The Na/HCO3 cotransporter NBCn1/SLC4A7 can affect glutamate neurotoxicity in primary cultures of rat hippocampal neurons. Here, we examined NMDA-induced neurotoxicity in NBCn1 knockout mice to determine whether a similar effect also occurs in the mouse brain. In primary cultures of hippocampal neurons from knockouts, NMDA had no neurotoxic effects, determined by lactate dehydrogenase release and nitric oxide synthase (NOS)-dependent cGMP production. Male knockouts and wildtypes (6–8 weeks old) were then injected with NMDA (75 mg/kg; ip) and hippocampal neuronal damages were assessed. Wildtypes developed severe tonic-clonic seizures, whereas knockouts had mild seizure activity (motionless). In knockouts, the NOS activity, caspase-3 expression/activity and the number of TUNEL-positive cells were significantly low. Immunochemical analysis revealed decreased expression levels of the NMDA receptor subunit GluN1 and the postsynaptic density protein PSD-95 in knockouts. Extracellular recording from hippocampal slices showed no Mg2+/NMDA-mediated epileptiform events in knockouts. In conclusion, these results show a decrease in NMDA neurotoxicity by NBCn1 deletion. Given that acid extrusion has been known to prevent pH decrease and protect neurons from acid-induced damage, our study presents novel evidence that acid extrusion by NBCn1 stimulates neurotoxicity.

Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO3 transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pHo) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pHo 7.4. At pHo 6.5, the intracellular pH of neurons was ~1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pHo 6.5. NBCn1 expression also increased by 40% in a prolonged Mg2+-free incubation at normal pHo. Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mM glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pHo 7.2 for 6 h) after glutamate exposure, acidic incubation (pHo 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg2+-free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pHo and Mg2+ depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg2+-free conditions.

In this study, we examined an ammonium conductance in the mouse thick ascending limb cell line ST-1. Whole cell patch clamp was performed to measure currents evoked by NH4Cl in the presence of BaCl2, tetraethylammonium, and BAPTA Application of 20 mmol/L NH4Cl induced an inward current (-272 ± 79 pA, n = 9). In current-voltage (I-V) relationships, NH4Cl application caused the I-V curve to shift down in an inward direction. The difference in current before and after NH4Cl application, which corresponds to the current evoked by NH4Cl, was progressively larger at more negative potentials. The reversal potential for NH4Cl was +15 mV, higher than the equilibrium potential for chloride, indicating that the current should be due to NH4(+) We then injected ST-1 poly(A) RNA into Xenopus oocytes and performed two-electrode voltage clamp. NH4Cl application in the presence of BaCl2 caused the I-V curve to be steeper. The NH4(+) current was retained at pH 6.4, where endogenous oocyte current was abolished. The NH4(+) current was unaffected by 10 μmol/L amiloride but abolished after incubation in Na(+)-free media. These results demonstrate that the renal cell line ST-1 produces an NH4(+) conductance.

The electroneutral Na/HCO3 cotransporter NBCn1 (SLC4A7) contributes to intracellular pH maintenance and transepithelial HCO3− movement. In this study, we expressed NBCn1 in Xenopus oocytes and examined the effect of NBCn1 on oocyte NH4+ transport by analyzing changes in membrane potential, current, and intracellular pH mediated by NH4Cl. In the presence of HCO3−/CO2, applying NH4Cl (20 mM) produced intracellular acidification of oocytes. The acidification was faster in oocytes expressing NBCn1 than in control oocytes injected with water. However, NH4Cl-mediated membrane depolarization was smaller in oocytes expressing NBCn1. In HCO3−/CO2-free solution, NH4Cl produced a smaller inward current in NBCn1-expressing oocytes (56% inhibition by 20 mM NH4Cl; measured at −60 mV), while minimally affecting intracellular acidification. The inhibition of the current by NBCn1 was unaffected when BaCl2 replaced KCl. Current-voltage relationships showed a positive and nearly linear relationship between NH4Cl-mediated current and voltage, which was markedly reduced by NBCn1. Large basal currents (before NH4Cl exposure) were produced in NBCn1-expressing oocytes due to the previously characterized channel-like activity of NBCn1. Inhibiting this channel-like activity by Na+ removal abolished NBCn1’s inhibitory effect on NH4Cl-mediated currents. The currents were progressively reduced over 72–120 h after NBCn1 cRNA injection, during which the channel-like activity was high. These results indicate that NBCn1 by its Na/HCO3 cotransport activity stimulates NH4+ transport, while reducing NH4+ conductance by its channel-like activity.

In this study, we examined the effect of bicarbonate transporters on ammonium/ammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mM ouabain and 0.2 mM bumetanide to minimize carrier-mediated NH4+ transport, and the intracellular accumulation of 14C-methylammonium/methylammonia (MA) was determined. In CO2∕HCO3−-free solution, cells at normal pH briefly accumulated 14C-MA over 7 min and reached a plateau. In CO2∕HCO3− solution, however, cells markedly accumulated 14C-MA over the experiment period of 30 min. This CO2∕HCO3−-dependent accumulation was reduced by the bicarbonate transporter blocker 4,4’-diisothiocyanatostilbene-2,2’-disulfonate (DIDS; 0.5 mM). Replacing Cl– with gluconate reduced the accumulation but the reduction was more substantial in the presence of DIDS. Incubating cells at pH 6.8 (adjusted with NaHCO3 in 5% CO2) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for controls. DIDS reduced 14C-MA accumulation in controls but had no effect after acidic incubation. Immunoblot showed that NBCn1 was upregulated after acidic incubation and in NH4Cl-containing media. The Cl/HCO3 exchanger AE2 was present but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated 14C-MA transport, which was abolished by replacing Na+. Two-electrode voltage clamp of oocytes exhibited negligible current after NH4Cl application. These results suggest that DIDS-sensitive HCO3− extrusion normally governs NH4+/NH3 uptake in the MTAL cells. We propose that, under acidic conditions, DIDS-sensitive HCO3− extrusion is inactivated while NBCn1 is upregulated to stimulate NH4+ transport.

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO3 transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO3 transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO3− currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO3-dependent pH recovery from a CO2-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO2/HCO3−-free solution that was abolished by Cl− removal from the bath. In the presence of CO2/HCO3−, however, the outward current produced by D555E decreased only slightly after Cl− removal. Starting from a Cl−-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO2/HCO3−. The substitution of Asp555 with an asparagine also produced a Cl− current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO3−. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp555 plays a role in HCO3− selectivity.

Recent studies have shown that accessory proteins that interact with the apical Na+/H+ exchanger NHE3 are a vital part of the dynamic nature of the Na+/H+ exchanger regulation. We have identified MAST205, a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa that interacts with NHE3. MAST205 contains a S/T kinase domain and a PDZ domain that mediates interaction with NHE3. Northern blot analysis showed that MAST205 is highly expressed in human and rat kidney. Expression in opossum kidney (OK) cells showed that MAST205 is predominantly expressed in the apical membrane of the cells. Immunohistochemical studies demonstrated the presence of MAST205 at the apical region of the renal proximal tubules. Heterologous expression of MAST205 in OK cells inhibited endogenous NHE3 activity, and this inhibition required the presence of the kinase domain of MAST205, since deletion of the kinase domain or a dominant-negative mutant of MAST205 did not affect the activity of NHE3. Consistent with these results, we found that MAST205 phosphorylated NHE3 under in vitro conditions. However, overexpression of MAST205 did not affect expression of NHE3 proteins, suggesting that the effect of MAST205 was not mediated by a decrease in NHE3 expression. These findings suggest that MAST205 regulates NHE3 activity and, although the precise mechanism is yet to be determined, MAST205 appears to inhibit NHE3 activity through a phosphorylation-dependent mechanism.

The electrogenic Na+–HCO3− cotransporter (NBCe1) plays a central role in intracellular pH (pHi) regulation as well as HCO3− secretion by pancreatic ducts and HCO3− reabsorption by renal proximal tubules. To understand the structural requirements for the electrogenicity of NBCe1, we constructed chimeras of NBCe1-A and the electroneutral NBCn1-B, and used two-electrode voltage clamp to measure electrogenic transporter current in Xenopus oocytes exposed to 5% CO2–26 mm HCO3−(pH 7.40). The chimera consisting of NBCe1-A (i.e. NBCe1-A ‘background’) with the cytoplasmic N-terminal domain (Nt) of NBCn1-B had a reversal potential of −156.3 mV (compared with a membrane potential Vm of −43.1 mV in a HCO3−-free solution) and a slope conductance of 3.0 μS (compared with 12.5 μS for NBCe1-A). Also electrogenic were chimeras with an NBCe1-A background but with NBCn1-B contributing the extracellular loop (L) between transmembrane segment (TM) 5 and 6 (−140.9 mV/11.1 μS), the cytoplasmic C-terminal domain (Ct; −123.8 mV/9.7 μS) or Nt + L + Ct (−120.9 mV/3.7 μS). Reciprocal chimeras (with an NBCn1 background but with NBCe1 contributing Nt, L, Ct or Nt + L + Ct) produced no measurable electrogenic transporter currents in the presence of CO2–HCO3−. pHi recovered from an acid load, but without the negative shift of Vm that is characteristic of electrogenic Na+–HCO3− cotransporters. Thus, these chimeras were electroneutral, as were two others consisting of NBCe1(Nt–L)/NBCn1(TM6–Ct) and NBCn1(Nt–L)/NBCe1(TM6–Ct). We propose that the electrogenicity of NBCe1 requires interactions between TM1–5 and TM6–13.

Postmortem studies reveal that the brain pH in schizophrenia patients is lower than normal. The exact cause of this low pH is unclear, but increased lactate levels due to abnormal energy metabolism appear to be involved. Schizophrenia patients display distinct changes in mitochondria number, morphology, and function, and such changes promote anaerobic glycolysis, elevating lactate levels. pH can affect neuronal activity as H+ binds to numerous proteins in the nervous system and alters the structure and function of the bound proteins. There is growing evidence of pH change associated with cognition, emotion, and psychotic behaviors. Brain has delicate pH regulatory mechanisms to maintain normal pH in neurons/glia and extracellular fluid, and a change in these mechanisms can affect, or be affected by, neuronal activities associated with schizophrenia. In this review, we discuss the current understanding of the cause and effect of decreased brain pH in schizophrenia based on postmortem human brains, animal models, and cellular studies. The topic includes the factors causing decreased brain pH in schizophrenia, mitochondria dysfunction leading to altered energy metabolism, and pH effects on the pathophysiology of schizophrenia. We also review the acid/base transporters regulating pH in the nervous system and discuss the potential contribution of the major transporters, sodium hydrogen exchangers (NHEs), and sodium-coupled bicarbonate transporters (NCBTs), to schizophrenia.

The previous reports on an addiction vulnerability marker in the human SLC4A7 gene encoding the Na/HCO3 transporter NBCn1 suggest that this pH-regulating protein may affect alcohol-related behavior and response. Here, we examined alcohol consumption and sensitivity to the sedative effects of alcohol in male NBCn1 knockout mice. These mice displayed lower pH in neurons than wildtype controls, determined by intracellular pH in hippocampal neuronal cultures. Neurons from knockout mice had a higher action potential threshold and a more depolarized membrane potential, thus reducing membrane excitability. In a two-bottle free choice procedure, knockout mice consumed more alcohol than controls and consistently increased alcohol consumption after repeated alcohol deprivation periods. Quinine and sucrose preference was similar between genotypes. Knockout mice showed increased propensity for alcohol-induced conditioned place preference. In loss of righting reflex assessment, knockout mice revealed increased sensitivity to alcohol-induced sedation and developed tolerance to the sedation after repeated alcohol administrations. Furthermore, chronic alcohol consumption caused NBCn1 downregulation in the hippocampus and striatum of mice and humans. These results demonstrate an important role of NBCn1 in regulation of alcohol consumption and sensitivity to alcohol-induced sedation.