Enhanced Formation of a HCO−3 Transport Metabolon in Exocrine Cells of Nhe1–/– Mice

Tetsuji Nakamoto,Seth L. Alper,James E. Melvin,Mireya Gonzalez-Begne,Andrew K. Stewart,Ha-Van Nguyen

doi:10.1074/jbc.m707266200

Abstract

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.

Highlights

Consistent with a functional linkage between these two exchangers, targeted disruption of the Nhe1 gene decreases fluid secretion [6]
ClϪ/HCO3Ϫ Exchanger Activity Is Enhanced in Nhe1-deficient Mice—Knocking out the expression of an ion transport pathway is frequently associated with up-regulation of other functionally related transport proteins [29, 38]
Naϩ/Hϩ exchanger null mutation might be expected to give rise to compensatory changes to enhance the movement of ClϪ and HCO3Ϫ by increasing ClϪ/HCO3Ϫ exchanger activity

Summary

Introduction

Consistent with a functional linkage between these two exchangers, targeted disruption of the Nhe gene decreases fluid secretion [6]. In mammalian cells carbonic anhydrases (Ca) appear to associate directly with ClϪ/HCO3Ϫ exchangers, including members of both the SLC4A and SLC26A gene families, to form functional complexes termed transport metabolons (16 –24). Such transport metabolons, through direct coupling of transmembrane bicarbonate transport with physically proximate (and possibly bound) carbonic anhydrase activity, may maximize transmembrane bicarbonate fluxes associated with the regulation of intracellular and extracellular or lumenal pH (25; but see Ref. 26). To gain further insight into the functional relationship between the ClϪ/HCO3Ϫ and Naϩ/Hϩ exchangers of the basolateral membrane of exocrine acinar cells, we examined the expression and activity of HCO3Ϫ metabolism and transport proteins in parotid acini of mice deficient in Nhe and Nhe. The interaction of murine carbonic anhydrase II (Car2) with Ae2 was enhanced in Nhe1-deficient mice, suggesting an in vivo correlation between increases in ClϪ/HCO3Ϫ exchanger activity and formation of a functional Car2-Ae2 bicarbonate transport metabolon

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Conclusion