Phosphorylation of Ser982 in the sodium bicarbonate cotransporter kNBC1 shifts the HCO3-: Na+ stoichiometry from 3:1 to 2:1 in murine proximal tubule cells (original) (raw)
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The Sodium Bicarbonate Cotransporter: Structure, Function, and Regulation
2006
The role of the Na ؉-coupled HCO 3 ؊ transporter (NBC) family is indispensable in acid-base homeostasis. Almost all tissues express a member of the NBC family. NBC has been studied extensively in the kidney and plays a role in proximal tubule HCO 3 ؊ reabsorption. Although the exact function of this transporter family on other tissues is not very clear, the ubiquitous expression of NBC family suggests a role in cell pH regulation. Altered NBC activity caused by mutations of the gene responsible for NBC protein expression results in pathophysiologic conditions. Mutations of NBC resulting in important clinical disorders have been reported extensively on one member of the NBC family, the kidney NBC (NBC1). These mutations have led to several structural studies to understand the mechanism of the abnormal NBC1 activity.
Biophysical Journal, 1998
The voltage dependence of the kinetics of the sodium bicarbonate cotransporter was studied in proximal tubule cells. This electrogenic cotransporter transports one Na ϩ , three HCO 3 Ϫ , and two negative charges. Cells were grown to confluence on a permeable support, mounted on a Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, di-nitro-stilbene-di-sulfonate-sensitive current was shown to be sodium and bicarbonate dependent and therefore was taken as flux through the cotransporter. Voltage-current relations were measured as a function of Na ϩ and HCO 3 Ϫ concentrations between Ϫ160 and ϩ160 mV under zero-trans and symmetrical conditions. The kinetics could be described by a Michaelis-Menten behavior with a Hill coefficient of 3 for HCO 3 Ϫ and 1 for Na ϩ. The data were fitted to six-state ordered binding models without restrictions with respect to the rate-limiting step. All ordered models could quantitatively account for the observed current-voltage relationships and the transinhibition by high bicarbonate concentration. The models indicate that 1) the unloaded transporter carries a positive charge; 2) the binding of cytosolic bicarbonate to the transporter "senses" 12% of the electric field in the membrane, whereas its translocation across the membrane "senses" 88% of the field; 3) the binding of Na ϩ to the cotransporter is voltage independent.
Stoichiometry of the rat kidney Na + -HCO 3 - cotransporter expressed in Xenopus laevis oocytes
Pfl�gers Archiv European Journal of Physiology, 1999
The rat kidney Na +-HCO 3 cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na + and HCO 3 as permeable ions. The current carried by rkNBC (I NBC) was identified by its response to changing bath Na + concentration(s) and quantified as the current blocked by 4,4'-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO 3 to Na + transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 µmol/l) blocks I NBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na + and HCO 3 concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO 3 concentration was 25 mmol/l throughout, but the Na + concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch.
Cloning and functional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney
The American journal of physiology, 1998
We have recently cloned the renal electrogenic Na(+)-bicarbonate contransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na(+)-bicarbonate cotransporter. NBC constitutes the major route for HCO3- reabsorption and assists in Na+ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in lambda gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of approximately 500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular m...
Proceedings of the National Academy of Sciences, 1993
Electrogenic cotransport of Na+ with HCO3- has been reported in numerous tissues. It has always been shown with a net transfer of negative charge, but in some situations achieves net outward transport of both species with a stoichiometry of at least three HCO3- ions per Na+ ion (3:1), and in other situations achieves net inward transport of both species and has a stoichiometry of at most two HCO3- ions per Na+ ion (2:1). This suggests either that there may be more than one protein responsible for Na(+)-HCO3- cotransport in different tissues or that if there is a single protein, its stoichiometry may differ depending on the orientation of net transport. The present study, using conventional or double-barreled ion-selective microelectrodes to follow basolateral membrane potential and intracellular pH or Na+ activity in Necturus proximal convoluted tubule in vivo, shows that the orientation of the basolateral Na(+)-HCO3- cotransporter can be reversed upon switching from a perfusate sim...
Journal of Clinical Investigation, 1991
HCO3 exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO-3 cotransporter. In these experiments, we have studied the effect of internal pH on the activity of the Na+:HCO-cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. Equilibrium thermodynamics predicts that in the presence of constant intravesicular concentration of Na+, an increasing concentration of HCO3 will be associated with an increasing driving force for Na+:HCO3 cotransport across the vesicles. Our experimental approach was to preequilibrate the membrane vesicles with 1 mM 22Na' at pH1 6.8-8.0 and known concentrations of HCO3. The vesicles were diluted 1:100 into Na+-free solution at pH 7.4 and the net flux of 22Na+ was assayed over 5 s. The results demonstrate that the net flux of Na+ was significantly higher at pH1 7.2 than pH, 8.0 despite much higher IHCO3I at pHi 8.0. This suggests that an internal pHsensitive site regulates the activity of the Na+:HCOcotransporter. This modifier site inhibits the cotransporter at alkaline pH despite significant base concentration and is maximally functional around physiologic pH. The combination of modifier sites on the luminal Na+/H+ exchanger and the basolateral Na+:HCOcotransporter should help maintain intracellular pH in a narrow range with changes in extracellular pH. (J.