The Renal Cortical Na + /HCO 3 − Cotransporter VI: The Effect of Chemical Modification in Cotransporter Activity

Abstract

The Na+/HCO3- cotransporter is the main system that mediates bicarbonate removal out of the proximal tubule cell into the blood. We have previously partially purified this protein and showed that chemical modification of the alpha-amino groups by fluorescein isothiocyanate (FITC) inhibited the activity of the Na+/HCO3- cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that this compound binds at or near the substrate transport sites of the cotransporter. We examined the effect of agents that modify the sulfhydryl group (dithiothreitol), carboxyl groups (n-n'dicyclohexyl carbodiimide) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which may be important for transport function. The sulfhydryl residues modifier, carboxyl group modifier, and tyrosine modifier significantly inhibited bicarbonate dependent 22Na uptake in basolateral membranes by 50-70% without altering the 22Na uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting that the tyrosine residues are not at the substrate binding sites. To determine the presence and role of glycosylation on the Na+/HCO3- cotransporter protein, we examined the effects of different glycosidases (endoglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransporter activity. All glycosidases caused a significant 50-80% inhibition of cotransporter activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na+/HCO3- cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl groups as well as glycosylation are important for expression of full functional activity of the cotransporter.

Short-term regulation of organic anion transporters

“…Glycosylation is a two-step process; first, a bulky sugar chain is covalently added to the NH 2 group on the side chain of an asparagine (Asn) residue (N-linked glycosylation) or to the OH group of serine (Ser) or threonine (Thr) side chains (O-linked glycosylation) of the proteins, and secondly, the added sugars undergo a series of modification. Glycosylation has been demonstrated to play critical roles in the regulation of membrane targeting (Lee et al, 2003; Tanaka et al, 2004), protein folding (Kameh et al, 1998; Zhou et al, 2005), the maintenance of protein stability (resistance to proteolysis) (Khanna et al, 2001; Buck et al, 2004), and providing recognition structures for interaction with diverse external ligands (Ott et al, 1992; Bernardo et al, 1997). For the OAT family, each step of glycosylation plays a distinct role in OAT function.…”