INVITED REVIEW
Structural determinants and significance of regulation of electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter stoichiometry

Eitan Gross1 and Ira Kurtz2

1 Departments of Urology and Physiology and Biophysics, Case Western Reserve University, and Veterans Affairs Medical Center, Cleveland, Ohio 44106; and 2 Division of Nephrology, David Geffen School of Medicine, University of California, Los Angeles, California 90095


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters play an important role in intracellular pH regulation and transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in various tissues. Of the characterized members of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter superfamily, NBC1 and NBC4 proteins are known to be electrogenic. An important functional property of electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters is their HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ coupling ratio, which sets the transporter reversal potential and determines the direction of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> flux. Recent studies have shown that the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ transport stoichiometry of NBC1 proteins is either 2:1 or 3:1 depending on the cell type in which the transporters are expressed, indicating that the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ coupling ratio can be regulated. Mutational analysis has been very helpful in revealing the molecular mechanisms and signaling pathways that modulate the coupling ratio. These studies have demonstrated that PKA-dependent phosphorylation of the COOH terminus of NBC1 proteins alters the transport stoichiometry. This cAMP-dependent signaling pathway provides HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-transporting epithelia with an efficient mechanism for modulating the direction of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> flux through the cotransporter.

bicarbonate; transport; sodium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

THE BICARBONATE/CARBON DIOXIDE (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2) system is the most important buffer system in the extracellular fluid space (8). CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> are in equilibrium according to the following relationship
CO<SUB>2</SUB> + H<SUB>2</SUB>O ↔ H<SUB>2</SUB>CO<SUB>3</SUB> ↔ HCO<SUP>−</SUP><SUB>3</SUB> + H<SUP>+</SUP> (1)
The uniqueness of this reaction is derived from the fact that the components of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> buffer system are under the physiological control of two organs: the lungs, which modulate the level of PCO2 in the blood by altering the CO2 excretion rate, and the kidneys, which control the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration by absorbing the filtered HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> load and maintaining whole body proton balance. The pKa of the H2CO3 left-right-arrow HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> + H+ reaction is 3.57, and it would appear that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> would not be an effective buffer. However, because dissolved CO2 is in equilibrium with H2CO3, Eq. 1 can be simplified to CO2 + H2O left-right-arrow HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> + H+ (pK'a 6.1). Although the pK'a is still significantly less than the normal extracellular pH of 7.4, this system is an effective buffer because the PCO2 can be regulated by changes in alveolar ventilation. An additional reaction HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> left-right-arrow H+ + CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> can occur; however, the pKa of this reaction is 10.1; therefore, the CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> concentration is ~1:500 the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration at a pH of 7.4.

An essential role of the kidney in maintaining the blood HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration at ~25 mM is to reclaim HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> filtered through the glomeruli. The renal proximal tubule is the site in the nephron where the greatest quantity of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is reabsorbed into the peritubular blood. Proximal tubule transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is mediated by the coupled secretion of protons into the lumen and the transport of an equal number of base equivalents across the basolateral membrane into the peritubular blood. Apical proton secretion is mediated by an apical membrane Na+/H+ exchanger (NHE3) and to a lesser degree, a vacuolar H+-ATPase (8). The majority of basolateral plasma membrane HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is mediated by electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport (7, 20, 45, 113, 125).

Unlike the proximal tubule, which absorbs HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> from the glomerular filtrate, the direction of transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is in the secretory mode in several organs, including the duodenum (5), pancreas (35, 51, 57, 66, 109), airway epithelium (111), and salivary glands (72, 126). In the pancreatic duct, the concentration of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> can reach 140 mM in humans and guinea pigs (12, 13). Given a total fluid secretion rate of ~1 liter/day, the human pancreas therefore secretes ~ 140 meq/day of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, which drives sodium and water into the lumen by electroosmotic coupling (30, 56, 127). Pancreatic and duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion play an important role in buffering the hydrochloric acid load from the stomach (5). Furthermore, recent evidence suggests that the duodenum has a basolateral electrogenic Na+- HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport process (5, 53, 59, 92) that mediates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> influx and protects the epithelium from gastric acid-induced injury (5). In addition, by elevating pancreatic ductal pH, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion may have an additional role in activating pancreatic enzymes (35).

In contrast to the proximal tubule, the cellular model for pancreatic duct transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is less well understood. In pancreatic ducts, a basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport process plays a major role in mediating basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> influx, with subsequent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion across the apical membrane (2, 41, 56, 57, 118, 127). This basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport process was also found to be electrogenic (41). The apical transport mechanism(s) responsible for apical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretions have not been completely delineated. The traditional model for apical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit in exchange for Cl- entry via the anion exchanger is still questionable, as none of the known anion exchanger (AE) isoforms has been found in the apical membrane of these cells, nor has the obligatory dependence on apical Cl- been demonstrated. Greeley et al. (39) reported stimulation of downregulated in adenoma (DRA; SLC26A3) and the anion transporter PAT1 (SLC26A6) in cultured pancreatic duct cells (CFPAC-1) transfected with CFTR. They also found high levels of apical DRA in native mouse pancreatic duct cells by immunocytochemistry. While the presence of DRA may account for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Cl- exchange activity in some species, it does not explain the independence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit on the presence of apical Cl- in guinea pigs and possibly humans (55). Alternatively, it is conceivable that PAT1 plays a role in apical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exits in these cells. More recently, Ishiguro et al. (55), using a Cl--sensitive fluorophore to measure intracellular Cl- concentrations, proposed a model in which the luminal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit mode switches from a mixed mechanism involving both anion exchange and an anion conductance (most probably CFTR) at a high luminal Cl- concentration to a single mechanism involving only CFTR at a low luminal Cl- concentration. A similar mechanism was proposed to account for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in the rat pancreatic duct (38, 87) and in airway submucosal gland cells (31). An important role for CFTR in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is also implicated by the finding of impaired HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in the pancreas of patients with cystic fibrosis (27). Shumaker et al. (110) have suggested that PKA stimulates basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> influx in pancreatic duct cells through the basolateral electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter by activating the apical CFTR Cl- channel and depolarizing the basolateral membrane potential. It was speculated that the reduced number of functional CFTR Cl- channels in the cystic fibrotic pancreas is responsible for impaired HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion due to an insufficient depolarization of the basolateral membrane potential.


    PROXIMAL TUBULE AND PANCREATIC DUCT CELL MODELS
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

Figure 1 illustrates the relative roles that the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter plays in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the renal proximal tubule and the pancreatic duct. These models represent the presently accepted cellular mechanisms for transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in these two epithelia. In the renal proximal convoluted tubule (PCT), the cotransporter supports transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by mediating basolateral efflux of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, whereas in the pancreas the cotransporter supports transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion by mediating basolateral influx of these ions. There are several questions that arise with regard to these transport models. First, how does the cotransporter in the proximal tubule transport Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> across the membrane against their respective concentration gradients? Second, does the same electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter contribute to transepithelial Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in both epithelia? Third, what is the mechanism(s) for the difference in Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> stoichiometry in renal proximal tubule vs. pancreatic duct cells?


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Cellular models for transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. In the renal proximal tubule (A), kNBC1 mediates Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux by coupling the transport of 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and 1 Na+ to the membrane potential. In the exocrine pancreas (B), pNBC1 mediates Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> influx by coupling the transport of 2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to the downhill flux of Na+.


    ELECTROGENIC NBC PROTEINS AND MEMBERS OF THE HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> TRANSPORTER SUPERFAMILY
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

NBC1 is a member of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter superfamily (BTS), which includes the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger proteins AE1-AE3 (6, 24); the Na+-driven Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers (40, 100, 124); the electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC3 (93, 94) [splice variant NBC2 (54); rat orthologue NBCn1(25)]; and the second known electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter isoform NBC4 (95, 96, 104). AE4, which was initially reported to be a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (118), may function as an electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (88, 89). kNBC1, encoded by an alternate promoter in the NBC1 (SLC4A4) gene (3), is the main NBC1 variant expressed in the basolateral membrane of the renal proximal tubule (2, 22, 99, 105), where it normally operates with a 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+ stoichiometry. pNBC1 [also referred to as hhNBC (26), dNBC1 (59), and NBC1b (114)] is the major NBC1 variant expressed in the basolateral membrane of pancreatic ducts (1, 41, 73), where it normally operates with a 2:1 stoichiometry. Sequence alignment of kNBC1 and pNBC1 reveals that the two variants are 93% identical to each other, except that the 41 NH2-terminal amino acids of kNBC1 are replaced by 85 in pNBC1. An additional NBC1 COOH-terminal variant (rb2NBC) cloned from rat brain mediates electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport; however, its transport stoichiometry has not been measured (17). Finally, the recently cloned NBC4c splice variant of NBC4, which shares the greatest similarity at the amino acid level with NBC1 proteins, functions as an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in mammalian epithelial cells and is expressed in several tissues, including brain, heart, kidney, testis, pancreas, liver, and muscle (95, 96, 104).


    THERMODYNAMICS OF ELECTROGENIC NA+-COUPLED HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> TRANSPORT
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

Electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters are an example of a secondary active cotransporter, which couples the transport of one solute(s) against its concentration gradient to the electrochemical gradient of another solute(s). In the proximal tubule, kNBC1 couples the uphill transport of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to the membrane potential. The latter is determined mainly by the concentration gradient of potassium, generated by the action of the Na+-K+-ATPase. Thermodynamics dictates that for the membrane potential to drive the transport in the efflux direction, under average steady-state intra- and extracellular concentrations of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> found in the proximal tubule, the cotransporter should carry a net charge of -2 equivalents, e.g., 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> + 1 Na+ or 1 CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> + 1 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> + 1 Na+. These stoichiometries place the reversal potential of the cotransporter positive relative to the membrane potential and therefore ensures efflux of these ions (Fig. 2). A 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+ stoichiometry was reported by us and by other groups for basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in the proximal tubule, although there are also reports of a 2:1 stoichiometry (see Table 1).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Plot of the reversal potential (Erev) vs. the stoichiometry (n) for an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. The plot was generated using Eq. 5 with a 10-fold Na+ concentration gradient (high outside) and no HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradient. The plot illustrates that a basolateral cotransporter with a 2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+ stoichiometry such as pNBC1 in the pancreatic ducts will mediate the basolateral influx of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. A transporter with a 3:1 stoichiometry such as kNBC1 in the renal proximal tubule will mediate basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter stoichiometry in various cells and expression systems

Several methods have been described in the literature for measurements of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry. However, before describing present techniques for measuring the stoichiometry, we will briefly discuss the basic thermodynamics of the cotransport process. The cotransport of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> across a membrane can be described by
Na<SUP>+</SUP><SUB>i</SUB> + <IT>n</IT>HCO<SUP>−</SUP><SUB>3i</SUB> ↔ Na<SUP>+</SUP><SUB>o</SUB> + <IT>n</IT>HCO<SUP>−</SUP><SUB>3o</SUB> (2)
where the subscripts i and o stand for intracellular and extracellular compartments, respectively. The transport stoichiometry of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter can be determined by finding Na+ and/or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration gradients and membrane potentials at which no flux through the transporter occurs because electrical and chemical driving forces balance each other. The transporter reaction (Eq. 2) is at equilibrium and no net flux occurs when
&Dgr;&mgr;Na<SUP>i−o</SUP> = n&Dgr;&mgr;HCO<SUP>o−i</SUP><SUB>3</SUB> (3)
where Delta µNai-o is the in-to-out electrochemical potential difference for Na+, nDelta µHCO3o-i is the out-to-in electrochemical potential difference for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and n is the number of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anions cotransported with each Na+ cation. Expressing the electrochemical potential differences in terms of the relevant ion concentrations and the membrane potential, Vm, yields
<FR><NU>[Na<SUP>+</SUP>]<SUB>i</SUB></NU><DE>[Na<SUP>+</SUP>]<SUB>o</SUB></DE></FR> exp <FR><NU><IT>FV</IT><SUB>m</SUB></NU><DE><IT>RT</IT></DE></FR> = <FENCE><FR><NU>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>o</SUB></NU><DE>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>i</SUB></DE></FR> exp <FR><NU><IT>FV</IT><SUB>m</SUB></NU><DE><IT>RT</IT></DE></FR></FENCE><SUP><IT>n</IT></SUP> (4)
where F, R, and T have their usual meaning, and brackets and subscripts i and o stand for the intra- and extracellular concentration of the indicated ions, respectively. Vm at which no flux (current) occurs is called reversal potential (Erev), and can be experimentally determined from the current-voltage (I-V) relationship. Once determined, Erev can be used to evaluate the actual HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-to-Na+ transport ratio, n, according to
E<SUB>rev</SUB><IT>=</IT><FR><NU><IT>RT</IT></NU><DE><IT>F</IT>(<IT>n−</IT>1)</DE></FR> ln <FR><NU>[Na]<SUB>i</SUB></NU><DE>[Na]<SUB>o</SUB></DE></FR> <FR><NU>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUP><IT>n</IT></SUP><SUB>1</SUB></NU><DE>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUP><IT>n</IT></SUP><SUB>o</SUB></DE></FR> (5)
Equation 5 suggests that Erev depends logarithmically on the intra-to-extracellular ratio of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations and inversely on the cotransport ratio (Fig. 2). The prelogarithmic factor in Eq. 5 equals 26 mV for n = 2. Thus a 10-fold change in extracellular Na+ concentration will result in an initial change in membrane potential of 26 mV, whereas a 10-fold change in extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration will be expected to result in a 120-mV change. Seki et al. (107) have measured the cotransport stoichiometry in microperfused proximal tubules from rabbits and found it to be 2:1. They also measured the initial rates of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+ fluxes, by measuring intracellular pH and Na+ concetnration with a microelectrode, in response to a 10-fold step reduction of extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, and found the former flux to be about twice as large as the latter, thus reconfirming the 2:1 stoichiometry determined independently by the membrane potential measurements. Soleimani et al. (112, 113) used a variation of the flux ratio method in measuring the uptake of 22Na+ into vesicles made from the basolateral membrane of rabbit proximal tubule cells. We have used an apically permeabilized preparation of high-resistance epithelial monolayers grown on filters, in combination with an ion-substitution protocol, to measure the I-V relationship of electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters at defined Na+ concentration gradients (41, 43-47, 104). We then determined the reversal potential of the cotransporter from the I-V relationships and used Eq. 5 to calculate the stoichiometry. Figure 3 illustrates the experimental protocol used to measure the I-V relationships of electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters. Because thermodynamics describes net charge movements but not the chemical identity of the ions that carry the charge, none of the methods described above can distinguish between a stoichiometry that involves the transport of 2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anions vs. 1 CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> divalent anion. Indeed, a 3:1 stoichiometry could result from the transport of 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+ or 1 CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>:1 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+.. Furthermore, a 2:1 stoichiometry could result from either the loss of one HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site or a change from the transport of 1 CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> and 1 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to 2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anions (76, 77, 90), possibly due to a conformational change in the cotransporter that then leads to altered substrate affinity. A distinction between these various transport modes is not presently possible. Because at an extracellular pH of 7.4 the concentration of CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> is ~500-fold lower than that of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and ~1,000-fold lower at an intracellular pH of 7.1, it might be predicted that the flux of CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> would be negligible compared with that of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. However, this assumption would not be correct if the binding constant of the cotransporter for CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> is ~500- to 1,000-fold higher then that for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Experimental protocol for measurement of current-voltage (I-V) relationship of an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in epithelial monolayers. A 5-fold Na+ concentration gradient was applied across apically permeabilized cell monolayers in an Ussing chamber by perfusing the basolateral compartment with a modified HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Ringer solution containing 50 mM Na+ and the apical compartment with a solution containing 10 mM Na+. A: voltage pulse protocol used to collect I-V relationships in the absence (solid line) and presence (dotted line) of 2 mM DNDS. Voltage was stepped from -100 mV to 100 mV with 10-mV steps. B: I-V relationships in the absence (filled circles) and presence (open circles) of DNDS obtained by averaging the current at each voltage over 5 s. C: I-V relationships in the absence (filled circles) and presence (open circles) of DNDS for a 5-fold Na+ concentration gradient but in the nominal absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. The difference between the currents in B and those measured in C at the corresponding voltages represent the DNDS-sensitive currents.


    REGULATION OF NBC1 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:NA+ STOICHIOMETRY
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

Table 1 summarizes the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometries reported in different cell types. As can be seen from Table 1, different stoichiometries have been reported for different species, different tissues/cell types in the same species, and even for the same cell type in the same species. While it is possible that the various stoichiometries might be due to differences in the techniques utilized in some of these studies (e.g., isotope fluxes into basolateral vesicles vs. basolateral membrane potential changes in response to alterations in peritubular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+ concentrations), additional data suggest that this explanation is insufficient. Specifically, when proximal tubules were incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing Ringer, the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of the cotransporter was 2:1 (67, 77). However, when the incubation medium was changed to DMEM + norepinephrine, the stoichiometry changed to 3:1. In addition, Planelles et al. (90) has shown that the stoichiometry shifts from 3:1 to 2:1 in Necturus proximal tubules exposed to peritubular isohydric hypercapnia. It was unclear until recently whether the shift in stoichiometry involved more then one cotransporter, each operating with a different HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry, or whether the same cotransporter protein could change its stoichiometry. This important question has been addressed by us at the molecular level with the cloning of the NBC1 electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters.

After the cloning of the pancreatic variant of the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (1), pNBC1, we found that it operated with an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of 2:1 in pancreatic duct cells (41). This result suggested that the stoichiometry shift observed previously in the proximal tubule (67, 77, 90) may involve different NBC1 variants. However, more recent data from our laboratories using heterologous mammalian expression systems have challenged this view. We have expressed kNBC1 and pNBC1 in two different cell lines, mPCT cells derived from a murine proximal tubule and mCD cells derived from the collecting duct (Ref. 43, Table 1). Both NBC1 variants exhibited a 3:1 stoichiometry in mPCT cells and a 2:1 stoichiometry in mCD cells. These findings are significant in two ways. First, they suggest that the difference in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry in the pancreas and the kidney is not related to the difference between the NH2 termini of pNBC1 and kNBC1, respectively. Second, the data indicate that the stoichiometry of each cotransporter can be regulated by cells and is not an inherent property of the transporter.


    PHOSPHORYLATION OF NBC1 PROTEINS
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

To address the possibility that a cell can regulate the transport stoichiometry of NBC1 proteins, we have studied second messengers known to regulate epithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport that could also be potential candidates as modulators of the cotransporter's stoichiometry. cAMP is a strong modulator of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in both the kidney and the exocrine pancreas. In the renal proximal tubule, cAMP regulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by decreasing the rate of apical Na+/H+ exchange and basolateral sodium HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux (68, 74, 102). In the guinea pig interlobular pancreatic duct, Ishiguro et al. (57) found that secretin, acetylcholine, and forskolin stimulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. The effect was mediated by stimulation of the basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter and was independent of Na+/H+ exchange or vacuolar H+-ATPase activity. In mPCT cells transfected with kNBC1, the cAMP agonist 8-bromoadenosine 3',5'-cyclic monophosphate caused the stoichiometry of kNBC1 to shift from 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+ to 2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+. Using single-site mutagensis and PKA pharmacological inhibitors, we found that effect to be mediated by PKA phosphorylation of Ser982 at the COOH terminus of kNBC1 (44). pNBC1 has an equivalent COOH-terminal consensus PKA phosphorylation site at Ser1026 and an additional site in its NH2 terminus (Thr49). Work in our laboratories is presently underway to determine the role of these sites in regulation of the stoichiometry of this NBC1 variant. Recently, Muller-Berger et al. (76) have reported that elevation of intracellular Ca2+ in Xenopus laevis oocytes expressing kNBC1 caused the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry to shift from 2:1 to 3:1. In light of our finding that phosphorylation of Ser982 shifts the stoichiometry of kNBC1 from 3:1 to 2:1, it is possible that elevation of intracellular Ca2+ in the oocyte activates a protein phosphatase that dephosphorylates kNBC1-Ser982. It would be interesting in this regard to determine the phosphorylation state of NBC1 proteins in X. laevis oocytes.

How does the proximal tubule cell maintain intracellular pH homeostasis if PKA-dependent phosphorylation induces a switch in the direction of kNBC1-mediated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport from efflux to influx? One possible mechanism is a parallel decrease in luminal proton efflux via downregulation of NHE3 activity (Fig. 1). Potential mediators of the cross talk between the basolateral and luminal membrane domains include the following. 1) Changes in intracellular pH: intracellular pH is a very potent modulator of NHE3 activity in the proximal tubule due to a proton binding regulatory site on the cytoplasmic face of the protein (14). There is an exponential increase in exchanger-mediated Na+ influx with decreasing intracellular pH below 7.0 and complete inhibition at a pH above 7.2. We found an ~2-fold increase in the current through the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in renal proximal tubule cells at pH 7.25-7.50 compared with that at pH 6.5 (47). 2) Protein-protein interaction: Kurashima et al. (68) and Zhao et al. (128) studied protein-protein interaction between NHE3 and NHERF-1 in CHO cells. PKA phosphorylated Ser605 in the intracellular COOH terminus of the protein, which was associated with inhibition of the exchanger. Using two-dimensional phosphopeptide maps of NHE3 immunoprecipitated from metabolically labeled cells and back-phosphorylation assays of NHE3 from the same cells, NHERF-1 was shown to be required for NHE3 phosphorylation by PKA. In a more recent study, Weinman et al. (123) used an NHERF-1-deficient cell line to show that cAMP-mediated inhibition of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport requires the presence of NHERF-1 protein in these cells, although neither the cotransporter nor NHERF-1 were direct targets of PKA phosphorylation (123). Furthermore, the cotransporter and NHERF-1 did not associate with each other on yeast-two-hybrid or coimmunoprecipitation assays nor did they colocalize on immunocytochemistry. It was hypothesized that NHERF-1 plays an important role in cAMP-mediated inhibition of the cotransporter in these cells by mediating PKA phosphorylation of a presently unidentified third protein. Taken together, these results suggest that in the proximal tubule cAMP may serve as a second messenger to coordinate changes in acid-base flux between the basolateral and apical membrane domains.


    MECHANISM OF PHOSPHORYLATION-INDUCED STOICHIOMETRY SHIFT
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

How does phosphorylation of Ser982 at the COOH terminus of kNBC1 shift its stoichiometry from 3:1 to 2:1? To address this question, one requires a better understanding of how HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> interacts with the cotransporter. Previously, we presented a mathematical model that describes the interaction of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+ with the cotransporter (46). The model consists of 6 states connected by 12 voltage-dependent rate constants and with separate binding and release steps for Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 4). Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> are assumed to bind to the cotransporter on one side and are released on the other side in an ordered (sequential) rather than a random manner. This simplification is further supported by the finding that the current through the cotransporter as a function of either Na+ or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration can be described by a Michaelis-Menten formalism (46). To further simplify the mathematical derivation of the model, we grouped the binding and release of all three negative charge equivalents into one step. Fitting the model to a series of I-V relationships obtained at different concentrations of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> indicated that the binding of 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anions (or 1 CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> and 1 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) to the cotransporter is voltage dependent, with an electrical coefficient of 0.2 at pH 7.5. This indicates that, on average, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> "senses" ~20% of the membrane's electric field on binding to the cotransporter or that the binding site for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is located about one-fifth of the electrical distance into the membrane. This result raises the possibility that the binding of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to the cotransporter might be regulated by modifying the electric field around its binding site.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4.   Six-state ordered-binding transport model of NBC1. The rate constants for the forward (fi) and backward (bi) reactions are modulated by voltage and/or ligand concentration as described previously (46). The binding of 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Bic) anions to the transporter is described as a single, lumped step (see text). The model does not distinguish the binding of 3 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anions vs. 1 CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> and 1 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

Is it conceivable that phosphorylation of Ser982 shifts the stoichiometry from 3:1 to 2:1 by modifying the local electric field around the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site and perhaps disrupting the binding of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>? Examination of the amino acid sequence of kNBC1 reveals that the region flanking Ser982 is highly charged with an acidic cluster downstream of Ser982 consisting of four aspartic acid residues, Asp984, Asp986, Asp988 and Asp989, and a poly-lysine cluster upstream of Ser982. The presence of a highly charged segment in the vicinity of an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site might alter the electric field around it and interfere with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding to the cotransporter (Fig. 5A). Whatever the mechanism might be by which the COOH terminus "interferes" with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding, this model requires that it can only occur after phosphorylation of Ser982. This could occur if phosphorylation induces a conformational change in the COOH terminus that enables the "plug" domain to occlude an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site. A similar concept was proposed to explain removal of fast N-type inactivation of voltage-dependent potassium channels (Kv) after phosphorylation by PKC (11). In these studies, the authors used a synthetic inactivation domain to demonstrate the loss of overall structural stability, and, after phosphorylation of Ser8, Ser15 and Ser21 resulted in the dissociation of the peptide from its binding site on the channel and loss of channel inactivation. Similarly, Hayashi et al. (48) found that growth-associated protein-43 undergoes a conformational change from random coil to alpha -helix on interaction with an acidic phospholipid membrane phase. This change in conformation, which is required for the binding of calmodulin, was abolished when the protein was phosphorylated by PKC. Thus one may speculate that phosphorylation of Ser982 in the COOH terminus of kNBC1 (and possibly Ser1026 in pNBC1) renders this region more flexible and capable of competing with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> for binding to the cotransporter.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Hypothetical model illustrating a potential mechanism for the PKA-induced shift in the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ transport stoichiometry of NBC1. A: PKA-dependent phosphorylation of kNBC1-Ser982 or pNBC1-Ser1026 permits negatively charged aspartic acid residues in the COOH terminus of NBC1 proteins to interact electrostatically with one putative HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site on the transporter, resulting in a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>: Na+ transport stoichiometry of 2:1. B: when kNBC1-Ser982 or pNBC1-Ser1026 is unphosphorylated, the putative HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site in the transporter is unblocked and available to bind HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, resulting in an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ transport stoichiometry of 3:1. Also depicted in grey is a second putative protein such as carbonic anhydrase II that may interact electrostatically with negatively charged residues within the COOH terminus of unphosphorylated NBC1 proteins.

Alternatively, it is possible that phosphorylation of Ser982 disrupts the interaction of kNBC1 with a second protein. The COOH-terminal acidic aspartic acid residues could interact electrostatically with one or more putative proteins in proximal tubule cells, thereby preventing the plugging of one putative HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site. The important structural features of protein-protein recognition sites include the number of contact residues involved (10), the interface area (97), the chemical nature of the interfaces (71), residue packing (61), electrostatic interactions (86), and steric strain (101). The hypothesis that a second protein can interact electrostatically with the COOH terminus of kNBC1, thereby preventing this acidic COOH-terminal region from plugging a putative HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport site, is based in part on accumulating structural and mutational evidence that reveals a central role for electrostatic contributions to protein-protein interaction For example, in the Ras-related protein Rap1A, Asp37, Asp38, and Asp57 mediate its electrostatic interaction with the Ras binding domain of the Ras effector molecule c-Raf1, a serine/threonine kinase, the crystal structure of which was recently solved (81). Dynamic electrostatic interactions involving aspartic acid residues have been reported that can mediate important biological effects. Specifically, agonist-dependent activation of the alpha 1-adrenergic receptor is postulated to involve the disruption of an interhelical electrostatic interaction between Asp125 and Lys331 in transmembrane domains three and seven, respectively (91). In addition, the cAMP-specific phosphodiesterase (PDE4) contains unique signature regions UCR1 and UCR2 that interact electrostatically (15). Importantly, the interaction is disrupted by PKA-dependent phosphorylation of UCR1 Ser54 that results in the activation of PDE4. Phospholamban PLB is a peptide that binds to the Ca2+-ATPase in sarcoplsmic reticulum of cardiac myocytes, thereby attenuating the pump rate (60). Phosphorylation of PLB by PKA relieves this inhibition by disrupting the interaction between the two proteins. Similarly, it is possible that the interaction of acidic COOH-terminal aspartic acid residues with one or more putative proteins in proximal tubule cells containing a band of basic residues is modulated by the PKA-dependent phosphorylation of Ser982 in the kNBC1 COOH terminus.


    ELECTROSTATIC PROTEIN-PROTEIN INTERACTIONS INVOLVING BTS PROTEINS
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

AE1, a member of the BTS that mediates Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, interacts electrostatically via asparate residues in its COOH terminus with basic residues in the NH2 terminus of carbonic anhydrase II (119-121). Functional studies have demonstrated that carbonic anhydrase II stimulates the transport function of AE1, AE2, and AE3 (114). Free cytosolic carbonic anhydrase II is apparently not sufficient to support maximal anion exchanger function. The electrostatic interaction between carbonic anhydrase II and the anion exchangers is thought to minimize the distance for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> diffusion between the two proteins (63, 114). In addition, the highly acidic NH2 terminus of AE1, which has 16 aspartic acid residues, is thought to interact strongly with basic residues in the catalytic center of fructose 1,6-bisphosphate aldolase (79). The first 31 residues of AE1 contain 5 aspartic acid and 11 glutamic acid residues, whereas the catalytic region required for interaction with aldolase is strongly basic. AE1 can be specifically displaced from fructose 1,6-bisphosphate aldolase by the anionic compounds ATP and 2,3-diphosphoglycerate (79). The highly charged NH2 terminus of AE1 also interacts electrostatically with the basic residues Lys191 and Lys212 in glyceraldehyde-3-phosphate dehydrogenase (98). The binding reaction is rapidly reversible and involves the anion NH2 terminus of AE1 and the basic active site of the enzyme. In addition, the glycolytic enzyme phosphofructokinase interacts electrostatically with the NH2 terminus of AE1 (50). Sequence alignment of kNBC1 with AE1 reveals a homologous acidic cluster at the COOH terminus of human AE1 (D887ADD). This acidic cluster is critical for binding of AE1 to carbonic anhydrase II (119). Furthermore, Sterling et al. (114) found that inhibition of carbonic anhydrase II with acetazolamide in HEK-293 cells transfected with AE1 inhibited the anion exchanger by 50-60%. Carbonic anhydrase II is expressed in the cytoplasm of proximal tubule cells and intercalated cells, and the loss of function mutations in the enzyme leads to proximal and distal renal tubular acidosis and depletion of collecting duct intercalated cells (21, 70, 80). It is possible that carbonic anhydrase II binds to the COOH terminus of kNBC1 in proximal tubule cells, creating a local increase in the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration in the vicinity of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding site of kNBC1. A local increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration might further stimulate the flux of the transporter when it operates in the 3:1 stoichiometry mode (Fig. 5B). It is yet to be determined whether carbonic anhydrase II binds to kNBC1 and whether such a functional interaction exists between the two proteins. We have recently shown that an acidic cluster in the COOH terminus of kNBC1 (D986NDD) is required for the PKA-dependent stoichiometry shift (42). Whether PKA-dependent phosphorylation of kNBC1-Ser982 disrupts the putative interaction between kNBC1 and carbonic anhydrase II remains to be determined. Carbonic anhydrase II is also highly expressed in pancreatic ducts (9, 36, 84). A similar model whereby carbonic anhydrase II interacts with pNBC1 may play an important role in modulating the rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in the pancreas.


    CHEMICAL PROBES FOR HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> BINDING SITES
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

Stilbene derivatives block ion transport mediated by several members of the BTS proteins (1, 6, 22, 40, 65, 99, 100, 103, 104, 108, 116, 117, 122), suggesting that these compounds might be useful in probing specific HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> binding sites in these proteins. NBC1 has two putative DIDS binding motifs: KMIK, at a site homologous to the KLXK (X = I, Y) in the AE proteins, and a KLKK motif at a more COOH-terminal site. NBC3, an electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (69, 93, 94), is missing the first motif but does contain the second putative DIDS binding motif (944-KLKK). NBC4, a new member of the BTS, is also missing the first of the two putative DIDS binding motifs but retains the second motif (863-KLKK). NBC4, cloned by Pushkin et al. (95, 96, 104) is presently represented by four variants, NBC4a-NBC4d. When expressed in mPCT cells, NBC4c was found to be electrogenic, with a stoichiometry of 3:1 (Table 1 and Ref. 104). These latter findings suggests that there is probably no direct correlation between the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry and the number of putative DIDS binding motifs. This conclusion is further augmented by the finding that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and DIDS do not compete on the same binding site in AE1 (103). Recently, the cyclooxygenase inhibitor tenidap has been shown to be a potent inhibitor of kNBC1 expressed in X. laevis oocytes (32). The inhibitory potency of kNBC1 by tenidap is similar to its inhibition of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (75). Whether tenidap can be utilized to probe the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or CO<UP><SUB>3</SUB><SUP>2−</SUP></UP> binding sites in NBC1 or other members of the BTS is yet to be determined.


    SUMMARY AND FUTURE DIRECTIONS
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

Significant progress has been made in characterizing the molecular mechanisms involved in regulating the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ transport stoichiometry of NBC1. The finding that phosphorylation of kNBC1 by PKA shifts its HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>: Na+ stoichiometry from 3:1 to 2:1 has important implications for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the renal proximal tubule where kNBC1 normally mediates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption by operating with a 3:1 stoichiometry. The previous finding that PKA also modulates the activity of NHE3 in these cells suggests that this second messenger may also mediate cross talk between the basolateral and apical membrane domains in proximal tubule cells. In the apical membrane, the interaction between PKA and NHE3 is mediated by NHERF. Although presently no NHERF isoforms have been localized to the basolateral membrane of these cells, it is still possible that the interaction between PKA and kNBC1 is also mediated by a third protein. PKA has been shown to be compartmentalized in many cell types via A kinase anchoring proteins (28). It would thus be of interest to determine whether A kinase anchoring proteins mediate PKA-kNBC1 or PKA-pNBC1 interaction. Additionally, kNBC1 and pNBC1 have several PKC consensus phosphorylation sites whose role in modulating their transport stoichiometry is presently being studied. The structural basis for the unique coupling between the transport of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters is still largely unknown. Identification of the amino acids that participate in the coordination of these ion ligands, and the distances between them in three-dimensional space, could greatly assist in our understanding of this question. Several photo- and chemically labile probes have been previously used to identify amino acids that participate in coordination of Na+ in other Na+ transport proteins such as NHE, ENaC, and the Na+-K+-ATPase (33, 58, 64). A similar approach may prove useful in identifying coordinating amino acids in NBC1 proteins. Recent advances in crystallography of membrane proteins (85) may also be applicable for structural studies of electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters. Finally, NMR and X-ray crystallography may also prove useful in determining structural/conformational changes in the COOH terminus of kNBC1 and pNBC1 after phosphorylation of Ser982 and Ser1026, respectively.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46976, DK-58563, and DK-07789 (to I. Kurtz). E. Gross is supported by Cystic Fibrosis Foundation Grant Gross01G0 and the American Heart Association Grant 9706507.


    FOOTNOTES

Address for reprint requests and other correspondence: I. Kurtz, Rm. 7-155 Factor Bldg., 10833 Le Conte Ave., Los Angeles, CA 90095 (E-mail: Ikurtz{at}mednet.ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00148.2002


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
PROXIMAL TUBULE AND PANCREATIC...
ELECTROGENIC NBC PROTEINS AND...
THERMODYNAMICS OF ELECTROGENIC...
REGULATION OF NBC1 HCO3-:NA+...
PHOSPHORYLATION OF NBC1...
MECHANISM OF PHOSPHORYLATION-...
ELECTROSTATIC PROTEIN-PROTEIN...
CHEMICAL PROBES FOR HCO3-/CO32-...
SUMMARY AND FUTURE DIRECTIONS
REFERENCES

1.   Abuladze, N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A, and Kurtz I. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem 273: 17689-17695, 1998[Abstract/Free Full Text].

2.   Abuladze, N, Lee I, Newman D, Hwang J, Pushkin A, and Kurtz I. Axial heterogeneity of sodium-bicarbonate cotransporter expression in the rabbit proximal tubule. Am J Physiol Renal Physiol 274: F628-F633, 1998[Abstract/Free Full Text].

3.   Abuladze, N, M, Song Pushkin A, Newman D, Lee I, Nicholas S, and Kurtz I. Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3. Gene 251: 109-122, 2000[ISI][Medline].

4.   Aiello, EA, Petroff MG, Mattiazzi AR, and Cingolani HE. Evidence for an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> symport in rat cardiac myocytes. J Physiol 512: 137-148, 1998[Abstract/Free Full Text].

5.   Akiba, Y, Furukawa O, Guth PH, Engel E, Nastaskin I, Sassani P, Dukkipatis R, Pushkin A, Kurtz I, and Kaunitz JD. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury. J Clin Invest 108: 1807-1816, 2001[Abstract/Free Full Text].

6.   Alper, SL. The band 3-related anion exchanger (AE) gene family. Annu Rev Physiol 53: 549-564, 1991[ISI][Medline].

7.   Alpern, RJ. Mechanism of basolateral membrane H+/OH-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process. J Gen Physiol 86: 613-636, 1985[Abstract].

8.   Alpern, R, and Rector RC, Jr. Renal acidification mechanisms. In: The Kidney, edited by Brenner BM, and Rector FC, Jr.. Philadelphia, PA: Saunders, 1996, p. 408-471.

9.   Alvarez, L, Fanjul M, Carter N, and Hollande E. Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1). J Histochem Cytochem 49: 1045-1053, 2001[Abstract/Free Full Text].

10.   Amit, AG, Mariuzza RA, Phillips SE, and Poljak RJ. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science 233: 747-753, 1986[ISI][Medline].

11.   Antz, C, Bauer T, Kalbacher H, Frank R, Covarrubias M, Kalbitzer HR, Ruppersberg JP, Baukrowitz T, and Fakler B. Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate. Nat Struct Biol 6: 146-150, 1999[ISI][Medline].

12.   Argent, BE, and Case RM. Pancreatic ducts. Cellular mechanism and control of bicarbonate secretion. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1994, p. 1473-1479.

13.   Argent, BE, and Gray MA. Regulation and formation of fluid and electrolyte secretion by pancreatic ductal epithelium. In: Biliary and Pancreatic Ductal Epithelia: Pathology and Pathophysiology, edited by Sirica AE, and Longnecker DS.. New York: Dekker, 1997, p. 349-377.

14.   Aronson, PS, Nee J, and Suhm MA. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature 299: 161-163, 1982[ISI][Medline].

15.   Beard, MB, Olsen AE, Jones RE, Erdogan S, Houslay MD, and Bolger GB. UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions. J Biol Chem 275: 10349-10358, 2000[Abstract/Free Full Text].

16.   Bevensee, MO, Apkon M, and Boron WF. Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. J Gen Physiol 110: 467-483, 1997[Abstract/Free Full Text].

17.   Bevensee, MO, Schmitt BM, Choi I, Romero MF, and Boron WF. An electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain. Am J Physiol Cell Physiol 278: C1200-C1211, 2000[Abstract/Free Full Text].

18.   Bok, D, Schibler MJ, Pushkin A, Sassani P, Abuladze N, Naser Z, and Kurtz I. Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye. Am J Physiol Renal Physiol 281: F920-F935, 2001[Abstract/Free Full Text].

19.   Bonanno, JA, and Giasson C. Intracellular pH regulation in fresh and cultured bovine corneal endothelium. II. Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. Invest Ophthalmol Vis Sci 33: 3068-3079, 1992[Abstract].

20.   Boron, WF, and Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO3 transport. J Gen Physiol 81: 53-94, 1983[Abstract].

21.   Breton, S, Alper SL, Gluck SL, Sly WS, Barker JE, and Brown D. Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice. Am J Physiol Renal Fluid Electrolyte Physiol 269: F761-F774, 1995[Abstract/Free Full Text].

22.   Burnham, C, Amlal H, Wang Z, Shull GE, and Soleimani M. Cloning and functional expression of a human kidney Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. J Biol Chem 72: 19111-19114, 1997.

23.   Camilion de Hurtado, MC, Perez NG, and Cingolani HE. An electrogenic sodium-bicarbonate cotransport in the regulation of myocardial intracellular pH. J Mol Cell Cardiol 27: 231-242, 1995[ISI][Medline].

24.   Casey, JR, and Reithmeier RA. Anion exchangers in the red cell and beyond. Biochem Cell Biol 76: 709-713, 1998[ISI][Medline].

25.  Choi I, Aalkjaer C, Boulpaep EL, and Boron WF. An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel. Nature 405: 571-575.

26.   Choi, I, Romero MF, Khandoudi N, Bril A, and Boron WF. Cloning and characterization of a human electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter isoform (hhNBC). Am J Physiol Cell Physiol 276: C576-C584, 1999[Abstract/Free Full Text].

27.   Choi, JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, and Muallem S. Aberrant CFTR-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in mutations associated with cystic fibrosis. Nature 410: 94-97, 2001[ISI][Medline].

28.   Coghlan, VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, and Scott JD. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267: 108-111, 1995[ISI][Medline].

29.   Deitmer, JW, and Schlue WR. An inwardly directed electrogenic sodium-bicarbonate co-transport in leech glial cells. J Physiol 411: 179-194, 1989[Abstract].

30.   De Ondarza, J, and Hootman SR. Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts. Am J Physiol Gastrointest Liver Physiol 272: G124-G134, 1997[Abstract/Free Full Text].

31.   Devor, DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 743-760, 1999[Abstract/Free Full Text].

32.   Ducoudret, O, Diakov A, Muller-Berger S, Romero MF, and Fromter E. The renal Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>cotransporter expressed in Xenopus laevis oocytes: inhibition by tenidap and benzamil and effect of temperature on transport rate and stoichiometry. Pflügers Arch 442: 709-717, 2001[ISI][Medline].

33.   Ellis-Davies, GC, Kleyman TR, and Kaplan JH. Photolabile amiloride derivatives as cation site probes of the Na,K-ATPase. J Biol Chem 271: 10353-10358, 1996[Abstract/Free Full Text].

34.   Fitz, JG, Persico M, and Scharschmidt BF. Electrophysiological evidence for Na+-coupled bicarbonate transport in cultured rat hepatocytes. Am J Physiol Gastrointest Liver Physiol 256: G491-G500, 1989[Abstract/Free Full Text].

35.   Freedman, SD, and Scheele GA. Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function. Ann N Y Acad Sci 713: 199-206, 1994[Abstract].

36.   Fujikawa-Adachi, K, Nishimori I, Sakamoto S, Morita M, Onishi S, Yonezawa S, and Hollingsworth MA. Identification of carbonic anhydrase IV and VI mRNA expression in human pancreas and salivary glands. Pancreas 18: 329-335, 1999[ISI][Medline].

37.   Gleeson, D, Smith ND, and Boyer JL. Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. J Clin Invest 84: 312-321, 1989[ISI][Medline].

38.   Gray, MA, Greenwell JR, and Argent BE. Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells. J Membr Biol 105: 131-142, 1988[ISI][Medline].

39.   Greeley, T, Shumaker H, Wang Z, Schweinfest CW, and Soleimani M. Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells. Am J Physiol Gastrointest Liver Physiol 281: G1301-G1308, 2001[Abstract/Free Full Text].

40.   Grichtchenko, II, Choi I, Zhong X, Bray-Ward P, Russell JM, and Boron WF. Cloning, characterization, and chromosomal mapping of a human electroneutral Na+-driven Cl-HCO3 exchanger. J Biol Chem 276: 8358-8363, 2001[Abstract/Free Full Text].

41.   Gross, E, Abuladze N, Pushkin A, Kurtz I, and Cotton CU. The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:1 Na+. J Physiol 531: 375-382, 2001[Abstract/Free Full Text].

42.   Gross, E, Fedotoff O, Pushkin A, Abuladze N, and Kurtz I. An aspartate cluster in the C-terminus of kNBC1 modulates the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry (Abstract). FASEB J 16: A795, 2002.

43.   Gross, E, Hawkins K, Abuladze N, Pushkin A, Hopfer U, and Kurtz I. The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent. J Physiol 531: 597-603, 2001[Abstract/Free Full Text].

44.   Gross, E, Hawkins K, Pushkin A, Abuladze N, Hopfer U, Sassani P, Dukkipati R, and Kurtz I. Phosphorylation of Ser982 in kNBC1 shifts the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry from 3:1 to 2:1 in proximal tubule cells. J Physiol 537: 659-665, 2001[Abstract/Free Full Text].

45.   Gross, E, and Hopfer U. Activity and stoichiometry of Na:HCO3 cotransport in immortalized renal proximal tubule cells. J Membr Biol 152: 245-252, 1996[ISI][Medline].

46.   Gross, E, and Hopfer U. Voltage and co-substrate dependence of the Na-HCO3 cotransportrer kinetics in renal proximal tubule cells. Biophys J 75: 810-824, 1998[Abstract/Free Full Text].

47.   Gross, E, and Hopfer U. Effects of pH on kinetic parameters of the Na-HCO3 cotransporter in renal proximal tubule. Biophys J 76: 3066-3075, 1999[Abstract/Free Full Text].

48.   Hayashi, N, Matsubara M, Titani K, and Taniguchi H. Circular dichroism and 1H nuclear magnetic resonance studies on the solution and membrane structures of GAP-43 calmodulin-binding domain. J Biol Chem 272: 7639-7645, 1997[Abstract/Free Full Text].

49.   Heyer, M, Muller-Berger S, Romero MF, Boron WF, and Fromter E. Stoichiometry of the rat kidney Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter expressed in Xenopus laevis oocytes. Pflügers Arch 438: 322-329, 1999[ISI][Medline].

50.   Higashi, T, Richards CS, and Uyeda K. The interaction of phosphofructokinase with erythrocyte membranes. J Biol Chem 254: 9542-9550, 1979[ISI][Medline].

51.   Hootman, SR, and de Ondarza J. Overview of pancreatic duct physiology and pathophysiology. Digestion 54: 323-330, 1993[ISI][Medline].

52.   Hughes, BA, Adorante JS, Miller SS, and Lin H. Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium. J Gen Physiol 94: 125-150, 1989[Abstract].

53.   Isenberg, JI, Ljungstrom M, Safsten B, and Flemstrom G. Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl--HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and NaHCO3 cotransport. Am J Physiol Gastrointest Liver Physiol 265: G677-G685, 1993[Abstract/Free Full Text].

54.   Ishibashi, K, Sasaki S, and Marumo F. Molecular cloning of a new sodium bicarbonate cotransporter cDNA from human retina. Biochem Biophys Res Commun 246: 535-538, 1998[ISI][Medline].

55.   Ishiguro, H, Naruse S, Kitagawa M, Mabuchi T, Kondo T, Hayakawa T, Case RM, and Steward MC. Chloride transport in microperfused interlobular ducts isolated from guinea-pig pancreas. J Physiol 539: 175-189, 2002[Free Full Text].

56.   Ishiguro, H, Steward MC, Lindsay AR, and Case RM. Accumulation of intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in interlobular ducts from guinea-pig pancreas. J Physiol 495: 169-178, 1996[Abstract].

57.   Ishiguro, H, Steward MC, Wilson RW, and Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol 495: 179-191, 1996[Abstract].

58.   Ismailov, II, Kieber-Emmons T, Lin C, Berdiev BK, Shlyonsky VG, Patton HK, Fuller CM, Worrell R, Zuckerman JB, Sun W, Eaton DC, Benos DJ, and Kleyman TR. Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel. J Biol Chem 272: 21075-21083, 1997[Abstract/Free Full Text].

59.   Jacob, P, Christiani S, Rossmann H, Lamprecht G, Vieillard-Baron D, Muller R, Gregor M, and Seidler U. Role of Na+HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion. Gastroenterology 119: 406-419, 2000[ISI][Medline].

60.   James, P, Inui M, Tada M, Chiesi M, and Carafoli E. Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. Nature 342: 90-92, 1989[ISI][Medline].

61.   Janin, J, and Chothia C. The structure of protein-protein recognition sites. J Biol Chem 265: 16027-16030, 1990[Free Full Text].

62.   Jensen, LJ, Schmitt BM, Berger UV, Nsumu NN, Boron WF, Hediger MA, Brown D, and Breton S. Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis. Biol Reprod 60: 573-579, 1999[Abstract/Free Full Text].

63.   Kifor, G, Toon MR, Janoshazi A, and Solomon AK. Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase. J Membr Biol 134: 169-179, 1993[ISI][Medline].

64.   Kleyman, TR, and Cragoe EJ. Amiloride and its analogs as tools in the study of ion transport. J Membr Biol 105: 1-21, 1988[ISI][Medline].

65.   Kopito, RR, Lee BS, Simmons DM, Lindsey AE, Morgans CW, and Schneider K. Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger. Cell 59: 927-937, 1989[ISI][Medline].

66.   Kuijpers, GA, Van Nooy IG, De Pont JJ, and Bonting SL. The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors. Biochim Biophys Acta 778: 324-331, 1984[ISI][Medline].

67.   Kunimi, M, Seki G, Hara C, Taniguchi S, Uwatoko S, Goto A, Kimura S, and Fujita T. Dopamine inhibits renal Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats. Kidney Int 57: 534-543, 2000[ISI][Medline].

68.   Kurashima, K, Yu FH, Cabado AG, Szabo EZ, Grinstein S, and Orlowski J. Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms. J Biol Chem 272: 28672-28679, 1997[Abstract/Free Full Text].

69.   Kwon, TH, Pushkin A, Abuladze N, Nielsen S, and Kurtz I. Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney. Am J Physiol Renal Physiol 278: F327-F336, 2000[Abstract/Free Full Text].

70.   Lai, LW, Chan DM, Erickson RP, Hsu SJ, and Lien YH. Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy. J Clin Invest 101: 1320-1325, 1998[Abstract/Free Full Text].

71.   Lee, B, and Richards FM. The interpretation of protein structures: estimation of static accessibility. J Mol Biol 55: 379-400, 1971[ISI][Medline].

72.   Luo, X, Choi JY, Ko SB, Pushkin A, Kurtz I, Ahn W, Lee MG, and Muallem S. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> salvage mechanisms in the submandibular gland acinar and duct cells. J Biol Chem 276: 9808-9816, 2001[Abstract/Free Full Text].

73.   Marino, CR, Jeanes V, Boron WF, and Schmitt BM. Expression and distribution of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in human pancreas. Am J Physiol Gastrointest Liver Physiol 277: G487-G494, 1999[Abstract/Free Full Text].

74.   McKinney, TD, and Myers P. Bicarbonate transport by proximal tubules: effect of parathyroid hormone and dibutyryl cyclic AMP. Am J Physiol Renal Fluid Electrolyte Physiol 238: F166-F174, 1980[Free Full Text].

75.   McNiff, P, Robinson RP, and Gabel CA. Reduction of intracellular pH by tenidap. Involvement of cellular anion transporters in the pH change. Biochem Pharmacol 50: 1421-1432, 1995[ISI][Medline].

76.   Muller-Berger, S, Ducoudret O, Diakov A, and Fromter E. The renal Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter expressed in Xenopus laevis oocytes: change in stoichiometry in response to elevation of cytosolic Ca2+ concentration. Pflügers Arch 442: 718-728, 2001[ISI][Medline].

77.   Muller-Berger, S, Nesterov VV, and Fromter E. Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide. Pflügers Arch 434: 383-391, 1997[ISI][Medline].

78.   Munsch, T, and Deitmer JW. Sodium-bicarbonate cotransport current in identified leech glial cells. J Physiol 474: 43-53, 1994[Abstract].

79.   Murthy, SN, Liu T, Kaul RK, Kohler H, and Steck TL. The aldolase-binding site of the human erythrocyte membrane is at the NH2 terminus of band 3. J Biol Chem 256: 11203-11208, 1981[Abstract/Free Full Text].

80.   Nagai, R, Kooh SW, Balfe JW, Fenton T, and Halperin ML. Renal tubular acidosis and osteopetrosis with carbonic anhydrase II deficiency: pathogenesis of impaired acidification. Ped Nephrol 11: 633-636, 1997[ISI][Medline].

81.   Nassar, N, Horn G, Herrmann C, Scherer A, McCormick F, and Wittinghofer A. The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue. Nature 375: 554-560, 1995[ISI][Medline].

82.   Newman, EA. Sodium-bicarbonate cotransport in retinal Muller (glial) cells of the salamander. J Neurosci 11: 3972-3983, 1991[Abstract].

83.   Newman, EA, and Astion ML. Localization and stoichiometry of electrogenic sodium bicarbonate cotransport in retinal glial cells. Glia 4: 424-428, 1991[ISI][Medline].

84.   Nishimori, I, FujikawaAdachi K, Onishi S, and Hollingsworth MA. Carbonic anhydrase in human pancreas: hypotheses for the pathophysiological roles of CA isozymes. Ann NY Acad Sci 880: 5-16, 1999[Abstract/Free Full Text].

85.   Nollert, P, Navarro J, and Landau EM. Crystallization of membrane proteins in cubo. Methods Enzymol 343: 183-199, 2002[ISI][Medline].

86.   Norel, R, Sheinerman F, Petrey D, and Honig B. Electrostatic contributions to protein-protein interactions: fast energetic filters for docking and their physical basis. Protein Sci 10: 2147-2161, 2001[Abstract/Free Full Text].

87.   Novak, I, and Greger R. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport. Pflügers Arch 411: 546-553, 1988[ISI][Medline].

88.   Parker, MD, Boron WF, and Tanner MJ. Characterization of human "AE4" as an electroneutral, sodium-dependent bicarbonate transporter (Abstract). FASEB J 16: A796, 2002.

89.   Parker, MD, Ourmozdi EP, and Tanner MJ. Human BTR1, a new bicarbonate transporter superfamily member and human AE4 from kidney. Biochem Biophys Res Commun 282: 1103-1109, 2001[ISI][Medline].

90.   Planelles, G, Thomas SR, and Anagnostopoulos T. Change of apparent stoichiometry of proximal-tubule Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport upon experimental reversal of its orientation. Proc Natl Acad Sci USA 90: 7406-7410, 1993[Abstract].

91.   Porter, JE, and Perez DM. Characteristics for a salt-bridge switch mutation of the alpha 1b adrenergic receptor. Altered pharmacology and rescue of constitutive activity. J Biol Chem 274: 34535-34538, 1999[Abstract/Free Full Text].

92.   Praetorius, J, Hager H, Nielsen S, Aalkjaer C, Friis UG, Ainsworth MA, and Johansen T. Molecular and functional evidence for electrogenic and electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters in murine duodenum. Am J Physiol Gastrointest Liver Physiol 280: G332-G343, 2001[Abstract/Free Full Text].

93.   Pushkin, A, Abuladze N, Lee I, Newman D, Hwang J, and Kurtz I. Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family. J Biol Chem 274: 16569-16575, 1999[Abstract/Free Full Text].

94.   Pushkin, A, Abuladze N, Lee I, Newman D, Hwang J, and Kurtz I. Mapping of the human NBC3 (SLC4A7) gene to chromosome 3p22. Genomics 57: 321-322, 1999[ISI][Medline].

95.   Pushkin, A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family. Biochim Biophys Acta 1493: 215-218, 2000[ISI][Medline].

96.   Pushkin, A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Two C-terminal variants of NBC4, a new member of the sodium bicarbonate cotransporter family: cloning, characterization, and localization. Int Un Biochem Mol Biol 50: 13-19, 2000.

97.   Rees, DC, and Lipscomb WN. Refined crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 A resolution. J Mol Biol 160: 475-498, 1982[ISI][Medline].

98.   Rogalski, AA, Steck TL, and Waseem A. Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell. J Biol Chem 264: 6438-6446, 1989[Abstract/Free Full Text].

99.   Romero, MF, Hediger MA, Boulpaep EL, and Boron WF. Expression cloning and characterization of a renal electrogenic Na/HCO3 cotransporter. Nature 387: 409-413, 1997[ISI][Medline].

100.   Romero, MF, Henry D, Nelson S, Harte PJ, Dillon AK, and Sciortino CM. Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter. J Biol Chem 275: 24552-24559, 2000[Abstract/Free Full Text].

101.   Ruhlmann, A, Kukla D, Schwager P, Bartels K, and Huber R. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region. J Mol Biol 77: 417-436, 1973[ISI][Medline].

102.   Ruiz, OS, and Arruda JAL Regulation of the renal Na-HCO3 cotransporter by cAMP and Ca-dependent protein kinases. Am J Physiol Renal Fluid Electrolyte Physiol 262: F560-F565, 1992[Abstract/Free Full Text].

103.   Salhany, JM. Allosteric effects in stilbenedisulfonate binding to band 3 protein (AE1). Cell Mol Biol 42: 1065-1096, 1996[ISI].

104.   Sassani, P, Pushkin A, Gross E, Gomer A, Abuladze N, Dukkipati R, Carpenito G, and Kurtz I. Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter. Am J Physiol Cell Physiol 282: C408-C416, 2002[Abstract/Free Full Text].

105.   Schmitt, BM, Biemesderfer D, Romero MF, Boulpaep EL, and Boron WF. Immunolocalization of the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in mammalian and amphibian kidney. Am J Physiol Renal Physiol 276: F27-F38, 1999[Abstract/Free Full Text].

106.   Sciortino, CM, and Romero MF. Cation and voltage dependence of rat kidney electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter, rkNBC, expressed in oocytes. Am J Physiol Renal Physiol 277: F611-F623, 1999[Abstract/Free Full Text].

107.  Seki G, Coppola S, and Fromter E. 1993. The Na-HCO3 cotransporer operates with a coupling ratio of 2 HCO3 to 1 Na in isolated rabbit renal proximal tubule. Pflügers Arch 425: 409-416, 1993.

108.   Sekler, I, Lo RS, Mastrocola T, and Kopito RR. Sulfate transport mediated by the mammalian anion exchangers in reconstituted proteoliposomes. J Biol Chem 270: 11251-11256, 1995[Abstract/Free Full Text].

109.   Sewell, WA, and Young JA. Secretion of electrolytes by the pancreas of the anaesthetized rat. J Physiol 252: 379-396, 1975[Abstract].

110.   Shumaker, H, Amlal H, Frizzell R, Ulrich CD., 2nd, and Soleimani M. CFTR drives Na+- nHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in pancreatic duct cells: a basis for defective HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in CF. Am J Physiol Cell Physiol 276: C16-C25, 1999[Abstract/Free Full Text].

111.   Smith, JJ, and Welsh MJ. cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J Clin Invest 89: 1148-1153, 1992[ISI][Medline].

112.   Soleimani, M, and Aronson PS. Ionic mechanism of Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in renal basolateral membrane vesicles. J Biol Chem 264: 18302-18308, 1989[Abstract/Free Full Text].

113.   Soleimani, M, Grassl SM, and Aronson PS. Stoichiometry of Na-HCO3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J Clin Invest 79: 1276-1280, 1987[ISI][Medline].

114.   Sterling, D, Reithmeier RA, and Casey JR. A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers. J Biol Chem 276: 47886-47894, 2001[Abstract/Free Full Text].

115.   Sun, XC, Bonanno JA, Jelamskii S, and Xie Q. Expression and localization of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter in bovine corneal endothelium. Am J Physiol Cell Physiol 279: C1648-C1655, 2000[Abstract/Free Full Text].

116.   Tsuganezawa, H, Kobayashi K, Iyori M, Araki T, Koizumi A, Watanabe S, Kaneko A, Fukao T, Monkawa T, Yoshida T, Kim DK, Kanai Y, Endou H, Hayashi M, and Saruta T. A new member of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney. J Biol Chem 276: 8180-8189, 2001[Abstract/Free Full Text].

117.   Van't Hof, W, Malik A, Vijayakumar S, Qiao J, van Adelsberg J, and Al-Awqati Q. The effect of apical and basolateral lipids on the function of the band 3 anion exchange protein. J Cell Biol 139: 941-949, 1997[Abstract/Free Full Text].

118.   Villanger, O, Veel T, and Raeder MG. Secretin causes H+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion from pig pancreatic ductules by vacuolar-type H+-adenosine triphosphatase. Gastroenterology 108: 850-859, 1995[ISI][Medline].

119.   Vince, JW, Carlsson U, and Reithmeier RA. Localization of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anion exchanger binding site to the amino-terminal region of carbonic anhydrase II. Biochemistry. 39: 13344-13349, 2000[ISI][Medline].

120.   Vince, JW, and Reithmeier RA. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. J Biol Chem 273: 28430-284377, 1998[Abstract/Free Full Text].

121.   Vince, JW, and Reithmeier RA. Identification of the carbonic anhydrase II binding site in the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anion exchanger AE1. Biochemistry 39: 5527-5533, 2000[ISI][Medline].

122.   Wang, CZ, Yano H, Nagashima K, and Seino S. The Na+-driven Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. Cloning, tissue distribution, and functional characterization. J Biol Chem 275: 35486-35490, 2000[Abstract/Free Full Text].

123.   Weinman, EJ, Evangelista CM, Steplock D, Liu MZ, Shenolikar S, and Bernardo A. Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> co-transporter in BSC-1 cells. J Biol Chem 276: 42339-42346, 2001[Abstract/Free Full Text].

124.   Wolosin, JM, Alvarez LJ, and Candia OA. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the toad lens epithelium is mediated by an electronegative Na+-dependent symport. Am J Physiol Cell Physiol 258: C855-C861, 1990[Abstract/Free Full Text].

125.   Yoshitomi, K, Burckhardt BC, and Fromter E. Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule. Pflügers Arch 405: 360-366, 1985[ISI][Medline].

126.   Young, JA, Cook DI, ew van Lennep, and Roberts M. Secretion by the major salivary glands. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, vol. 1, p. 773-815.

127.   Zhao, H, Star RA, and Muallem S. Membrane localization of H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters in the rat pancreatic duct. J Gen Physiol 104: 57-85, 1994[Abstract].

128.   Zhao, H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, and Moe OW. Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase A and NHE-3 phosphoserines 552 and 605. J Biol Chem 274: 3978-3987, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(5):F876-F887