Increased neuronal excitability and seizures in the Na+/H+ exchanger null mutant mouse

Xiang Q. Gu1, Hang Yao1, and Gabriel G. Haddad1,2

Departments of 1 Pediatrics (Section of Respiratory Medicine) and 2 Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mice lacking the Na+/H+ exchanger isoform 1 (NHE1) manifest neurological diseases that include ataxia, motor deficits, and a seizure disorder. The molecular basis for the phenotype has not been clear, and it has not been determined how the lack of NHE1 leads, in particular, to the seizure disorder. We have shown in this work that hippocampal CA1 neurons in mutant mice have a much higher excitability than in wild-type mice. This higher excitability is partly based on an upregulation of the Na+ current density (608.2 ± 123.2 pA/pF in NHE1 mutant vs. 334.7 ± 63.7 pA/pF in wild type in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2). Alterations in Na+ channel characteristics, including steady-state inactivation (shift of 18 mV in the depolarization direction in the mutant), recovery from inactivation (tau h = 5.22 ± 0.49 ms in wild-type neurons and 2.20 ± 0.20 ms in mutant neurons), and deactivation (at -100 mV, tau d = 1.75 ± 0.53 ms in mutant and 0.21 ± 0.05 ms in wild-type neurons) further enhance the differences in excitability between mutant and wild-type mice. Our investigation demonstrates the existence of an important functional interaction between the NHE1 protein and the voltage-sensitive Na+ channel. We hypothesize that the increased neuronal excitability and possibly the seizure disorder in mice lacking the NHE1 is due, at least in part, to changes in Na+ channel expression and/or regulation.

sodium channels; sodium-hydrogen exchanger mutant mice


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MICE LACKING the Na+/H+ exchanger isoform 1 (NHE1) have a specific, easily recognizable phenotype (3, 7). These mice show ataxia, have recurrent seizures by the age of 2-3 wk, and 60-70% of them die early. Seizures are frequent (up to 120/h), and electrocorticographic recordings consist of generalized, bilaterally symmetrical 3- to 4-Hz spike-wave activity. Studies so far have not identified the reason(s) for these phenotypic manifestations, including seizures, in the NHE1 null mouse.

That a mutation in a membrane protein, such as NHE, leads to seizure activity is not surprising. Indeed, a number of mutations in various membrane protein genes, such as the Ca2+ channel beta -subunit (Cchb4) or the voltage-gated K+ channel subunit (Kv1.1), have been shown to produce convulsions (1, 5, 6, 10, 25). Furthermore, mutations in ion channel proteins, including the voltage-sensitive Na+ channel and a Ca2+ channel (CACNA1A), have been identified to cause myotonia and ataxia (2, 23). Of interest to this work is that the ataxia of humans suffering from a mutation in CACNA1A is responsive to acetazolamide and pH changes (2).

Although a mutation in the NHE1 protein could conceivably result in the development of a seizure disorder, it is not readily apparent how a mutation in a protein responsible for a nonelectrogenic exchange between Na+ and H+ can lead to such a disorder. If Na+/H+ exchange were eliminated, one could hypothesize that there would be a major drop in intracellular pH (pHi), which, in turn, could alter the function of a number of channels and possibly lead to changes in neuronal excitability. However, recent studies from our laboratory on CA1 hippocampal neurons did not show any major shift in resting pHi (24). Surprisingly, in these same studies, we identified in the NHE null mutant CA1 cells abnormalities other than in the NHE protein. These abnormalities consisted of alterations in expression or regulation of membrane proteins whose activity depended on the presence of extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, e.g., the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (20, 24). Therefore, in light of our previous findings on NHE mutant nerve cells (20, 24), we have asked two questions about these mice in regard to hippocampal neurons. First, are the NHE null CA1 hippocampal neurons more excitable than the wild-type CA1 neurons in such a way that the increased excitability predisposes mice to seizure? And second, if this is the case, what is the cellular or molecular basis for this increase in neuronal excitability in the null cells? Although the repetitive seizures that these mice exhibit are generalized in nature, we focused our work on cells from the hippocampus for several reasons. For example, it is well known that the hippocampal formation participates in, propagates, or initiates seizures. Furthermore, hippocampal neurons have been well studied and characterized by many groups of investigators including us (4, 8, 17, 18). Finally, the NHE1 is highly expressed in the hippocampus (17, 20), and hence abnormalities that ensue from the NHE mutation would have a high likelihood of being detected.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NHE1 Mutant Mice Genotyping

We performed genotyping on all presumed mutant mice to confirm the phenotype. Genomic DNA was obtained from mice tails and used for PCR amplification with the primers 5'-TCGCCTCAGGAGTAGTGATGCG-3' (sense) and 5'-CGTCTTGTGCAGGGCATGA-3' (antisense), respectively, corresponding to base pairs 1397-1418 and 1800-1819 of mouse NHE1 cDNA sequence (accession no. U51112). The amplification program was set at 94°C for 1.5 min, 60°C for 1 min, and 72°C for 1.5 min for 30 cycles. DNA was subjected to the endonuclease Spe1 to differentiate between wild-type and homozygote mutant genotype. The basis for the difference in band pattern was a single nucleotide difference (change from A in the wild type to a T in the mutant, resulting in a TAG and a stop codon). This transforms the surrounding sequence from CAACAAGTTCC to CAACTAGTTCC. Spe1, which cuts in between A and C in the sequence ACTAG, cleaves the mutant molecule (2 bands) but not the wild type (1 band).

Preparation of CA1 Cells

B6SJL,+/swe (slow-wave epilepsy) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) (7). These heterozygous mice were mated in our institution, and the resulting homozygous NHE1 mutant (25%) and wild-type (25%) F1 mice progeny were used. Although homozygous mutant mice had a clear phenotype consisting of locomotor ataxia in the hindlimbs and a slow, wide-based gait and coarse truncal instability starting at ~2-3 wk of age, a PCR-based test was always performed to confirm the genotype (see below). Mice 21-30 days old were used, and their hippocampi were removed and sliced into 7-10 transverse 400-µm-thick sections. The slices were immediately transferred to a container with 25 ml of fresh, oxygenated, and slightly stirred HEPES buffer at room temperature. After 30 min of exposure to trypsin (0.08%) and 20 min of protease (0.05%) digestion, the slices were washed several times with HEPES buffer and left in oxygenated solution. The CA1 region was then dissected out and triturated in a small volume (0.25 ml) of HEPES buffer. The Yale Animal Care and Use Committee approved these studies.

Solutions

For the current-clamp experiments, the external HEPES solution-bathing neurons contained (in mM) 130 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose adjusted to pH 7.4 with NaOH. The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution contained 125 NaCl, 3.1 KCl, 1.25 NaH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose bubbled with 5% CO2 and 95% O2. The pipette solution for whole cell patch electrodes contained (in mM) 138 KCl, 0.2 CaCl2, 1 MgCl2, 10 HEPES (Na+ salt), and 10 EGTA adjusted to pH 7.4 with Tris. Neither ATP nor GTP was added to the pipette solution. The external HEPES and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solutions for the voltage-clamp experiments contained the same salts as those for the current-clamp experiments except for 10 mM tetraethylammonium, 5 mM 4-aminopyridine, and 0.1 mM CdCl2 that replaced equimolar concentrations of NaCl. The internal pipette solution for the whole cell voltage-clamp experiments was also similar to the internal solution for the current-clamp experiments, except for CsF or CsCl instead of KCl. The HEPES-buffered solutions for the enzymatic preparation and trituration of the CA1 cells contained (in mM) 125 NaCl, 3 KCl, 1.2 MgSO4, 1.25 NaH2PO4, 30 HEPES, and 10 glucose. Osmolarity of all solutions was adjusted to 290 mosmol/kgH2O. All chemicals were purchased from Sigma.

Recording Criteria

Morphological criteria. CA1 cells were used if they had a smooth surface, a three-dimensional contour, and were pyramidal in shape. Similar criteria have been used by us (9) and others (16) on freshly triturated neurons. The CA1 cells studied were obtained from 21- to 30-day-old mice.

Electrophysiological criteria. 1) Neurons were considered for recording if the seal resistance was >5 GOmega . 2) Only neurons with a holding current of <0.1 nA (command potential -100 mV) were used in the study. 3) Series resistance was <10 MOmega in neurons studied. The series resistances were compensated at a 90% level with the Axopatch 1C amplifier (Axon Instruments). Under these conditions, the error caused by uncompensated series resistances was <3 mV. To obtain adequate voltage clamp and minimize the space-clamp problem, only small neurons with short processes were used in Na+ current amplitude (INa) measurements. In addition, only cells with current-voltage curves that were smoothly graded over the voltage range of activation (approximately -50 to -10 mV) were used, as we have done in the past (9, 18).

Electrophysiological Recording

Electrodes were pulled on a Flaming/Brown micropipette puller (P-87; Sutter Instrument) from filamented borosilicate capillary glass (1.2-mm outer diameter, 0.69-mm inner diameter; World Precision Instruments). The electrodes were fire-polished, and resistances were 2-5 MOmega for voltage-clamp experiments and 7-9 MOmega for current-clamp experiments in the above-mentioned solutions. Action potentials (APs) were recorded in the current-clamp mode, and input resistance (Rm) was measured at -70 mV as a slope of the current trace evoked by a ramp voltage from -160 to 100 mV in the voltage-clamp mode. Least-squares regression analysis for 100 data points was performed to derive a relationship between voltage and current. Membrane potential (Vm) was measured in the current-clamp mode with no holding current. Current traces in voltage clamp were leak subtracted. Junction nulls were performed for each individual cell with the Axopatch 1C amplifier. One-tailed Student's t-test was performed for mean comparisons. Numbers in the text were given as means ± SE of the mean. All recordings were performed at room temperature (22-24°C).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neuronal Properties and Membrane Excitability in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2

All CA1 neurons fired APs in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution when they were held at -75 mV and given depolarizing currents in the current-clamp mode. However, the mutant CA1 neurons were more excitable than the wild-type ones, and the rheobase was substantially smaller in the NHE mutant than in the wild-type neurons (Fig. 1, A-C). For example, to generate one AP, 64.0 ± 15.9 pA (n = 16) was required in the wild-type neurons, but only 20.3 ± 3.5 pA (n = 8, P = 0.04) was needed in the mutant neurons (Fig. 1C). Wild-type CA1 neurons also had a more hyperpolarized Vm (-52.8 ± 3.0, n = 21, in wild type vs. -44.6 ± 3.2, n = 13, in mutant, P = 0.04; Fig. 1D) and a 30% lower Rm (402.0 ± 96.3 MOmega , n = 7) than in mutant neurons (546.2 ± 132.8 MOmega , n = 6). Hence, the differences in excitability between mutant and wild type in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution could at least be partly explained by alterations in neuronal properties such as Vm and Rm.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Voltage traces with evoked action potentials (APs) in wild-type (A) and Na+/H+ exchanger isoform 1 (NHE1) mutant (B) neurons, mean rheobase (C), and membrane potential (Vm; D) in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. In A and B, evoked APs were collected in the current-clamp mode with 10 depolarizing currents, starting with 5 pA and using 5-pA increments. Voltage traces in A and B start at the top and follow downward incrementally. In C, the minimum currents used to evoke an AP were averaged from 16 wild-type and 8 NHE mutant neurons in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. In D, Vm was collected in current clamp with no holding current (n = 21 and 13 for wild-type and NHE1 null mutant neurons, respectively). *Statistical difference at P = 0.04 for both C and D.

Neuronal Properties in HEPES

On the basis of our previous findings in which we showed that the differences between wild-type and mutant mice were related to the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 in the bathing solution, we also studied these cells in HEPES-containing solutions and removed all HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. These studies allowed us to determine whether the differences between the two groups of neurons were related to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent mechanisms. Interestingly, Vm and Rm were almost identical in wild type and mutant, and the differences in Vm and Rm identified in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution were totally eliminated in HEPES (Fig. 2). Indeed, there was no significant difference in Vm and Rm between wild type and mutant (Vm was -42.3 ± 2.0 mV, n = 19, and -41.3 ± 2.1 mV, n = 21; Rm was 608.3 ± 329.5 MOmega , n = 11, and 503.7 ± 85.2 MOmega , n = 23 for wild type and mutant, respectively) in HEPES solution. Intriguingly, however, CA1 neurons in the NHE null mutant were still much more excitable than wild-type neurons, in spite of the lack of difference in Vm and Rm. In fact, the rheobase in the wild-type neurons was double that of the NHE1 mutant neurons in HEPES (24.8 ± 4.8, n = 12, vs. 12.9 ± 2.0, n = 6, P = 0.05; Fig. 2A).


View larger version (6K):
[in this window]
[in a new window]
 
Fig. 2.   Rheobase (A) and Vm (B) in HEPES. In A, the minimum currents used to evoke an AP were averaged from 12 wild-type and 6 NHE mutant neurons in HEPES. In B, Vm was collected in current clamp with no holding current (n = 19 and 21 for wild-type and NHE1 null neurons, respectively). *Statistical difference at P = 0.05 for A.

Na+ Current Magnitude

Although the higher excitability of mutant neurons compared with wild-type neurons could be explained, at least in part, by the differences in Vm and Rm in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, the same reasoning could not be used in HEPES conditions because the differences in passive properties between the two groups were not observed. Hence, we examined other properties, such as the Na+ channel properties in both mutant and wild type to uncover the basis for the increased excitability in the mutant. When we held CA1 neurons at -130 mV, depolarizing pulses to -20 mV evoked an inward current that reached a peak in less than a millisecond and decayed quickly to zero current. TTX (1 µM) blocked the current almost totally. On the basis of its voltage dependency, characteristics of fast activation and inactivation, and TTX sensitivity, we considered this to be a voltage-sensitive fast Na+ current.

In HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, the average peak Na+ current in NHE mutant neurons was 3.0 ± 0.6 nA (n = 15). The wild-type neurons had a much smaller peak Na+ current of 1.3 ± 0.3 nA (n = 18, P = 0.01; Fig. 3A). Because the difference in current magnitude between both groups of neurons could be related to neuronal size, we controlled for surface area. The Na+ current density was also much larger in the mutant (608.2 ± 123.2 pA/pF, n = 15) than in the wild type (334.7 ± 63.7 pA/pF, n = 18, P = 0.02; Fig. 3B). Hence, the magnitude of the Na+ current was larger in the mutant than in the wild type, and this provided an important explanation for the abnormally high excitability in the mutant. Interestingly, a similar major difference in peak Na+ current between mutant and wild type was also found in HEPES with a Na+ current density of 626.1 ± 78.2 pA/pF (n = 25) in the NHE mutant CA1 neurons compared with 423.9 ± 55.3 pA/pF (n = 22, P = 0.02) in the wild type.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 3.   Na+ current amplitude (INa; A) and Na+ current density (B) in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. Peak Na+ currents were obtained in voltage clamp, and current densities were derived from the ratio of peak Na+ current over whole cell capacitance. n = 18 and 15 in A and B for wild-type and NHE1 null mutant neurons. *Statistical difference at P = 0.01 for A and P = 0.02 for B.

Na+ Current Characteristics

We next examined the characteristics of the Na+ current to determine whether there are other differences between the mutant and the wild type that could be important in determining excitability.

Activation. With CA1 neurons held at -130 mV, depolarizing voltages were given from -70 to +80 mV with 10-mV increments. For both the wild-type and the NHE null neurons, the threshold for Na+ channel activation in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 was -60 mV, and the Na+ current reached a peak at approximately -30 mV. The midpoint for the voltage-conductance relationship was almost the same in both groups [-50.35 mV (slope factor = 4.06) for wild-type neurons, n = 7; -48.23 mV (slope factor = 5.40) for NHE1 null neurons, n = 6].

Steady-state inactivation. Steady-state inactivation of the Na+ current was studied using a prepulse potential from -130 to -20 mV and then stepping Vm to -20 mV in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. Although there was no difference in the activation kinetics between mutant and wild-type neurons, there was a major difference in the steady-state inactivation characteristics (Fig. 4, A-C). Figure 4C shows the Boltzmann fit of the relationship between the ratio of a peak current at a prepulse potential to the maximum current of all peak currents (I/Imax) and the prepulse potential as well as the relationship between the ratio of a conductance at a Vm to the maximum conductance of all conductances (g/gmax) and Vm. The half voltage of steady-state inactivation (hinfinity 1/2) was -82.52 mV (slope factor = 7.79, n = 7) and -64.98 mV (slope factor = 6.37, n = 6) for the wild-type and mutant neurons, respectively. Therefore, since there was no difference in activation but a major difference in the steady-state inactivation between wild-type and mutant neurons, the NHE mutant neurons seemed to have a much larger window current than wild-type neurons (Fig. 4C).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Steady-state inactivation of the Na+ current in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. In wild-type (A) and NHE1 null mutant neurons (B), current traces were collected at -20 mV from prepulse potentials from -130 to -20 mV with an increment of 10 mV and a duration of 502 ms. In C, steady-state inactivation curves were plotted as normalized current (I/Imax) against prepulse potential and fitted by a Boltzmann equation for the averaged, normalized current against the prepulse potentials (n = 6 and 6, for wild-type and NHE1 null mutant neurons, respectively). Na+ current activation is also shown in C. Activation was derived from current traces (collected from -70 to +80 mV with a 10-mV increment from a holding potential of -130 mV in the voltage-clamp mode) divided by the driving force in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. Curve fitting in C was obtained from the averaged data using the Boltzmann equation; n = 7 and 6 for wild-type and NHE1 null neurons, respectively; m, activation curve; h, steady-state inactivation curve; g/gmax, normalized conductance. Scales in A and B, 50 pA/pF and 10 ms.

Recovery from inactivation. To examine the recovery from inactivation, we used a two-pulse protocol, as shown in Fig. 5. In HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution, the recovery from inactivation in NHE null mutant neurons was much faster than in wild-type CA1 neurons. For example, if two pulses were 2.7 ms apart, the ratio of the second peak Na+ current over the first was 0.29 ± 0.03 (n = 6) for the wild-type neurons and 0.45 ± 0.06 (n = 4, P = 0.02) for the mutant neurons (Fig. 5C). The time constant (tau h) for recovery in the wild type was, in fact, more than double that of the mutant neurons (5.22 ± 0.49 ms, n = 6 and 2.20 ± 0.20 ms, n = 4, P = 0.001).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Traces representing recovery from inactivation of the Na+ current in wild-type (A) and NHE1 null (B) neurons in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. The two-pulse voltage protocol was shown under the traces in A. Two identical pulses were delivered with increasing intervals (t) in between each pair of pulses. C: ratio (IpeakE/IpeakC) of the current evoked from the second pulse over the one evoked from the first was plotted against interval (t in ms) in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 for wild-type (n = 6) and NHE1 null (n = 4) neurons. Except for the one comparison at t = 29.7 ms, all data points in wild type were statistically smaller than those of NHE1 mutant neurons. Scales in A and B, 50 pA/pF and 10 ms.

Deactivation characteristics. In addition, we examined the deactivation properties, i.e., the transition from the open to the resting closed state for both mutant and wild-type neurons in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution. We held CA1 neurons at -100 mV, depolarized them for 1 ms to -10 mV, and repolarized them to -70 or -100 mV (Fig. 6A, inset). The average tau h for deactivation, tau d at -100 mV, was 4 to 8 times smaller for the wild-type neurons (0.21 ± 0.05 ms, n = 5) than for the mutant neurons (1.75 ± 0.53 ms, n = 4, P = 0.007; Fig. 6). At -70 mV, similar results were obtained. Hence, in physiological HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, there were major differences between mutant CA1 neurons and wild type, not only in the magnitude of the Na+ current but also in its characteristics.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Traces representing deactivation of the Na+ current in wild-type (A) and NHE1 null (B) neurons in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. The voltage protocol is shown under the trace of A. C: time constants for deactivation (tau d) are plotted for the 2 groups (wild type, n = 5; NHE1 null, n = 4). *Statistical difference at P = 0.007 and P = 0.02 for tau d at -100 and -70 mV, respectively. Scales in A and B, 200 pA and 10 ms.

Na+ current characteristics in HEPES. It is interesting to note that not all the differences between wild-type and mutant cells that were present in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 persisted in HEPES. For example, the differences in peak Na+ current between wild-type and mutant neurons persisted, and the mutant still showed a much larger current density in both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 and HEPES. Also, tau d at -100 mV was severalfold smaller in wild- type (0.18 ± 0.01 ms, n = 5) than in mutant neurons (1.22 ± 0.46 ms, n = 4, P = 0.02). Other differences were much more apparent in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 and did not persist in HEPES. For instance, in HEPES, the steady-state inactivation characteristics were almost identical between wild-type and mutant neurons (midpoints -62.89 mV, slope factor 6.00, n = 5, and -64.98 mV, slope factor 6.37, n = 6, respectively).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have made two major observations. First, we have shown that NHE1 mutant neurons are much more excitable than wild-type neurons. Second, we have uncovered one of the putative mechanisms for the increased excitability in these mutant neurons.

It is clear from our data that CA1 hippocampal neurons are much more excitable in the mutant than in the wild-type mouse. Although we have presented data mainly on evoked generation of action potentials in these cells, even spontaneous activity was more readily observed in mutant than in wild-type neurons. At no time did we find any mutant cell that was less than or equally excitable to the wild type. Because mutant neurons were more depolarized and had a higher input resistance than wild-type neurons in physiological solutions containing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, we first thought that the main reason for the increased excitability in the mutant neurons was related to the differences in Vm and Rm between the two groups of neurons. However, when cells were studied in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, differences in these passive properties no longer existed, but differences in excitability persisted between wild-type and mutant neurons. Hence, we sought other reasons to explain the increased excitability in the mutant neurons. Although there is a number of important questions that stem from these findings, one of these would be whether the firing threshold is lower in the mutant neurons because of an alteration in expression or regulation of the voltage-sensitive Na+ channels. Our data clearly show that there were at least two major differences in the Na+ channels between the mutant and the wild-type neurons, one being related to the size of the current itself and the other one being related to the properties of the channel. The increased current density in the mutant neuron would indicate that the mutant has an increase in the single-channel conductance, an increase in the open probability of the channel, or an increase in the expression of the Na+ channel protein in the neuronal membrane. The changes in the characteristics of the Na+ channels strongly suggest that these channels are more available for recruitment in the steady state, recover to the closed state after inactivation at a faster rate, and stay in the open state longer than the wild type before they transition to the closed state after a brief period of activation. These major differences lead us to believe that the increased Na+ current density in the mutant neurons occurs either on the basis of alterations in the Na+ channel regulation or an increased expression of the protein itself, or both.

In physiological solutions containing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, there seems to be multiple causes for the increased neuronal excitability in the NHE1 mutant cells. Namely, mutant neurons are more depolarized, have higher input resistance, and have abnormally high Na+ current and abnormal Na+ current characteristics. Although it is clear that the differences in the Na+ current between mutant and wild-type neurons are related to alterations in the Na+ channel, the differences in Vm and Rm are probably related to changes in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent mechanisms (15, 24). These data are indeed consistent with our previous findings (24) indicating that NHE1 mutant nerve cells cannot recover as well as wild-type neurons from an acid load, not only in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 but actually in the presence of mainly HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2.

Of interest also is that there were electrophysiological differences between HEPES solution and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution in both wild-type and mutant cells. For example, the rheobase for wild-type and NHE1 mutant nerve cells was higher in the presence than in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. This was mainly caused by a shift in both the voltage-conductance curve and the steady-state inactivation curve to the left in such a way that the window current was smaller, i.e., the availability of Na+ channels in the range of -120 to -40 mV was decreased in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 (15).

Our results in this study demonstrate that the lack of NHE1 has major implications on nerve cell function not only through Na+ and H+ exchange. This work shows that the absence of the NHE1 protein moiety itself may lead to abnormalities in other membrane proteins. In this regard, we hypothesize that interactions exist between NHE1 and an electrogenic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent transporter (e.g., Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with 1:2 stoichiometry) and, as this work demonstrates, between NHE1 and the voltage-sensitive Na+ channel. Supporting this idea are other observations from our laboratory demonstrating that the expression of the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter is decreased in the NHE mutant hippocampus compared with those in the wild type (12). Hence, we would like to propose the notion that the absence of NHE1 protein disturbs the interactions between certain specific proteins that form a functional complex in the neuronal membrane or their functional regulation. Of relevance to this idea is the fact that the NHE regulatory factor (NHERF), which has PSD-95/Dlg/ZO-1 (PDZ) domains, has been shown to "organize" membrane proteins, including the NHE3 protein, into functional units (14). Whether this occurs among NHERF, NHE1, and the voltage-sensitive Na+ channels is not known at present. Such interactions in neurons are analogous to those in epithelial cells between the cystic fibrosis transmembrane conductance regulator, an outward rectifier Cl- channel, and the epithelial Na+ channel (13, 14, 19, 21, 22).

Another important issue to highlight in this model is that the phenotype of abnormal gait and seizures appears only after a few weeks from birth. Because of this, we raise the issue of whether the lack of NHE1 affected central neurons in the first few weeks of life by altering the function of other membrane proteins (such as the Na+ channel), leading to abnormal cell function and excitability. While this is certainly possible, we favor other explanations for at least two reasons: 1) the NHE1 is normally (in wild type) very minimally expressed in the first 7-14 days of life (12), and 2) our data on pHi in mutant vs. wild-type cells at 3-4 wk of age did not show major differences (<0.1 pHi unit) (24).

An alternative explanation for our results is that the lack of NHE1 in neurons leads, by an unknown mechanism, to increased neuronal excitability. This increased neuronal excitability may, in turn, lead to other changes in these neurons, such as the alterations that we have seen in the Na+ channel, whether it is an increased expression or alteration in current and its kinetics. This study cannot distinguish between this possibility and the other explanation in which the alterations in Na+ channel lead to increased excitability.

Although the nature of network properties between neurons is crucial in inducing and maintaining behaviors such as seizures, myotonia, ataxia, and related neurological disorders, changes in membrane and cellular properties are of major importance in "priming" neurons for increased discharge and increased excitability. Clearly, the alterations in the passive properties as well as in the Na+ channel of the NHE1 mutant neurons can be causally related to the abnormally high discharge and higher excitability of the mutant neurons. Because our observations have all been made in CA1 neurons, our findings take on added significance because of the relationship between seizure generation and hippocampal formation. However, it is important to realize that the hippocampus may only be a participant in the seizures of the NHE1 mutant mouse and, especially, that they are generalized, absence seizures with a tonic and clonic component. It is possible that thalamic and neocortical circuits, for example, are critical in the neurological disorder of this mouse in light of the relatively slow periodicities on the electrocorticogram (11). Hence, one corollary conclusion that could be drawn from this work is that neurons from regions other than the hippocampus (e.g., thalamus) may have similar cellular defects, as described in this work, especially in locations where the NHE1 is highly expressed (17, 20).

Although this work provides an explanation for the increased excitability in the mutant neurons via an upregulation in the Na+ current and alterations in the current kinetics, we cannot rule out other possible alterations in other membrane proteins that could affect cell physiology and function. Indeed, our recent Western blot analysis (R. M. Douglas and G. G. Haddad, unpublished observations) of the hippocampus from mutant mice shows altered expression in Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter expression. Also, it is possible that other channels may be affected, because Vm is different in mutant than in wild-type mice. Clearly, additional work is needed to dissect out all the potential changes, which could be many, that have taken place in the mutant mice.

This work illustrates another important idea with respect to mice with specific phenotypes that are ascribed to specific mutations and lack of corresponding proteins. We have demonstrated in this work that the phenotype in this mouse may not be related directly to the missing gene and its product but rather to the consequences of the lack of this NHE protein. We believe that the absence of this protein induces changes in other proteins that, in turn, have their own impact on neuronal function.

In summary, we have shown that the NHE mutant mice, which demonstrate a seizure disorder as part of their phenotype, have an abnormally high hippocampal neuronal excitability. This increased excitability is based on 1) an upregulation of the Na+ current and alterations in the channel characteristics, including steady-state inactivation and recovery from inactivation and deactivation, and 2) alterations observed in the passive properties of CA1 neurons. This investigation is the first to document the existence of an important functional link between the NHE1 and the voltage-sensitive Na+ channel. Furthermore, the changes in the Na+ channel that we have documented in this work may be critical for the increased excitability of neurons in NHE1 mutant mice.


    ACKNOWLEDGEMENTS

We thank Dr. Edward Novotny for valuable suggestions and comments on this manuscript.


    FOOTNOTES

This work was supported by National Institutes of Health Grants P01-HD-32573 and NS-35918 (to G. G. Haddad).

Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics, Section of Respiratory Medicine, Yale Univ. School of Medicine, 333 Cedar St., Rm. 508, New Haven, CT 06510 (E-mail: gabriel.haddad{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 February 2001; accepted in final form 30 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adelman, JP, Bond CT, Pessia M, and Maylie J. Episodic ataxia results from voltage-dependent potassium channels with altered functions. Neuron 15: 1449-1454, 1995[ISI][Medline].

2.   Battistini, S, Stenirri S, Piatti M, Gelfi C, Righetti PG, Rocchi R, Giannini F, Battistini N, Guazzi GC, Ferrari M, and Carrera P. A new CACNA1A gene mutation in acetazolamide-responsive familial hemiplegic migraine and ataxia. Neurology 53: 38-43, 1999[Abstract/Free Full Text].

3.   Bell, SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, Shull GE, and Scott WJ. Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. Am J Physiol Cell Physiol 276: C788-C795, 1999[Abstract/Free Full Text].

4.   Bevensee, MO, Cummins TR, Haddad GG, Boron WF, and Boyarsky G. pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. J Physiol (Lond) 494: 315-328, 1996[Abstract].

5.   Boland, LM, Price DL, and Jackson KA. Functional consequences of potassium channel mutations identified in families with inherited episodic ataxia (Abstract). Biophys J 72: A140, 1997.

6.   Burgess, DL, Jones JM, Meisler MH, and Noebels JL. Mutation of the Ca2+ channel (subunit gene Cchb4) is associated with ataxia and seizures in the lethargic (Ih) mouse. Cell 88: 385-392, 1997[ISI][Medline].

7.   Cox, GA, Lutz CM, Yang CL, Biemesderfer D, Bronson RT, Fu A, Aronson PS, Noebels JL, and Frankel WN. Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice. Cell 9: 139-148, 1997.

8.   Cummins, TR, Donnelly DF, and Haddad GG. Effect of metabolic inhibition on the excitability of isolated hippocampal CA1 neurons: developmental aspects. J Neurophysiol 66: 1471-1482, 1991[Abstract/Free Full Text].

9.   Cummins, TR, Xia Y, and Haddad GG. Functional properties of rat and human neocortical voltage-sensitive sodium currents. J Neurophysiol 71: 1052-1064, 1994[Abstract].

10.   D'Adamo, MC, Liu Z, Adelman JP, Maylie J, and Pessia M. Episodic ataxia type-1 mutations in the hKv1.1 cytoplasmic pore region alter the gating properties of the channel. EMBO J 17: 1200-1207, 1998[Abstract/Free Full Text].

11.   Destexhe, A, McCormick DA, and Sejnowski TJ. Thalamic and thalamocortical mechanisms underlying 3 Hz spike-and-wave discharges. Prog Brain Res 121: 289-307, 1999[Medline].

12.   Douglas, RM, Schmitt BM, Xia Y, Bevensee MO, Biemesderfer D, Boron WF, and Haddad GG. Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: ontogeny of protein expression in the rat brain. Neuroscience 102: 217-228, 2001[ISI][Medline].

13.   Egan, ME, Flotte TR, Afione S, Solow R, Zeitlin PL, Carter B, and Guggiono WB. Correction of defective PKA regulation of outwardly rectifying chloride channels by insertion of cystic fibrosis transmembrane regulator into CF airway epithelial cells. Nature 358: 581-584, 1992[ISI][Medline].

14.   Fanning, AS, and Anderson JM. Protein modules as organizers of membrane structure. Curr Opin Cell Biol 11: 432-437, 1999[ISI][Medline].

15.   Gu, XQ, Yao H, and Haddad GG. Effect of extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> on Na+ channel characteristics in hippocampal CA1 neurons. J Neurophysiol 84: 2477-2483, 2000[Abstract/Free Full Text].

16.   Hamill, O, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[ISI][Medline].

17.   Ma, E, and Haddad GG. Expression and localization of Na+/H+ exchangers in rat central nervous system. Neuroscience 79: 591-603, 1997[ISI][Medline].

18.   O'Reilly, JP, Cummins TR, and Haddad GG. Oxygen deprivation inhibits Na+ current in rat hippocampal neurones via protein kinase C. J Physiol (Lond) 503: 479-488, 1997[Abstract].

19.   Pilewski, JM, and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215-S255, 1999[Medline].

20.   Schmitt, BM, Berger UV, Douglas RM, Bevensee MO, Hediger MA, Haddad GG, and Boron WF. Na/HCO3 cotransporters in rat brain: expression in glia, neurons and choroid plexus. J Neurosci 20: 6839-6848, 2000[Abstract/Free Full Text].

21.   Schweibert, EM, Benos D, Egan ME, Stutts MJ, and Guggino WB. CFTR is indeed a "conductance regulator". Physiol Rev 79: S145-S166, 1999[Medline].

22.   Schwiebert, EM, Egan ME, Hwang TH, Fulmer SB, Allen SS, Cutting GR, and Guggino WB. CFTR regulates outwardly rectifying chloride channels through autocrine stimulation of outwardly rectifying chloride channels. Cell 81: 1063-1073, 1995[ISI][Medline].

23.   Steinlein, OK, and Noebels JL. Ion channels and epilepsy in man and mouse. Curr Opin Genet Dev 10: 286-291, 2000[ISI][Medline].

24.   Yao, H, Ma E, Gu XQ, and Haddad GG. Intracellular pH regulation of CA1 neurons in Na+/H+ isoform 1 mutant mice. J Clin Invest 104: 637-645, 1999[Abstract/Free Full Text].

25.   Zerr, P, Adelman JP, and Maylie J. Episodic ataxia mutations in Kv1.1 alter potassium channel function by dominant negative effects or haploinsufficiency. J Neurosci 18: 2842-2848, 1998[Abstract/Free Full Text].


Am J Physiol Cell Physiol 281(2):C496-C503
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society