Departments of 1 Pediatrics (Section of Respiratory Medicine) and 2 Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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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
HCOh = 5.22 ± 0.49 ms in wild-type
neurons and 2.20 ± 0.20 ms in mutant neurons), and deactivation
(at
100 mV,
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
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INTRODUCTION |
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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
-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
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METHODS |
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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 HCORecording 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 G. 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 M
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 M ![]() |
RESULTS |
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Neuronal Properties and Membrane Excitability in
HCO
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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
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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 HCOIn HCO
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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
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
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).
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Recovery from inactivation.
To examine the recovery from inactivation, we used a two-pulse
protocol, as shown in Fig. 5. In
HCOh) 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).
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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 HCO100 mV, depolarized them for 1 ms to
10 mV, and repolarized them to
70 or
100 mV (Fig.
6A, inset). The
average
h for deactivation,
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
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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
HCOd 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
62.89 mV, slope factor 6.00, n = 5, and
64.98 mV,
slope factor 6.37, n = 6, respectively).
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DISCUSSION |
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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
In physiological solutions containing
HCO
Of interest also is that there were electrophysiological differences
between HEPES solution and HCO120 to
40 mV was decreased in the presence
of HCO
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 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
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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Edward Novotny for valuable suggestions and comments on this manuscript.
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FOOTNOTES |
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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.
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