Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry
Disruption of the Epilepsy KCNQ2 Gene Results in Neural Hyperexcitability Hirotaka Watanabe, *Eiichiro Nagata, *Arifumi Kosakai, Motonao Nakamura, Masahiro Yokoyama, *Kortaro Tanaka, and Hitoshi Sasai Pharmaceutical Frontier Research Laboratories, Japan Tobacco, Yokohama; and *Department of Neurology, School of Medicine, Keio University, Tokyo, Japan
Abstract: Benign familial neonatal convulsion (BFNC) is a common idiopathic epilepsy with autosomal dominant inheritance. Recently, two novel voltage-dependent potassium channel genes, KCNQ2 and KCNQ3, were identified by positional cloning as being responsible for BFNC. Heterotetramers of the products of these genes form Mchannels and regulate the threshold of electrical excitability of neurons. We disrupted the mouse KCNQ2 gene via gene targeting to study the relationship between KCNQ2 and epilepsy. Homozygous pups (KCNQ2 ⫺/⫺) died within a few hours after birth owing to pulmonary atelectasis that was not due to the status of epileptic seizures, although their development was morphologically normal. Heterozygous mice had decreased expression of KCNQ2 and showed hypersensitivity to pentylenetetrazole, an inducer of seizure. These data indicate that the decreased expression of KCNQ2 might cause a hyperexcitability of the CNS, which accounts for the mechanism of BFNC. Key Words: Benign familial neonatal convulsion—Epilepsy—KCNQ2— M-channel—Knockout mouse. J. Neurochem. 75, 28 –33 (2000).
et al., 1999). Like shaker-type channels, potassium channels belonging to the KCNQ family have six transmembrane regions and one potassium-selected pore region. These KCNQ family proteins can also function as homotetramers or heterotetramers like other potassium channels. Both KCNQ2 and KCNQ3 are coexpressed in most neural cells and are thought to function as heterotetramers (Tinel et al., 1998; Yang et al., 1998). Recently, Wang et al. (1998) reported that these heterotetramers, consisting of KCNQ2 and KCNQ3, form M-channels and regulate the subthreshold electrical excitability of neurons. Although it is likely that BFNC is caused by neural hyperexcitability due to dysfunction of M-channels, there has been no direct evidence that an alteration in a KCNQ gene can actually cause epilepsy. We speculate that this epilepsy may be due to a half dosage of functional KCNQ2 (dosage effect) or to a dominant-negative effect by protein encoded by the mutant allele. We are also interested in the role of KCNQ2 in brain development because expression is detected in neural cells of the CNS and the sympathetic nervous system and is particularly high in 11.5 days postcoitus (dpc) embryos (Nakamura et al., 1998). To address this question, we isolated the mouse KCNQ2 gene and generated KCNQ2-knockout mice by homologous recombination in embryonic stem (ES) cells. We found that homozygous mutants developed normally in utero but died within the first postnatal day. Furthermore, although heterozygous mice showed normal behavior and morphology compared with wild-type mice, they showed increased sensitivity to an epileptic inducer.
Epileptic disorders affect ⬎0.5% of the general population, and about half of these are idiopathic generalized epilepsies such as benign familial neonatal convulsion (BFNC) (Baraitser, 1990). The symptoms of BFNC usually start within 1 week after birth and end spontaneously by several months of age. The prognosis of BFNC is generally good, although ⬃10% of individuals suffer seizures later on in life (Psenka and Holden, 1996). The loci for BFNC were formerly mapped to 20q13.3 (Leppert et al., 1989) and 8q24 (Lewis et al., 1993; Steinlein et al., 1995). Recently the genes responsible for BFNC were identified by positional cloning and are novel voltage-dependent potassium channel genes called KCNQ2 (Biervert et al., 1998; Singh et al., 1998) and KCNQ3 (Charlier et al., 1998). Potassium channels in neural cells have several important roles in establishing the resting membrane potential and in determining action potentials. Four KCNQ family genes have been isolated to date: KCNQ1 (Wang et al., 1996), KCNQ2, KCNQ3, and KCNQ4 (Kubisch
Received November 10, 1999; revised manuscript received March 6, 2000; accepted March 8, 2000. Address correspondence and reprint requests to Dr. H. Sasai at Pharmaceutical Frontier Research Laboratories, Japan Tobacco, Inc., 13-2, Fukuura 1-chome, Kanazawa-Ku, Yokohama, Kanagawa, 2360004, Japan. E-mail:
[email protected] The first two authors contributed equally to this work. Abbreviations used: BFNC, benign familial neonatal convulsion; dpc, days postcoitus; ES, embryonic stem; PTZ, pentylenetetrazole.
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CHARACTERIZATION OF BFNC GENE NULL MUTANT MICE
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FIG. 1. Targeted disruption of the murine KCNQ2 gene. a: Schematic representation of the targeting vector (top), the wild-type KCNQ2 allele (middle), and the predicted targeted allele (bottom). Several exons that were previously identified (Nakamura et al., 1998) are indicated by solid boxes with numbers in italics. The probes used for determining the genotype are indicated by solid bars. Restriction enzyme sites are indicated as follows: B, BamHI; V, EcoRV; I, EcoRI; and X, XhoI. b: Verification of genotypes by Southern blot analysis. The tail DNAs were completely digested with EcoRV and hybridized with a 1.5-kb EcoRV–BamHI probe shown in (a). Here, #578, #579, and #581 were heterozygous, and #580 was wild-type. c: Genotype verification by the PCR method. Tail DNAs were amplified either with a primer pair specific to the wild-type allele (left lane of a sample) or with one specific to targeted allele (right lane of the identical sample) in separate tubes. d: Semiquantitative RT-PCR analysis of KCNQ (KCNQ2 and KCNQ3) gene expression in whole embryo at 11 dpc and adult brain. The amount of wild-type KCNQ2 transcripts changed according to the copy number of wild-type alleles; however, the expression of KCNQ3 was unchanged. Primers for the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were used as an internal control. The number of PCR cycles was set to be less than the number at which PCR products reach saturation.
MATERIALS AND METHODS Construction of the targeting vector Mouse genomic clones that include the KCNQ2 gene were isolated previously (Nakamura et al., 1998). To delete the region containing exons 3–5 from the mouse KCNQ2 gene, we cloned a 2.7-kb BamHI–EcoRV fragment of its genomic clone (mKg3-10) and a 10.6-kb Sall–XhoI fragment of another genomic clone (mKg1-1) into the pMC1neo vector (Stratagene). As a negative selection marker, the HSV-TK gene from pMC-TK (Mansour et al., 1988) was added downstream of the 3⬘-homologous region as shown in Fig. 1a. The targeting vector was linearized with NotI and electroporated into ES cells (E14.1). Then the ES cells were exposed to selection medium containing 250 g/ml G418 and 2 M ganciclovir for 10 days. Subsequently, the drug-resistant colonies were subjected to PCR or Southern analysis to obtain the targeted clones.
Generation of KCNQ2-deficient mice ES cells in which KCNQ2 was disrupted were injected into the blastocysts derived from C57BL/6 mice, and the resulting
male chimeras were bred with C57BL/6 females. Germline transmission of the mutant KCNQ2 allele was determined by using PCR primers (G29, 5⬘-TTTTCCACCATCAAGGAG-3⬘; Neo right, 5⬘-TGGCGGACCGCTATCAGGAC-3⬘) that amplify between the first intron of KCNQ2 and the inserted neo resistance gene (Fig. 1a). To obtain KCNQ2-null mice, heterozygous F1 mice were intercrossed.
Genotype analysis To genotype animals, we used two sets of PCR primer pairs, which allowed us to discriminate between the wild-type allele and the KCNQ2-targeted allele. The mutant allele gives a 700-bp band in PCR with primers G29 and Neo right, and the wild-type allele gives a 1.2-kb band with the PCR primers G29 and m4R (5⬘-CATCCGCAAGATTTGCAAGA-3⬘). Each PCR procedure, in which one or the other primer pair was used, was done in two separate tubes. The tail DNAs were used as templates for PCR genotyping, and the amplified samples were characterized by agarose gel electrophoresis. To confirm the results of PCR analysis, Southern blot analysis was done by
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hybridizing the EcoRV-digested genomic DNA with a 1.5-kb EcoRV–BamHI probe (shown in Fig. 1a), which resulted in a 5.0-kb band for the wild-type allele and a 4.2-kb band for the KCNQ2-targeted allele.
RT-PCR for KCNQ2 and KCNQ3 expression Total RNA from tissues was prepared with TRIZOL reagent (GibcoBRL), and cDNA was synthesized with a first-strand cDNA synthesis kit (GibcoBRL). The primers used to target the KCNQ2 mRNA were G6 (5⬘-ACTGCCTGGTACATTGGCTT-3⬘) and m5R (5⬘-CCCCGTAGCCAATGGTCGTC-3⬘). This primer set, which amplifies between exon 5 and exon 6, was used to cover all the splicing variants of KQT2 shown by Nakamura et al. (1998). The primer pair for KCNQ3 was KQ3-11 (5⬘-CACCGTCAGAAGCACTTTGAG-3⬘) and KQ3-21 (5⬘-CCTTTAGTATTGCTACCACGAGG-3⬘), which amplifies between exon 7 and exon 8 (Schroeder et al., 1998). All the PCR primer pairs were complementary to two different exons separated by at least one intron to avoid amplification of genomic DNA. PCR procedures were carried out using rTaq (TaKaRa), and PCR products were analyzed by 2% agarose gel electrophoresis.
Histology We intercrossed the heterozygous mice. Neonatal pups were killed and dissected within 1 day after birth, and whole organs were fixed with 4% paraformaldehyde in phosphate-buffered saline at 4°C. Then these tissues were dehydrated with ethanol followed with chloroform and paraffin, embedded in paraffin blocks, and sectioned at 8 m with a microtome. The sections were mounted on gelatin-treated slides and stained with hematoxylin and eosin.
EEG analysis Wild-type and heterozygous mice at 3 weeks of age were selected for EEG analyses. Mice were anesthetized with 4% halothane, and stainless steel needle probes [length, 10 mm; radius, 0.15 mm (29 gauge); DANTEC] were implanted into the CA1 region of the hippocampus, cerebral cortex, and cerebellum and glued to the skull bone with dental cement (Durelon). The needle in the CA1 region of hippocampus was located at 1.5 mm from the bregma and 1 mm from the midline, and the needle in the motor cortex was located at 1 mm from the bregma and 2.5 mm from the midline. We injected 6 g/g pentylenetetrazole (PTZ; Sigma) intraperitoneally every minute for induction of status epilepticus. After the EEG study, we evaluated the genotypes and took the brain for microscopic morphological analysis to confirm if the stainless steel needles were implanted in the right positions.
RESULTS AND DISCUSSION We previously isolated mouse KCNQ2 genomic clones and studied their genomic structure (Nakamura et al., 1998). On the basis of this information, we constructed a targeting vector in which a crucial transmembrane region of KCNQ2 was replaced with a neomycin resistance gene (Fig. 1a). ES cells were electroporated with the targeting vector, followed by PCR screening for targeted clones. Of 218 drug-resistant colonies, one clone (Kv84) showed homologous recombination at the KCNQ2 gene. In this targeted clone, the region including exons 3–5, which codes for the second transmembrane region to a part of the pore region, was deleted as J. Neurochem., Vol. 75, No. 1, 2000
TABLE 1. Genotypes of animals Stage (total no.) Weaning a (66) Embryo 11–14 dpc (14) 19–20 dpc (27) Neonatalb P1 (9) P2 (4)
Wild-type (16)
Heterozygous (50)
Homozygous (0)
6 5
5 16
3 6
2 1
3 3
4c 0
a
Animals at weaning were 4 – 8 weeks old. Two heterozygous females were bred with heterozygous males. The neonatal pups were analyzed at postnatal day (P) 1 and P2. c Some of these homozygous pups were already dead. These dead animals were analyzed in the same way as live pups. b
expected (Fig. 1a). The Kv84 clone was also verified by Southern blot analysis using a 1.5-kb EcoRV–BamHI probe (data not shown). The germline transmission of mutant KCNQ2 was successful, and several heterozygote F1 pups were used to generate F2 animals. The genotypes of the F2 progeny were verified by PCR (data not shown) or Southern analysis (Fig. 1b). We found no homozygous mice (KCNQ2 ⫺/⫺) among 66 F2 mice ⬎4 weeks, so we genotyped the embryos to investigate when the KCNQ2 ⫺/⫺ population had disappeared (Table 1). Homozygous embryos were observed at around 12 and 19 dpc (Fig. 1c), and the Mendelian ratio was as expected, indicating that homozygosity was not lethal at the embryonic stage. When we observed the pups just after delivery, two of four KCNQ2 ⫺/⫺ pups were alive at day 1. At day 2, however, no homozygous pups were alive, indicating that KCNQ2 ⫺/⫺ animals died within 1 day after birth. To determine whether or not the KCNQ2 gene was properly disrupted in these mice, we performed semiquantitative RT-PCR using mRNA from whole embryos at 11.5 dpc, when KCNQ2 is normally highly expressed (Nakamura et al., 1998). Expression of KCNQ2 was completely absent in the homozygous mice and half the normal level in the heterozygous mice (Fig. 1d). We found that splicing of RNA derived from the KCNQ2targeted allele occurred between exon 2 and exon 6. The region deleted in this study, from exon 3 to exon 5, represents a loss of 429 bp from the open reading frame (Nakamura et al., 1998), which encodes a crucial portion of the potassium channel. Semiquantitative RT-PCR using a primer pair that was based on sequences in exon 2 and exon 6 indicated only faint expression of the modified form of KCNQ2 in homozygotes and heterozygotes (data not shown). On the other hand, KCNQ3 expression was not significantly different in the three genotypes, indicating that KCNQ3 expression was not affected by the impaired expression of KCNQ2. The homozygous newborn pups had a normal gross appearance, and some of them were able to survive for a few hours after birth. There was not any abnormality in the brain development or any abnormal findings in the
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FIG. 2. Paraffin sections of the neonatal lung region. Wild-type (a and d), living homozygous (b and e), and already dead homozygous (c and f) newborn mouse pups were dissected followed by tissue section preparation. The homozygous pups showed modest (b) or severe (c) atelectasis compared with normal alveoli maturation in wild-type pups (a). A high-magnification of b showed that the alveolar epithelial layer is thickened in homozygous pups (e). On the other hand, wild-type alveoli showed normal expansion (d). Bars ⫽ 50 m.
electrocardiogram (data not shown), even though KCNQ2 is highly expressed in the CNS and sympathetic nervous system (Yokoyama et al., 1996; Nakamura et al., 1998; Tinel et al., 1998; Wang et al., 1998; Yang et al., 1998). We could not observe any sign of seizure or distinct physical manifestations while these pups were alive. However, pathological investigation revealed that alveolar expansion was smaller in the homozygous pups than in wild-type and heterozygous pups (Fig. 2), suggesting that the homozygous pups died of pulmonary atelectasis. Pulsive catecholamine release from the adrenal gland at birth is regarded as a crucial event for initiating and maintaining respiration, releasing lung surfactants, and controlling pulmonary compliance (Hollingsworth and Gilfillan, 1984; Slotkin and Seidler, 1988). It has been shown that KCNQ2 is an important component of the M-current that might modulate membrane depolarization
of the adrenal chromaffin cells. Therefore, we speculate that the fine mechanisms of pulsive catecholamine release at birth was disturbed by continuous depolarization of the adrenal chromaffin cells due to KCNQ2 deficiency, which led to respiratory distress. Indeed, it has been reported that inhibition of the potassium current in adrenal chromaffin cells leads to membrane depolarization followed by an increase in intracellular Ca2⫹ concentration and catecholamine release (Kanno, 1977; Baker and Knight, 1978; Gonzalez-Garcia et al., 1993). However, further studies of catecholamine release in newborn homozygous mice will be needed to test our hypothesis. Next, we studied the EEGs of heterozygous animals in which the expression of normal KCNQ2 was reduced to half of that in wild-type mice (Fig. 1d). The basal EEG showed no apparent differences between wild-type and heterozygous mice (data not shown). PTZ induced seiJ. Neurochem., Vol. 75, No. 1, 2000
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FIG. 3. Electroencephalographic analysis of PTZ-induced seizure. a: Representative EEG recordings of PTZ-induced epileptic seizure. Left hippocampus (LH), right hippocampus (RH), left cortex (LC), right cortex (RC), and cerebellum (Ce) were analyzed with a reference electrode, which was set in the nasal bone. LH-LC, RH-RC, LH-RH, and LC-RC indicate EEGs obtained by subtracting the latter EEG from the former EEG. EMG is the electromyogram, which was measured in the left forefoot muscle. All brain regions showed four sharp waves (arrowheads), a sign of seizure, followed by a status epilepticus (arrow) after several administrations of PTZ. b: Number of PTZ injections to induce seizure shown in (a). Wild-type and heterozygous mice (each n ⫽ 5) were prepared for EEG measurements and injected with PTZ intraperitoneally. The number of PTZ injections at which mice first started to show seizure is plotted (F). The mean ⫾ SE (bars) values are also given (Œ). The heterozygous mice tended to require fewer PTZ injections to induce seizure compared with the wild-type mice.
zures in both groups (Fig. 3a), in which there were several presageful sharp waves (arrowheads) followed by an explosion of seizures (arrow). The PTZ-induced seizure was a generalized seizure, which simultaneously occurred in all regions of the cerebrum (Fig. 3a). The wild-type mice required a greater number of PTZ injections to induce seizures (13.0 ⫾ 3.74, mean ⫾ SE; n ⫽ 5) than the heterozygous mice (5.6 ⫾ 1.75; n ⫽ 5; Fig. 3b). This result suggested that half of the normal KCNQ2 molecules in heterozygous mice are vulnerable to PTZ, although one wild-type mouse manifested seizures without PTZ injections. It has been shown that the mutant KCNQ2 found in BFNC resulted in a mild, 20 – 40% reduction in Mchannel current in a study in which Xenopus oocytes were coinjected with KCNQ2/KCNQ3 (Schroeder et al., 1998; Lerche et al., 1999). It has also been shown that a 50% reduction in KCNQ2 expression caused a mild reduction in M-channel currents. Because heterozygous mice express a half dose of KCNQ2, it is likely that the CNS of these mice is hyperexcitable owing to the decreased function of M-channels. Consistent with this, we found that young KCNQ2 ⫹/⫺ mice (around 3 weeks of age) had a much higher sensitivity to PTZ than wild-type mice. A similar phenotype was reported in the case of inositol 1,4,5-trisphosphate receptor (type 1) ⫹/⫺ mice, whose CNS shows hyperexcitability due to a reduction in intracellular Ca2⫹ concentration (Nagata et al., 1998). However, we cannot exclude the possibility that the modified form of KCNQ2 from the targeted allele in KCNQ ⫹/⫺ mice had a dominant-negative effect on the normal KCNQ2/KCNQ3 heterotetramer, although the J. Neurochem., Vol. 75, No. 1, 2000
level of mutant KCNQ2 mRNA was not as high as expected. In spite of the fact that KCNQ2 ⫹/⫺ mice did not appear to show epileptic behaviors, our results are the first indication that the human epilepsy gene causes high susceptibility to drug-induced seizures in an animal model. The mild phenotype of BFNC patients who become free from symptoms within several months might explain the subtle phenotype found in KCNQ2 ⫹/⫺ mice. It is likely that the heterozygous mutation in KCNQ2 found in BFNC resulted in moderate dysfunction of the M-channels, which leads to mild epilepsy when the CNS is still immature and highly excitable. This finding is similar to the finding that febrile epilepsy, which is common in infants, becomes rare in older children, probably owing to the development of negative regulation of the excitability of the CNS (Singh et al., 1999). However, it has been reported that 10% of BFNC patients suffer from prolonged symptoms of epilepsy. This might be because of their continuous hyperexcitability due to severe mutations, which possibly generate dominant-negative proteins of the M-channel. However, there has been no case report of a dominant-negative mutation in BFNC. Singh et al. (1998) reported one BFNC family, kindred 1,547, with a large deletion in the KCNQ2 locus. In addition, Biervert and Steinlein (1999) reported three families with no mutation in the coding region of KCNQ2 despite tight linkage to 20q13.3, which implied a deletion of KCNQ2. These two clinical reports suggest that most BFNC cases are caused by a dosage effect rather than by a dominant-negative effect. KCNQ2 ⫹/⫺ mice in which expression of KCNQ2 is
CHARACTERIZATION OF BFNC GENE NULL MUTANT MICE only half that in normal mice may represent the main form of BFNC and are thought to be a good model of BFNC. The availability of KCNQ2 heterozygous mice and homozygous pups will enable further studies to elucidate the mechanism of BFNC. Acknowledgment: We want to thank Dr. Y. Kubo (Tokyo Metropolitan Institute for Neuroscience) for discussions on the manuscript, A. Kushi, K. Akiyama, and Y. Yamasaki for helpful advice on mouse breeding, and S. Nakanishi for histology techniques. We also thank M. Kumai, K. Ishii, H. Nakano, M. Yamasaki, and I. Sugamuma for help in generating and caring for the deficient mice used in this study and S. Inoue and M. Saito for their expert technical assistance. We thank Dr. M. Takase for the EEG work and helpful discussions.
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