Han Soo Yang

Neuronal expression of sodium/bicarbonate cotransporter NBCn1 (SLC4A7) and its response to chronic metabolic acidosis

The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves Na(+) and HCO(3)(-) into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HCl-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hippocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-d-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl-d-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excitotoxicity.

The electrogenicity of the rat sodium-bicarbonate cotransporter NBCe1 requires interactions among transmembrane segments of the transporter.

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.

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride 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.

Sodium/bicarbonate cotransporter NBCn1/slc4a7 increases cytotoxicity in magnesium depletion in primary cultures of hippocampal neurons

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.

Sodium–bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport

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 (14C‐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 experimental 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. Incubation of 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 control cells. The presence of DIDS reduced 14C‐MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting 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 medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS‐sensitive HCO3− extrusion is inactivated, while NBCn1 is upregulated to stimulate NH4+ transport.

Inhibition of rat Na+–HCO3– cotransporter (NBCn1) function and expression by the alternative splice domain

The Na+–HCO3– cotransporter NBCn1 (SLC4A7) has multiple variants depending upon splice domains in the cytoplasmic amino‐ and carboxy‐termini of the protein. In this study, we examined the role of the amino‐terminal splice domain containing 123 amino acids (cassette II) in the regulation of NBCn1 function and expression. Polymerase chain reaction detected NBCn1 mRNAs containing cassette II in a variety of tissues. Two variants, NBCn1‐B containing cassette II and NBCn1‐E lacking cassette II, were expressed in Xenopus oocytes and assessed by two‐electrode voltage clamp to measure the ionic current mediated by the transporters. The two variants showed similar current–voltage (I–V) relations when measured 3–4 days after RNA injection. Replacment of Cl− with gluconate did not affect the I–V relations. When exposed to solutions containing 20–50 mm Na+, the current produced by NBCn1‐B was slightly more positive than that produced by NBCn1‐E. The two currents were similar at 100 mm Na+. The slope conductances for the two variants were progressively increased at higher Na+ levels, and the increases were parallel and superimposed. Measured at different time points after RNA injection, NBCn1‐B produced lower conductance than NBCn1‐E at 24–48 h. Protein expression of NBCn1‐B was also low at these time points as determined by immunoblot of oocyte membrane preparation. Expressed in opossum kidney (OK) cells, NBCn1‐E caused a 1.5‐fold increase in ouabain‐sensitive production of p‐nitrophenol from p‐phenyl phosphate compared with control preparations, whereas NBCn1‐B had negligible effect. We conclude that the primary function of cassette II is to reduce NBCn1 protein expression.

PSD-95 Interacts with NBCn1 and Enhances Channel-like Activity without Affecting Na/HCO3 Cotransport

Background/Aims: The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3– movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results: In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion: Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.