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/CO₂) system is the most important buffer system in the extracellular fluid space (8). CO₂ and HCO are in equilibrium according to the following relationship

(1)

The uniqueness of this reaction is derived from the fact that the components of the HCO 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 concentration by absorbing the filtered HCO load and maintaining whole body proton balance. The pK_a of the H₂CO₃  HCO + H⁺ reaction is 3.57, and it would appear that HCO would not be an effective buffer. However, because dissolved CO₂ is in equilibrium with H₂CO₃, Eq. 1 can be simplified to CO₂ + H₂O  HCO + 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  H⁺ + CO can occur; however, the pK_a of this reaction is 10.1; therefore, the CO concentration is ~1:500 the HCO concentration at a pH of 7.4.

An essential role of the kidney in maintaining the blood HCO concentration at ~25 mM is to reclaim HCO filtered through the glomeruli. The renal proximal tubule is the site in the nephron where the greatest quantity of HCO is reabsorbed into the peritubular blood. Proximal tubule transepithelial HCO 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 absorption is mediated by electrogenic Na⁺-HCO cotransport (7, 20, 45, 113, 125).

Unlike the proximal tubule, which absorbs HCO from the glomerular filtrate, the direction of transepithelial HCO 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 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, which drives sodium and water into the lumen by electroosmotic coupling (30, 56, 127). Pancreatic and duodenal HCO 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 cotransport process (5, 53, 59, 92) that mediates HCO influx and protects the epithelium from gastric acid-induced injury (5). In addition, by elevating pancreatic ductal pH, HCO 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 transport is less well understood. In pancreatic ducts, a basolateral Na⁺-HCO cotransport process plays a major role in mediating basolateral HCO influx, with subsequent HCO secretion across the apical membrane (2, 41, 56, 57, 118, 127). This basolateral Na⁺-HCO cotransport process was also found to be electrogenic (41). The apical transport mechanism(s) responsible for apical HCO secretions have not been completely delineated. The traditional model for apical HCO 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/Cl exchange activity in some species, it does not explain the independence of HCO 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 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 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 secretion in the rat pancreatic duct (38, 87) and in airway submucosal gland cells (31). An important role for CFTR in HCO secretion is also implicated by the finding of impaired HCO secretion in the pancreas of patients with cystic fibrosis (27). Shumaker et al. (110) have suggested that PKA stimulates basolateral HCO influx in pancreatic duct cells through the basolateral electrogenic Na⁺-HCO 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 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 cotransporter plays in HCO transport in the renal proximal tubule and the pancreatic duct. These models represent the presently accepted cellular mechanisms for transepithelial HCO transport in these two epithelia. In the renal proximal convoluted tubule (PCT), the cotransporter supports transepithelial HCO absorption by mediating basolateral efflux of Na⁺ and HCO, whereas in the pancreas the cotransporter supports transepithelial HCO 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 across the membrane against their respective concentration gradients? Second, does the same electrogenic Na⁺-HCO cotransporter contribute to transepithelial Na⁺-HCO cotransport in both epithelia? Third, what is the mechanism(s) for the difference in Na⁺:HCO 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 transport. In the renal proximal tubule (A), kNBC1 mediates Na+ and HCO efflux by coupling the transport of 3 HCO and 1 Na+ to the membrane potential. In the exocrine pancreas (B), pNBC1 mediates Na+ and HCO influx by coupling the transport of 2 HCO to the downhill flux of Na+.

	ELECTROGENIC NBC PROTEINS AND MEMBERS OF THE HCO 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 transporter superfamily (BTS), which includes the Cl/HCO exchanger proteins AE1-AE3 (6, 24); the Na⁺-driven Cl/HCO exchangers (40, 100, 124); the electroneutral Na⁺-HCO cotransporter NBC3 (93, 94) [splice variant NBC2 (54); rat orthologue NBCn1(25)]; and the second known electrogenic Na⁺-HCO cotransporter isoform NBC4 (95, 96, 104). AE4, which was initially reported to be a Cl/HCO exchanger (118), may function as an electroneutral Na⁺-HCO 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: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 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 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 TRANSPORT

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Electrogenic Na⁺-HCO 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 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 found in the proximal tubule, the cotransporter should carry a net charge of 2 equivalents, e.g., 3 HCO + 1 Na⁺ or 1 CO + 1 HCO + 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:1 Na⁺ stoichiometry was reported by us and by other groups for basolateral Na⁺-HCO 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 cotransporter. The plot was generated using Eq. 5 with a 10-fold Na+ concentration gradient (high outside) and no HCO gradient. The plot illustrates that a basolateral cotransporter with a 2 HCO:1 Na+ stoichiometry such as pNBC1 in the pancreatic ducts will mediate the basolateral influx of HCO. A transporter with a 3:1 stoichiometry such as kNBC1 in the renal proximal tubule will mediate basolateral HCO efflux.

View this table: [in this window] [in a new window]   Table 1.   Electrogenic Na+-HCO cotransporter stoichiometry in various cells and expression systems

Several methods have been described in the literature for measurements of HCO: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 across a membrane can be described by

(2)

where the subscripts i and o stand for intracellular and extracellular compartments, respectively. The transport stoichiometry of the Na⁺-HCO cotransporter can be determined by finding Na⁺ and/or HCO 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

(3)

where µNa^io is the in-to-out electrochemical potential difference for Na⁺, nµHCO₃^oi is the out-to-in electrochemical potential difference for HCO, and n is the number of HCO anions cotransported with each Na⁺ cation. Expressing the electrochemical potential differences in terms of the relevant ion concentrations and the membrane potential, V_m, yields

(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. V_m at which no flux (current) occurs is called reversal potential (E_rev), and can be experimentally determined from the current-voltage (I-V) relationship. Once determined, E_rev can be used to evaluate the actual HCO-to-Na⁺ transport ratio, n, according to

(5)

Equation 5 suggests that E_rev depends logarithmically on the intra-to-extracellular ratio of Na⁺ and HCO 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 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 and Na⁺ fluxes, by measuring intracellular pH and Na⁺ concetnration with a microelectrode, in response to a 10-fold step reduction of extracellular HCO 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 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 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 anions vs. 1 CO divalent anion. Indeed, a 3:1 stoichiometry could result from the transport of 3 HCO:1 Na⁺ or 1 CO:1 HCO:1 Na^+.. Furthermore, a 2:1 stoichiometry could result from either the loss of one HCO binding site or a change from the transport of 1 CO and 1 HCO to 2 HCO 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 is ~500-fold lower than that of HCO and ~1,000-fold lower at an intracellular pH of 7.1, it might be predicted that the flux of CO would be negligible compared with that of HCO. However, this assumption would not be correct if the binding constant of the cotransporter for CO is ~500- to 1,000-fold higher then that for HCO.

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 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 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/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: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: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 and Na⁺ concentrations), additional data suggest that this explanation is insufficient. Specifically, when proximal tubules were incubated in HCO-containing Ringer, the HCO: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: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 cotransporters.

After the cloning of the pancreatic variant of the electrogenic Na⁺-HCO cotransporter (1), pNBC1, we found that it operated with an HCO: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: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 transport that could also be potential candidates as modulators of the cotransporter's stoichiometry. cAMP is a strong modulator of HCO transport in both the kidney and the exocrine pancreas. In the renal proximal tubule, cAMP regulates HCO absorption by decreasing the rate of apical Na⁺/H⁺ exchange and basolateral sodium HCO efflux (68, 74, 102). In the guinea pig interlobular pancreatic duct, Ishiguro et al. (57) found that secretin, acetylcholine, and forskolin stimulate HCO secretion. The effect was mediated by stimulation of the basolateral Na⁺-HCO 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:1 Na⁺ to 2 HCO: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: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 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 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 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 interacts with the cotransporter. Previously, we presented a mathematical model that describes the interaction of HCO 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 (Fig. 4). Na⁺ and HCO 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 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 indicated that the binding of 3 HCO anions (or 1 CO and 1 HCO) to the cotransporter is voltage dependent, with an electrical coefficient of 0.2 at pH 7.5. This indicates that, on average, HCO "senses" ~20% of the membrane's electric field on binding to the cotransporter or that the binding site for HCO is located about one-fifth of the electrical distance into the membrane. This result raises the possibility that the binding of HCO 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 (Bic) anions to the transporter is described as a single, lumped step (see text). The model does not distinguish the binding of 3 HCO anions vs. 1 CO and 1 HCO.

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 binding site and perhaps disrupting the binding of HCO? 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 binding site might alter the electric field around it and interfere with HCO binding to the cotransporter (Fig. 5A). Whatever the mechanism might be by which the COOH terminus "interferes" with HCO 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 binding site. A similar concept was proposed to explain removal of fast N-type inactivation of voltage-dependent potassium channels (K_v) 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 -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 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: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 binding site on the transporter, resulting in a HCO: Na+ transport stoichiometry of 2:1. B: when kNBC1-Ser982 or pNBC1-Ser1026 is unphosphorylated, the putative HCO binding site in the transporter is unblocked and available to bind HCO, resulting in an HCO: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 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 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 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 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 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<