (NBC) isoforms from several species 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, Caenorhabditis elegans (ceNBC).
Materials and Methods
To study the expression of ceNBC, we used the Xenopus oocyte expression system. First, we subcloned this cDNA into a Xenopus expression vector. This vector contains the 5' untranslated region (UTR) and 3'UTR of Xenopus beta-globin. We linearize the plasmid and use this cDNA as a template to synthesize copy RNA (cRNA) in vitro using mMessage (Ambion, Austin, TX). Placing our cDNA between these UTR's allows the Xenopus oocyte to efficiently translated the injected cRNA as well as makes the cRNA very stable in the oocyte. The transcribed cRNAis injected into oocytes for expression. Using microelectrodes we monitored membrane voltage (Vm) and intracellular pH (pHi) of oocytes expressing ceNBC. Our experimental assay is to switch from a non-HCO3 solution to a HCO3 solution in the presence or in the absence of Na+.
A C. elegans genomic clone (F52B5.1) was identified when the Ambystoma 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 sequence 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, phosphorylation sites (9 protein kinase C, 9 casein kinase II, and 3 protein kinase A).
Bath addition of CO2 / HCO3 caused these ceNBC oocytes did acidify normally, i.e., acidification resulting from the hydration CO2 and subsequent dissociation of H2CO3 to form HCO3 and H+. Addition of even 5% CO2 / 33 mM HCO3 (pH 7.5) to oocytes injected with the ceNBC cRNA, did not elicit a membrane hyperpolarization. This hyperpolarization is typical of our vertebrate NBC clones (see Romero et al, Nature 387:409, 1997; Romero et al, Am. J. Physiol. 274:F425, 1998). However for ceNBC, in the continued presence of CO2/HCO3, there is a detectable pHi recovery not observed in water-injected control oocytes. Removal of CO2/HCO3 caused pHi to return and 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.
Discussion and Conclusion
Physiologically, there are 3 varieties of Na+/ HCO3 cotransporter which have been described: (1) in the kidney an electrogenic 1 Na : 3 HCO3 cotransporter, (2) in the brain and gut an electrogenic 1 Na : 2 HCO3 cotransporter, and (3) in the heart, an electroneutral 1 Na : 1 HCO3 cotransporter. So far, from all these tissues, we have only have molecular evidence for electrogenic Na/HCO3 cotransporters, NBCs (see Romero and Boron, Ann. Rev. Physiol. 61, in press, 1999).
By using GenBank and the EST database, we have identified a sequence from the round worm, Caenorhabditis elegans ~40 % similar to the NBCs or ceNBC (GenBank # AF004926). Sequence and hydropathy analysis indicates that this ceNBC is similar to both the NBCs and the AEs in basic membrane topology, and thus 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. Currently, there does not appear to be a mammalian ortholog to ceNBC. Additionally, we have recently cloned an electrogenic Na/HCO3 cotransporter from human heart (Choi et al, FASEB J. 12: A1031, 1997), it is unclear if an electroneutral NaHCO3 even exists in mammalian tissues.
For C. elegans the role is ceNBC is also unclear. Presently, we do not know the cell or tissue distribution of ceNBC. Thus, it is difficult to even speculate on the physiologic role ceNBC might play. Nevertheless, exploring the role and genetics of ceNBC could provide clues of where to find neutral Na/HCO3 cotransporters in mammalian systems and / or a tool for studying acid-base transport defects.
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|Romero, M.F.; Boron, W.F.; (1998). Identification and Expression of an Electroneutral Na/HCO3 Cotransporter from Caenorhabditis elegans (ceNBC).. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/fliegel/romero0784/index.html|
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