Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

Christian Barmeyer,Henry J Binder,Laurence Weitgasser,Vazhaikkurichi M Rajendran,Danny Atway,Jeff Huaqing Ye,John P Geibel,Lukas M Hingsammer,Carol Soroka,Peter Geibel,Luis Eduardo M Quintas

doi:10.1371/journal.pone.0062864

Abstract

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.

Highlights

Na-HCO3 cotransporters (NBC) are critical for regulating intracellular pH, HCO3 absorption and HCO3 secretion [1,2,3,4,5,6]
The results presented in this study indicate that the colonic Na-HCO3 cotransport activities are encoded by NBC have been classified as electroneutral (NBCn)- and NBC isoforms [i.e. electrogenic (NBCe)-like proteins that are expressed in a tissue-specific pattern, as only NBCn-like transcripts are localized in distal colon, while both NBCn-like and NBCe-like transcripts are present in proximal
To identify whether any existing NBC-like isoforms are expressed in the rat colon, northern blot analyses of proximal and distal colon mRNA were performed using full length NBCe1A and NBCn1A cDNA as probes [1,14,15]

Summary

Introduction

Na-HCO3 cotransporters (NBC) are critical for regulating intracellular pH (pHi), HCO3 absorption and HCO3 secretion [1,2,3,4,5,6]. NBC have been classified as electroneutral (NBCn) and electrogenic (NBCe) that mediate Na and HCO3 cotransport with a stoichiometry of 1:1 and 1:.1, respectively (Table-1) [1,4,6]. Both NBCn and NBCe have been shown responsible for HCO3 movement and pHi regulation in both epithelial and nonepithelial cells [1,4,6]. We demonstrated a novel NBC that is sensitive to both amiloride and DIDS in basolateral membrane vesicles (BLMV) of rat distal colon [7]. Both NBC and Na-H exchange functions are present in colonic BLMV, NBC has been identified as critical for pHi regulation since acid intravesicular pH activated the former but not the latter [8]

Methods

Results

Conclusion