H+ exchanger isoform 1 null mutant mouse brain

Neuroscience 122 (2003) 37– 46

EXPRESSION OF Naⴙ/Hⴙ AND HCO3ⴚ-DEPENDENT TRANSPORTERS IN Naⴙ/Hⴙ EXCHANGER ISOFORM 1 NULL MUTANT MOUSE BRAIN J. XUE,a,c R. M. DOUGLAS,a,c D. ZHOU,a,c J. Y. LIM,a W. F. BORONb AND G. G. HADDADa,b,c,d*

Key words: NHE1 null, mouse, brain, acid-base transporters.

a Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA

Intracellular pH (pHi) can change rapidly and transiently in response to neuronal activity and neuronal activation by transmitters, hormones, growth factors or other messengers (Deitmer and Rose, 1996). These alterations in pHi, in turn, have an impact on CNS function via changes in ion channel conductance, synaptic transmission, intracellular coupling via gap junctions and metabolic enzyme activity (Busa and Nuccitelli, 1984; Moody, 1984; Chesler, 1990; Takahashi and Copenhagen, 1996). Hence, the maintenance of pHi homeostasis appears to be crucial for proper neuronal function. Because of its high metabolic rate, the CNS produces a large amount of metabolic acid, particularly under stressful conditions, such as excessive activity, hypoxia/ischemia and hyperglycemia. This feature renders neurons more susceptible to injury if subjected to wide swings in acid or alkaline loads. Thus, the mechanisms for acid-extrusion or acid-loading in neurons would be important for pHi homeostasis and brain function. A substantial number of studies have shown that acid– base membrane transporter proteins are key players in pHi regulation in the CNS (Boyarsky et al., 1993; Raley-Susman et al., 1993; Schwiening and Boron, 1994; Bevensee et al., 1996). Based on stoichiometry and direction of ionic movement, these acid– base regulatory proteins can be broadly divided into two groups. On the one hand, acid extruders, such as Na⫹/H⫹ exchangers (NHEs), Na⫹-dependent Cl⫺–HCO3⫺ exchangers and some Na⫹/HCO3⫺ cotransporters (NBCs), participate in the pHi recovery from acid loads. Acid loaders, on the other hand, such as Cl⫺– HCO3⫺ exchanger (AE) and some Na⫹/HCO3⫺ cotransporters, are responsible for the pHi recovery from alkaline loads (Bevensee and Boron, 1998). We and others have recently demonstrated the presence of NHEs and NBCs at both mRNA and protein levels in the CNS (Ma and Haddad, 1997; Schmitt et al., 2000; Douglas et al., 2001). The existence of a naturally occurring mouse mutant totally lacking the NHE1 isoform (the mostly densely and ubiquitously expressed NHE of the family; Cox et al., 1997) has recently facilitated the study of the effect of this mutation on pHi homeostasis. Our results have revealed that the absence of the NHE1 protein caused a lower steady-state pHi in hippocampal CA1 neurons, a reduced recovery rate from an acid load in HEPES buffer, which was exaggerated in HCO3⫺ solution, as well as increased neuronal excitability (Yao et al., 1999; Gu et al., 2001). The reduction of acid-extruding capability in

b

Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA c

Department of Pediatrics (Section of Respiratory Medicine), Albert Einstein College of Medicine, and Children’s Hospital at Montefiore, Rose F. Kennedy Center Room 845, 1410 Pelham Parkway South, Bronx, NY 10461, USA

d Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Abstract—Acid– base transporters, such as the sodium– hydrogen exchangers (NHEs) and bicarbonate-dependent transporters, play an important role in the regulation of intracellular pH (pHi) in the CNS. Previous studies from our laboratory have shown that the absence of the major NHE isoform 1 (NHE1) reduced the steady-state pHi and recovery rate from an acid load in the hippocampal neurons not only in HEPES but also in HCO3ⴚ solutions (Yao et al., 1999). The purpose of the current study was to determine whether the NHE1 null mutation affects the expression of pH-regulatory transporters in the mouse CNS. Immunoblotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) were performed to examine the protein and mRNA levels of NHE1– 4, electrogenic sodium-bicarbonate cotransporter 1 variants (NBCe1), and brain-specific anion exchanger 3 (AE3) in four brain regions (cerebral cortex, hippocampus, cerebellum and brainstem– diencephalon). NHE1 null mutant mice were compared with their wild type controls at the average age of approximately 4 weeks. Our results revealed that the NHE1 null mutation caused a significant increase in NHE3 in the cerebellum (84% for protein, 105% for mRNA), an increase in NBCe1 expression in the brainstem– diencephalon (approximately 40 –50% for protein, 9 –15% for mRNA), as well as a decrease in AE3 in the hippocampus (approximately 60% for protein, 24% for mRNA). We conclude that the NHE1 null mutation does alter the expression of other membrane transporters at both protein and mRNA levels. The alteration is region-specific. An increase in acid extruders (e.g. NHE3) and a decrease in acid loaders (e.g. AE3) suggest that there are some compensatory mechanisms that occur in NHE1 null mutant mice. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. *Correspondence to: G. G. Haddad, Department of Pediatrics, Albert Einstein College of Medicine, and Children’s Hospital at Montefiore, Rose F. Kennedy Center Room 845, 1410 Pelham Parkway South, Bronx, NY 10461, USA. Tel: ⫹1-718-430-4127; fax: ⫹1-718-4302199. E-mail address: [email protected] (G. G. Haddad). Abbreviations: AE, anion exchanger; AE3, anion exchanger isoform 3; BD, brainstem-diencephalon; CB, cerebellum; CX, cerebral cortex; HC, hippocampus; HEPES, N-(2-hydroxyethyl) piperazine-N⬘-(2ethanesulphonic acid); NBC, sodium-bicarbonate cotransporter; NBCe1, electrogenic sodium-bicarbonate cotransporter 1; NHE, sodium– hydrogen exchanger; pHi, intracellular pH.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00598-0

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J. Xue et al. / Neuroscience 122 (2003) 37– 46

Table 1. Rabbit polyclonal anti-NBC antibodies

Anti-RK-NBC (anti-MBP-NBC-5) Anti-NBC(K1A) Anti-NBC(B1B)

Residues

Recognize

Reference

108 Residues (928–1035) of NBCe1-A (rkNBC; access code: AAC40034) 46 Residues (1034–1079) of NBCe1-B (rb1NBC; access code: AAF87553) 61 Residues (1034–1094) of NBCe1-C (rb2NBC; access code: AAF87312)

NBCe1-A, NBCe1-B, NBCe1-C (only 62 of 108 residues) NBCe1-A (kidney), NBCe1-B (pancreas/heart) NBCe1-C (predominantly in brain)

Schmitt et al., 1999; Soleimani and Burnham, 2001 Bevensee et al., 2000

mutant hippocampal CA1 neurons in HEPES buffer indicated that there was little, if any, compensatory up-regulation of non-HCO3⫺-dependent mechanisms for acid extrusion in the hippocampus (HC). It was also interesting that acid-extruding ability became even worse in the presence of HCO3⫺ in the bath solution. Clearly, a number of questions can be raised from these results. First, what membrane protein expression is altered when NHE1 is mutated? Second, what is the basis of the interactions between NHE1 and other membrane proteins? Third, how does cell function change in the absence of NHE1? To start addressing some of these issues, we investigated NHE1 mutant mice with respect to the expression of acid– base membrane transporters such as the NHEs and the HCO3⫺-dependent transporters.

EXPERIMENTAL PROCEDURES Animals B6.SJL, ⫹/swe (slow wave epilepsy) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; Cox et al., 1997). These heterozygous mice were mated in our institution, and the resulting homozygous NHE1 null mutant (25%) and wild-type (25%) F1 mice progeny were chosen at the age of postnatal day 20 –39 in the current study. Homozygous mutant mice exhibited a neurological phenotype including locomotor ataxia in the hind limbs and a slow, wide-based gait and coarse truncal instability starting at 2–3 wk of age. Genotyping was performed on these mice to confirm the phenotype. These animals were handled with extreme care and the minimum number of animals that was absolutely required was used. This study was approved by the Yale Animal Care and Use Committee.

Antibodies The mouse monoclonal antibody to NHE1 (anti-NHE1; mAb 4E9) was derived from a MBP fusion protein containing the amino acids 514 – 818 of porcine NHE1 (Chemicon, Temecula, CA, USA). Rabbit polyclonal antibody to NHE2 (anti-NHE2; Ab597) recognized the cytoplasmic tail at amino acids 723– 809 of the NHE2 protein (Tse et al., 1994). Mouse monoclonal antibody to NHE3 (anti-NHE3; 4F5) was directed against the C-terminal amino acids 702– 832 of rabbit NHE3 protein (Biemesderfer et al., 1997). Rabbit polyclonal antibody to NHE4 (anti-NHE4) was developed against a 17 amino-acid peptide within the cytoplasmic C-terminal of the rat NHE4 protein (Chemicon). The Na⫹-coupled HCO3⫺ transporters are represented by at least five mammalian genes and, together with the three AEs (AE1–3), are part of the bicarbonate transporter superfamily. The first Na⫹-coupled HCO3⫺ transporter to be cloned was the electrogenic Na/HCO3 cotransporter NBCe1 (also known as NBC1). NBCe1 is transcribed as at least three variant mRNAs. NBCe1-A was first cloned from kidney (Romero et al., 1998) and was later found in brain, eye, and many other tissues (Soleimani and Burn-

Bevensee et al., 2000

ham, 2001). NBCe1-B was first cloned from pancreas (Abuladze et al., 1998), and was later found in heart (Choi et al., 1999), eye, and other tissues. NBCe1-A and NBCe1-B differ only in their amino-terminal sequences. NBCe1-C was found predominantly in brain (Bevensee et al., 2000) and it differs from NBCe1-B only in the presumed cytoplasmic C-terminus, where a 97-bp deletion causes the final 46 of NBCe1-B to be replaced by 61 novel amino acids in NBCe1-C. In the current study we have utilized three rabbit polyclonal anti-NBC antibodies that detect all reported variants of NBCe1 (Table 1; Boron, 2001; Soleimani and Burnham, 2001). (1) AntiRK-NBC was raised against the carboxy terminal 108 residues shared by NBCe1-A and NBCe1-B, also partially (62 residues) shared by NBCe1-C (Schmitt et al., 2000; Soleimani and Burnham, 2001). Thus anti-RK-NBC should in principle recognize all three variant NBCe1 polypeptides. (2) Anti-NBC(K1A) recognizes the carboxy-terminal 46 residues shared by NBCe1-A and NBCe1-B, but not the carboxy-terminal 61 amino acids unique to NBCe1-C (Bevensee et al., 2000). (3) Anti-NBC(B1B) recognizes the unique carboxy-terminal 61 amino acids (1034 –1094) of NBCe1-C, but not the 46 carboxy-terminal amino acids shared by NBCe1-A and NBCe1-B (Bevensee et al., 2000). Rabbit polyclonal antibody to AE isoform 3 (anti-AE3; ␣ SA8) is directed against C-terminal 12 residues of mouse AE3, which detects a protein of 180 kDa and does not cross-react with the abundant AE2 of choroids plexus (Yannoukakos et al., 1994). NHE2 antibody was kindly provided by Dr. Mark Donowitz of Johns Hopkins University (Baltimore, MD, USA) and NHE3 antibody was donated by Dr. Daniel Biemesderfer of Yale University School of Medicine (New Haven, CT, USA). The three NBCe1 antibodies were those developed in the laboratory of Dr. Walter F. Boron (New Haven, CT, USA). The AE3 antibody was a gift from Dr. Seth Alper of Harvard Medical School (Boston, MA, USA).

Genotyping of NHE1 null mutant mice A single A–T change at nucleotide 1693 (Cox et al., 1997) introduced a premature stop (TAG) codon within the open reading frame of NHE1. Therefore, the C-terminus of NHE1 is not present in null mutant mice. This spontaneous mutation transformed the surrounding sequence from CAACAAGTTCC (WT) to CAACTAGTTCC (Null) and created a cleavage site for the endonuclease SpeI. SpeI cut in between A and C in the sequence ACTAG and formed the basis for the present genotypic analysis. Genotyping was performed according to a method we previously described (Yao et al., 1999). Briefly, genomic DNA was obtained from mouse tails and used for PCR amplification. The primers were: sense, 5⬘-TCGCCTCAGGAGTAGTGATGCG-3⬘ and antisense, 5⬘-TCATGCCCTGCACAAAG ACG-3⬘, corresponding to base pairs 1397–1418 and 1800 –1819 of mouse NHE1 cDNA sequence (accession no. U51112). The amplification profile was set at 94 °C for 1.5 min, 60 °C for 1 min, and 72 °C for 1.5 min for 30 cycles. The resulting PCR products were digested by endonuclease SpeI. PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide and UV transillumination. Cleavage is indicative of a mutant allele (two bands at the size of

J. Xue et al. / Neuroscience 122 (2003) 37– 46

39

Fig. 1. Lack of NHE1 expression in NHE1 null mutant mice. (A). Genotypic analysis. A region encompassing the mutation was amplified from genomic tail DNA by PCR. The resultant PCR products were then subjected to SpeI digestion and separated on 1.5% agarose gels. The endonuclease SpeI cleaved the mutant allele into two pieces: 1.8 kb and 0.2 kb, but not the wild type (2.0 kb). (B). Immunoblot analysis. Expression of NHE1 protein was determined using C-terminal specific anti-NHE1 monoclonal antibody. A 110 kDa protein was recognized by this antibody in the wild type mice. As expected, no band was detectable in the homozygous null mutants.

1.8 kb and 0.2 kb), whereas wild-type alleles remained uncleaved (one band at the size of 2.0 kb; Fig. 1A).

Immunoblotting Tissue acquisition. To harvest brain tissue, mice were deeply anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ, USA) and decapitated with a guillotine. The brains were quickly removed from the cranium and placed in ice-cold lysis buffer (200 mM mannitol, 80 mM HEPES, 41 mM KOH, 1 ␮M pepstatin A, 1 ␮M leupeptin, 230 ␮M phenylmethylsulfonyl fluoride and 1 mM ethylenediamine tetrahydrochloride; pH 7.5; Sigma, St. Louis, MO, USA). Four brain regions were dissected out: cerebral cortex (CX), HC, cerebellum (CB) and brainstem– diencephalon (BD). The tissues of corresponding regions from four animals were pooled, weighed and transferred to lysis buffer (4⫻volume/ weight) for the subsequent microsomal preparation. Membrane fractionation. Crude microsomes were prepared from each of the four brain regions according to a method described by Grassl and Aronson (1986). Briefly, tissues were homogenized by 10 –20 strokes at 2000 r.p.m. with a Teflon-glass homogenizer (Thomas Scientific, Swedesboro, NJ, USA). The

homogenate was then centrifuged at 1000⫻g, 4 °C for 10 min to remove cellular debris. The supernatant was re-centrifuged at 100,000⫻g, 4 °C in a Beckman L8 –70 M ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA) for 1 h. The resulting pellet was re-suspended in 200 –500 ␮l of lysis buffer and stored at ⫺80 °C until usage. SDS-PAGE and Western blotting. Protein concentrations of the pooled membranes were determined using a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of membrane protein of each region was resolved on 10% pre-cast NuPAGE Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) and electro-transferred onto polyvinylidene fluoride membranes (Immobilin-P; Millipore, Bedford, MA, USA). Subsequently, membranes were incubated in blocking solution (5% non-fat dry milk, 0.1% Tween 20; in phosphate-buffered saline) at room temperature for 1 h and then probed with various primary antibodies at 4 °C overnight. The affinity-purified mouse monoclonal anti-NHE1 IgG (dilution factor of 1:1000), rabbit polyclonal anti-NHE2 IgG (1: 2000), mouse monoclonal anti-NHE3 hybridoma supernatant (1: 1000), affinity-purified rabbit polyclonal anti-NHE4 IgG (1:500), affinity-purified rabbit polyclonal anti-AE3 IgG (1:2000), rabbit

40

J. Xue et al. / Neuroscience 122 (2003) 37– 46

Table 2. The primers for RT-PCR Gene Primer set access code

PCR product size, bp

Reference

NHE3

AF139194

229

Praetorius et al., 2000

NBC

AF004017

AE3

M28383

␤-Actin NM 007393

Sense: 5⬘-TGGCCTTCATTCGCTCC-3⬘ (nt 3–19) Anti-sense: 5⬘-TACTCCTGCCGAGGCTTG-3⬘ (nt 214–231) Sense: 5⬘-GCGATTATTTTTCCAGTCATGATC-3⬘ (nt 2808–2831)

325 for NBCe1-A Schmitt et al., 1999; or-B Bevensee et al., 2000; Soleimani and Burnham, 2001 Anti-sense: 5⬘-ATCAGCATGATGTGTGGCGTTCAAGG-3⬘ (nt 3107–3132) 228 for NBCe1-C Sense: 5⬘-CCTCATTGCCTTCTTCTTGC-3⬘ (nt 2883–2902) 320 Kopito et al., 1989 Anti-sense: 5⬘-CAATGAGCGCAGTGATCTGT-3⬘ (nt 3183–3202) Sense: 5⬘-TGTTACCAACTGGGACGACA-3⬘ (nt 305–324) 392 Tokunaga et al., 1986 Anti-sense: 5⬘-TCTCAGCTGTGGTGGTGAAG-3⬘ (nt 677–696)

polyclonal anti-NBCe1 serum (anti-RK-NBC, 1:400; antiNBC(K1A), 1:1000; anti-NBC(B1B), 1:5000) were applied, respectively. After five rinses (3, 3, 15, 5, and 5 min) with blocking buffer, the membranes were incubated with peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1:2000; Zymed, South San Francisco, CA, USA) for 1 h at room temperature. The membranes were rinsed again, utilizing the same protocol as above, and protein signals were detected using an ECL chemiluminescence system (Amersham, Chalfont, UK). For normalization, all the membranes were stripped and re-probed with affinity-purified goat polyclonal antibody to actin at the dilution of 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Additionally, competition studies of NHE2, NHE3, AE3, and NBCe1 series with their respective antigens, i.e. synthetic peptides, were performed to verify the specificity of these antibodies within the CNS (data not shown).

PCR primers. The specific primers for NHE3, NBCe1, and AE3 were designed with the aid of Primer 3.0 based on published sequences of corresponding mRNAs (Table 2).

Densitometry. Scanning densitometry of immunoblot films was performed on a Personal Densitometer SI scanner (Molecular Dynamics, Sunnyvale, CA, USA) and analyzed with the aid of ImageQuaNT image analysis software (Molecular Dynamics).

Semi-quantitative PCR. PCR reactions were conducted in 50 ␮l mixture containing 3 ␮l cDNA from RT, 50 mM potassium chloride, 20 mM Tris–HCl pH 8.4, 1.5 mM magnesium chloride, 0.2 mM for dNTP mix, 0.2 ␮M each 5⬘ and 3⬘ primers and 2U of TaqDNA polymerase (Invitrogen). For NBCe1 and AE3, the reaction was started at 94 °C for 4 min and amplified for 30 cycles of 30 s at 94 °C, 1 min at 60 °C and 1 min at 72 °C, finally extended at 72 °C for 10 min. For NHE3, the amplification profile is: 95 °C for 4 min, 30 cycles of 30 s at 94 °C, 30 s at 50 °C increasing with 2 °C for each of the first five cycles to 60 °C and 30 s at 72 °C, final extension at 72 °C for 10 min. The PCR products were separated on 2% agarose gel, visualized with ethidium bromide staining, captured and analyzed by Chemi Doc system (BioRad). ␤-Actin was used as an internal control to confirm equal loading of the samples. cDNA from kidney was chosen as positive control. The bands of predicted molecular weight for each PCR product were excised from the gels and purified using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA) for sequence analysis.

RT-PCR

Statistical analyses

Total RNA isolation and reverse transcription. Total RNA was extracted from brain samples with TRIzol reagent (Gibco BRL, Grand Island, NY) according to the manufacturer’s instructions. Briefly, the distinct brain tissues were homogenized with 1 ml of TRIzol reagent per 100 mg of tissue. Total RNA was separated from DNA and protein using a phenol/chloroform phase separation and the RNA was precipitated with isopropanol. The RNA pellet was then washed with 80% ice-cold ethanol, dried and dissolved in RNase-free water. The yield of total RNA was determined by measuring the absorption at 260 nm (GeneQuant RNA/ DNA calculator, Amersham Biosciences Corp., Piscataway, NJ, USA). Two micrograms of DNase-I treated total RNA was reverse transcribed to first strand cDNA in a 20 ␮l reaction volume which contained 500 ng of Oligo-(dT)12–18 primer, 200 units of Superscript II reverse transcriptase, 0.5 mM dNTP Mix, 10 mM DTT, 2U RNase inhibitor, and First Strand Buffer (Invitrogen). The mixture of Oligo-(dT)12–18 primer, total RNA, dNTP mix, and DEPC water was heated at 65 °C for 5 min first, chilled on ice for 2 min. Then other components were added and incubated at 42 °C for 50 min. The subsequent incubation at 70 °C for 15 min was used to inactivate the reverse transcriptase. Finally, 2 U of RNase H was added and incubated at 37 °C for 20 min to digest remaining RNA. Negative control reactions excluding reverse transcriptase were performed to demonstrate no contamination by genomic DNA in the RNA samples.

Data were represented as ratios of protein or mRNA to the corresponding actin and reported as means⫾S.E.M. (n⫽3– 6). Because n was relatively small we resorted to a non-parametric Wilcoxon rank sum test to determine significant differences of means between NHE1 mutant and age-matched wild type mice in four distinct brain regions. Differences in means were considered statistically significant if P⬍0.05.

RESULTS Protein expression of NHEs in NHE1 null mutant mouse CNS Lack of NHE1 protein expression in mutant mice. In the wild type mice, anti-NHE1 antibody detected a strong band at a molecular weight of 110 kDa (Fig. 1B). The expression of this protein (normalized to actin) appeared to be robust throughout all brain regions. The BD and CB exhibited relatively high protein levels compared with CX and HC (CB⬵BD⬎CX⬵HC). As anticipated, the NHE1 null mutant mouse showed no NHE1 expression in any CNS preparation. NHE2. Anti-NHE2 antibody recognized a band of 85 kDa in the CNS. The expression of NHE2 protein was

J. Xue et al. / Neuroscience 122 (2003) 37– 46

CX C

HC

N

C

N

CB C

C

C

N

80 kDa

NHE3

43 kDa

Actin

•

5

HC

CB

BD

REGIONS Fig. 2. Expression of NHE3 proteins in NHE1 null mutant mice. Immunoblotting was employed to compare protein levels of NHE3 between NHE1 null mutant (N) and age-matched wild type controls (C) in four brain regions: CX, HC, CB, BD. The upper panel represents immunoblots of NHE3 proteins and their corresponding actins. The molecular weight (kDa) of each protein is shown in the left column. The lower panel represents the results of densitometric analyses. The x axis shows the different brain regions. The y axis depicts the relative protein expression level, as a ratio of protein to its actin density per 30 ␮g of total membrane protein. Values are means⫾S.E.M. (n⫽5). Statistical significance (Wilcoxon rank sum test, P⬍0.05) is indicated by a dot.

generally low in the CNS, suggesting that NHE2 was not a major isoform of NHE in the brain. No obvious difference was observed between the wild type and NHE1 null mutant mice (data not shown). NHE3. Anti-NHE3 antibody revealed a band at a molecular weight of 80 kDa (Fig. 2, upper panel). Interestingly, the expression of NHE3 protein was found almost exclusively in the CB (Fig. 2, lower panel). There were only faint bands detected in other regions of the mouse CNS. These results are in agreement with our previous in situ hybridization study (Ma and Haddad, 1997). Within the CB, there was a significant increase (84%) of NHE3 protein expression in the null mutant relative to wild type controls. NHE4. NHE4 appeared as a band of 65 kDa in the CNS. For the wild type mice, the predominant expression of this protein (normalized to actin) occurred in the CB, followed by the CX, BD, and HC (CB⬎CX⬎BD⬎HC). Though the NHE1 null mutant had a decrease of NHE4 in the cortex and CB as well as an increase in the BD, no significant changes were observed between the wild type vs. NHE1 null mutant mice in any of the brain regions studied (data not shown). Protein expression of HCO3ⴚ transporters in NHE1 null mouse CNS NBCs detected by anti-RK-NBC antibody. The antiRK-NBC antibody detected a band—presumably reflecting a combination of NBCe1-A, NBCe1-B and/or NBCe1-C—

N

CB C

BD N

C

N RK-NBC Actin

•

C N

0.3

0.2

0.1

0.0

CX

C

43 kDa 0.4

C N

10

0

HC N

130 kDa

NBC / ACTIN (AU)

15

NHE3 / ACTIN (AU)

CX

BD N

41

CX

HC

CB

BD

REGIONS

Fig. 3. Expression of NBCe1 proteins that are recognized by anti-RKNBC in NHE1 null mutant and wild-type control mice. Immunoblotting was employed to compare protein levels between NHE1 null mutant (N) and age-matched wild-type controls (C) in four brain regions: CX, HC, CB, BD. The upper panel represents immunoblots of proteins recognized by antiRK-NBC and their corresponding actins. The molecular weight (kDa) of each protein is shown in the left column. The lower panel represents the results of densitometric analyses. The x axis shows the different brain regions. The y axis depicts the relative protein expression level, as a ratio of protein to its actin density per 30 ␮g of total membrane protein. Values are means⫾S.E.M. (n⫽6). Statistical significance (Wilcoxon rank sum test, P⬍0.05) is indicated by a dot.

with an apparent molecular weight of 130 kDa (Fig. 3, upper panel). This value was moderately higher than the predicted molecular weight of approximately 116 kDa for the three NBCe1 protein variants, presumably reflecting the glycosylation of the protein’s third extracellular loop (Choi et al., 2003). We found that, in wild-type controls, the anti-RK-NBC signal (Fig. 3, lower panel) was strongest in the CB and BD, with much less expression in the CX and HC (CB⬵BD⬎CX⬵HC). The NHE null mutants differed significantly from the controls only for the BD, where the detected protein level was 43% higher. NBCs detected by the anti-NBC(K1A) antibody. AntiNBC(K1A) detected a band—presumably reflecting a combination of NBCe1-A and/or NBCe1-B—with an apparent molecular weight of 130 kDa (Fig. 4A, upper panel). For wild-type controls, the regional expression profile for antiNBC(K1A) (Fig. 4A, lower panel) was similar to that for anti-RK-NBC, except that the BD signal was somewhat lower (CB⬎BD⬎CX⬵HC). NHE1 null mutation did not lead to statistically significant changes in any of the regions tested. NBCs detected by anti-NBC(B1B) antibody. AntiNBC(B1B) detected a band—representing the brainspecific NBCe1-C isoform—with a molecular weight of 130 kDa (Fig. 4B, upper panel). Compared with the proteins detected by the other two antibodies, the expression of NBCe1-C was more homogeneous among the brain regions for the wild-type controls (Fig. 4B, lower panel), though the CB still had the highest expres-

42

J. Xue et al. / Neuroscience 122 (2003) 37– 46

(A).

CX C

HC

N

C

CB

N

C

CX

BD N

C

130 kDa

Actin

N

C

N

CB C

BD N

C

N AE3

180 kDa 43 kDa

0.75

C N

AE3 / ACTIN (AU)

0.25

CX

HC

CB

BD

CX C

HC

N

C

N

CB C

N

BD C

C N

0.3 0.2 0.1 0.0

REGIONS

(B).

Actin

0.4

0.50

0.00

• CX

HC

CB

BD

REGIONS

N NBC(B1B)

130 kDa

Actin

43 kDa 0.75

B1B / ACTIN (AU)

HC

NBC(K1A)

43 kDa

K1A / ACTIN (AU)

C

N

C N

•

0.50

0.25

0.00

CX

HC

CB

BD

REGIONS

Fig. 4. Expression of NBCe1 proteins that are recognized by antiNBC(K1A) (A) and anti-NBC(B1B) (B) in NHE1 null mutant and wildtype control mice. Immunoblotting was employed to compare protein levels between NHE1 null mutant (N) and age-matched wild type controls (C) in four brain regions: CX, HC, CB, BD. The upper panels represent immunoblots of proteins recognized by anti-NBC(K1A) or anti-NBC(B1B) proteins and their corresponding actins. The molecular weight (kDa) of each protein is shown in the left column. The lower panel represents the results of densitometric analyses. The x axis shows the different brain regions. The y axis depicts the relative protein expression level, as a ratio of protein to its actin density per 30 ␮g of total membrane protein. Values are means⫾S.E.M. (n⫽6). Statistical significance (Wilcoxon rank sum test, P⬍0.05) is indicated by a dot.

sion level. As was the case in the anti-RK-NBC study, the NHE1 null mutants differed significantly from the controls only for the BD, where the NBCe1-C protein levels were 48% greater. AE3 Anti-AE3 antibody showed a band at molecular weight of 180 kDa (Fig. 5, upper panel). In the wild type mice, CX and BD had relatively high levels of AE3 protein compared with HC and CB (CX⬵BD⬎HC⬵CB; Fig. 5, lower panel). Except for the CB, an overall decline in AE3 protein expression was found in other brain regions. A 61% reduction in HC in null mutants was statistically significant relative to their wild type controls.

Fig. 5. Expression of AE3 proteins in NHE1 null mutant mice. Immunoblotting was employed to compare protein levels of AE3 between NHE1 null mutant (N) and age-matched wild type controls (C) in four brain regions: CX, HC, CB, BD. The upper panel represents immunoblots of AE3 proteins and their corresponding actins. The molecular weight (kDa) of each protein is shown in the left column. The lower panel represents the results of densitometric analyses. The x axis shows the different brain regions. The y axis depicts the relative protein expression level, as a ratio of protein to its actin density per 30 ␮g of total membrane protein. Values are means⫾S.E.M. (n⫽6). Statistical significance (Wilcoxon rank sum test, P⬍0.05) is indicated by a dot.

Expression of acid– base transporter mRNAs in NHE1 null mutant mouse CNS To test if the mRNA levels of acid– base membrane transporters were altered, RT-PCR analysis was performed with the isoform specific primers. NHE3. The sequence of the mouse NHE3 (274 bp) was partially cloned from duodenum (Praetorius et al., 2000). The primers and PCR conditions in the present work were adopted from Praetorius et al. (2000). A 229 bp transcript was detected in the CB. The data revealed that there was a 105% significant increase of NHE3 mRNA in the NHE1 null mutation as compared with controls (Fig. 6A). NBCe1. The primers for NBCe1 were designed in a way that the resulting PCR product encompassed the 97 bp deletion that distinguishes NBCe1-A/B on the one hand (product size: 325 bp) from NBCe1-C on the other (product size: 228 bp). A very modest but statistically significant increase of NBCe1 mRNAs (9% for NBCe1-A/B and 15% for NBCe1-C) was observed in the BD of NHE1 null mutants compared with wild type controls (Fig. 6B). AE3. A 320 bp PCR product was amplified with the specific primers of mouse AE3. Our data show that there is a 24% significant decrease in AE3 mRNA in NHE1 null mutation relative to control in the HC (Fig. 6C). The sequences of the PCR products were analyzed and showed 96 –98% homology with their corresponding published mRNAs.

J. Xue et al. / Neuroscience 122 (2003) 37– 46

(C) Hippocampus C

(A) Cerebellum C

43

N

N

229 bp

NHE3

320 bp

AE3

392 bp

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392 bp

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15000 Integrated Density Value

Integrated Density Value

7500

5000

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10000

5000

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(B) Brainstem-Diencephalon C

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325 bp 228 bp

RK- and K1A-NBC B1B-NBC

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B1B-NBC Integrated Density Value

RK- and K1A-NBC Integrated Density Value

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0 C

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Fig. 6. mRNA expression of acid– base transporters in NHE1 null mutants (N) relative to wild type age-matched controls (C). RT-PCR was performed to assay mRNA levels. (A) NHE3 in CB; (B) NBCe1 in BD; (C) AE3 in HC. The upper panel represents PCR bands of acid– base transporters and their corresponding actins. The molecular weight (bp) of each PCR product is shown in the left column. The lower panel shows the results of densitometric analyses. The x axis represents the controls (C) vs NHE null mutants (N). The y axis depicts the integrated density values of the PCR bands. Values are means⫾S.E.M. (n⫽3). Statistical significance (Wilcoxon rank sum test, P⬍0.05) is indicated by an asterisk.

In summary, the expression of NHEs and HCO3⫺dependent transporters were region- and subtype-specific in the mouse CNS. For instance, NHE3 protein was predominantly localized in the CB. The NBCe1 proteins recognized by anti-RK-NBC, anti-NBC(K1A) and antiNBC(B1B) had similar regional expression pattern, and their levels in CB and BD appeared to be greater than in CX and HC. The NHE1 null mutants caused an elevation of NHE3 in the CB, an increase of the NBCe1 series in BD, as well as a decrease of AE3 in the HC at both mRNA and protein levels compared with wild type controls.

DISCUSSION The major findings in the current study were: (1) the expression of acid– base membrane transporters show spa-

tial heterogeneity within the CNS; and (2) the expression of acid– base transporters in the NHE1 null mutant is altered in a region- and isoform-specific manner, occurring at both transcriptional and translational levels. NHE1, the “housekeeping” and ubiquitously expressed NHE, mediates an electroneutral 1:1 exchange of intracellular H⫹ for extracellular Na⫹, and in so doing regulates pHi homeostasis and cell volume (Wakabayashi et al., 1997; Counillon and Pouyssegur, 2000; Putney et al., 2002). In 1997, Cox et al. reported that a spontaneous point mutation introduces a stop codon in the coding sequence of NHE1, resulting in a truncated, inactive transporter. Our genotyping and immunoblotting data confirmed that no NHE1 was expressed in these mutant mice. The NHE1 null mutation is not lethal and the mutant mice have

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J. Xue et al. / Neuroscience 122 (2003) 37– 46

no obvious acid– base disorder, or kidney or intestinal dysfunction, suggesting that the expression of compensatory pH-regulatory systems, such as bicarbonate-dependent transporters, may take place in these mice. However, these mutants do show epilepsy and selective neuronal death in the CB and brainstem. Therefore, we argued that compensation may not be sufficient in the mutant CNS, or if there were any compensation in the CNS of NHE1 mutants, this compensation would be restricted to specific regions. So far our electrophysiological and optical data (Yao et al., 1999) have revealed that, in hippocampal CA1 neurons, the absence of NHE1 causes a lower steadystate pHi and a markedly reduced recovery rate from an acid load, which becomes even worse in HCO3⫺ solutions. These data indicate that the ability of maintaining acid– base balance is jeopardized in the mutant hippocampal neurons. The present data in this paper have shed some light on the endowment of mutant cells in terms of pH regulatory membrane proteins. For instance, we have shown that the AE3 was down-regulated in the mutant HC. AE3, a brain isoform of the AE family, operates as an acid loader and facilitates electroneutral exchange of Cl⫺ and HCO3⫺ across the plasma membrane. By virtue of its activity, the AE3 protein contributes to the regulation of pHi, [Cl⫺] and cell volume (Kopito, 1990; Sterling and Casey, 1999; Alper et al., 2001). Although we do not have evidence for the functional significance of the reduced expression of AE3 protein in mutant hippocampal neurons, this down-regulation of AE3 could compensate for the loss of acid extruder activity of NHE1. With this reduction in AE3, one would anticipate a decreased ability of the mutant neuron to acid load at higher pHi, which matches well with its decreased ability to increase pHi because of the null NHE1 activity. Brain regions other than the HC also seem to have alterations in other membrane proteins. For example, the NHE3, which is primarily expressed in the CB, is increased in the mutant CB in a major way. This up-regulation of cerebellar NHE3 protein is particularly important since in the CB, especially in the Purkinje cells which are enriched with NHE3 (Ma and Haddad, 1997), NBCe1 immunoreactivity is absent (Schmitt et al., 2000) and the recovery from an acid load appears to depend solely on Na⫹/H⫹ exchangers rather on HCO3⫺ dependent transporters (Gaillard and Dupont, 1990). In contrast to the CB, the BD in the mutant has increased Na⫹/HCO3⫺ cotransporters. However, no apparent changes were observed in the expression of the other NHE isoforms or Na⫹/HCO3⫺ cotransporters in the cortex and HC of mutant mice. Hence, the alterations in the expression of these membrane proteins, and in turn, the ability of these neurons to respond to various stresses, depend on the type of nerve cell being affected. The reasons for the specificities in terms of alterations in the NHE1 null mutant neuron are not clear, but are not part of the scope of this work. In this work, we showed that in the absence of NHE1, the expression of a number of membrane transporters was altered at both protein and mRNA levels. Regarding the respective changes in mRNAs and proteins, it is worth

noting that up-regulation of NHE3 in the NHE1 null mouse showed a similar increase for mRNA and protein. It indicates that these changes may have been initiated at the transcriptional level. However, for the bicarbonate transporter family, such as sodium-bicarbonate cotransporters and AE3, the changes in their mRNAs were much smaller than those in their respective proteins. This suggests to us that NHE1 null mutation affects the expression of these transporters not only at a transcriptional level but also at a translational level. This latter could be related to an increased efficiency of the translational process, an increased stability of mRNAs, or a decreased degradation of the proteins. It is not clear at present how the NHE1 null mutation influences the mRNA expression of other acid– based membrane transporters. From other perspective in terms of the protein alterations in the NHE1 mutant, it has been found that NHE1 interacts with actin-binding proteins of the ezrin, radixin, moesin family (Denker et al., 2000), and NHE3 is linked to ezrin through adapter proteins such as NHE regulatory factor and NHE3 kinase A regulatory protein (Lamprecht et al., 1998). Furthermore, the AE3 protein associates with the cytoskeleton anchor protein ankyrin (Morgans and Kopito, 1993). Therefore, it is interesting to speculate that the NHE isoform 1 may interact with other proteins in the submembrane cellular compartment. The lack of NHE1 protein in a protein complex moiety may disrupt the stability of the whole protein complex, leading to modifications in the levels of other proteins. Another interesting phenomenon observed in this study is that the BD appears to be a rather sensitive region in the NHE1 null mutant animal. For example, the NHE1 null mutation caused the NBCe1 proteins detected by antiRK-NBC, as well as the NBCe1-C protein detected by anti-NBC(B1B), to increase in a major way in the BD. A unifying hypothesis for the data obtained with the three NBCe1 antibodies is that it is predominantly the brainspecific NBCe1-C variant that increases in the NHE1 null mutant. As expressed in Xenopus oocytes, all three NBCe1 variants have an apparent Na⫹:HCO3⫺ stoichiometry of 1:2 (Bevensee et al., 1996; Choi et al., 1999; Romero et al., 1998), which causes them to mediate a net influx of HCO3⫺ and thus function as acid extruders (i.e. mediating the extrusion of acid equivalents). As normally expressed in renal proximal tubules, NBCe1-A functions with an apparent Na⫹:HCO3⫺ stoichiometry of 1:3, which thus causes the transporter to mediate a net efflux of HCO3⫺ and thus function as an acid loader. However, this is the only known example in which a naturally expressing NBCe1 variant operates with a 1:3 stoichiometry. Thus, it is likely that the NBCe1 variants expressed in the brain generally mediate the net uptake of HCO3⫺ and, like NHE1, function as acid extruders in cells such as astrocytes (Bevensee et al., 1997a,b). To the extent that NHE1 and an acid-extruding NBCe1 are present in the same cell, this coexpression is an illustration of redundancy in a critically important control system (i.e. pHi regulation). Thus, it would not be surprising if such a cell were to

J. Xue et al. / Neuroscience 122 (2003) 37– 46

respond to a NHE1 knockout mutation by increasing the amount of NBCe1 protein. The implications of the NHE1 null mutation for cellular function and the consequential changes of other acid– base transporters are not clear at present. For example, the gene encoding NBCe1 is only one of at least five that are known to encode Na⫹-coupled HCO3⫺ transporters that mediate net HCO3⫺ uptake (i.e. acid extrusion). It will be interesting to learn whether the NHE1 null mutation causes increases in these other transporters: NBCe2 (or NBC4, a second electrogenic Na⫹/HCO3⫺ cotransporter), NBCn1 (an electroneutral Na⫹/HCO3⫺ cotransporter), NDCBE (a Na⫹-driven Cl⫺-HCO3⫺ exchanger), and NCBE (whose function is controversial, but may be either a Na⫹driven Cl⫺-HCO3⫺ exchanger or an electroneutral Na/ HCO3 cotransporter). Such studies will require the explicit molecular definition of individual acid– base transporters, as well as specific pharmacological agents or other approaches for specifically blocking these transporters. Another example pertains to the Cl⫺/HCO3⫺ exchanger, with the decrease in AE3 expression, it is possible that the extracellular pH might be reduced in the NHE1 null mutants. Whether this is beneficial or not can not be certain since this potential effect depends on the level of extracellular pH. It is interesting to note, however, that NHE3 increased its expression in the NHE1⫺/⫺ CB and, in the brain, NHE3 is mostly expressed in the CB (Ma and Haddad, 1997; current data). Furthermore, there is cell death in the deep cerebellar nuclei in the NHE1 null mutants and these mice suffer from cerebellar ataxia (Cox et al., 1997). How the increase in NHE3 or other consequential changes are related to the cerebellar pathology is not clear. In summary, we demonstrate in this work that in the NHE isoform 1 null mouse, by the age of 4 weeks, a time when the CNS phenotype is fully apparent, there are 1) major alterations in expression of acid– base membrane transporters that can affect pH and cell volume regulation, 2) these alterations occur at both transcriptional and translational levels, and 3) the changes show major regional variability in the CNS, which suggests that these transporters may play distinct roles in various brain regions. Acknowledgements—We would like to thank Hillary Sunamoto and Cate Muenker for the assistance with this project. This work was supported by NIH grants PO1HD32573 and RO1HL66327.

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(Accepted 25 July 2003)