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Summary: Purpose: Previous quantitative trait loci (QTL) mapping studies from our laboratory identified a 6.6 Mb segment of distal chromosome 1 that contains a gene (or genes) having a strong influence on the difference in seizure susceptibility between C57BL/6 (B6) and DBA/2 (D2) mice. A gene transfer strategy involving a bacterial artificial chromosome (BAC) DNA construct that contains several candidate genes from the critical interval was used to test the hypothesis that a strain-specific variation in one (or more) of the genes is responsible for the QTL effect.
Methods: Fertilized oocytes from a seizure-sensitive congenic strain (B6.D2-Mtv7a/Ty-27d) were injected with BAC DNA and three independent founder lines of BAC-transgenic mice were generated. Seizure susceptibility was quantified by measuring maximal electroshock seizure threshold (MEST) in transgenic mice and nontransgenic littermates.
Results: Seizure testing documented significant MEST elevation in all three transgenic lines compared to littermate controls. Allele-specific RT-PCR analysis confirmed gene transcription from genome-integrated BAC DNA and copy-number-dependent phenotypic effects were observed.
Conclusions: Results of this study suggest that the gene(s) responsible for the major chromosome 1 seizure QTL is found on BAC RPCI23-157J4 and demonstrate the utility of in vivo gene transfer for studying quantitative trait genes in mice. Further characterization of this transgenic model will provide new insight into mechanisms of seizure susceptibility.
C57BL/6 (B6) mice are highly resistant to seizures induced experimentally whereas DBA/2 (D2) mice are highly susceptible (Engstrom and Woodbury, 1988; Kosobud and Crabbe, 1990; Ferraro et al., 2002). This observation has served as the basis for much research aimed at gaining insight into the pathogenesis of human epilepsy; however, the biological factors that mediate this strain difference are still unknown. Previous work has demonstrated that gene variation plays a large role in B6 and D2 strain-specific seizure responses and several chromosomal regions have been implicated. In particular, a segment of distal chromosome 1 has been shown to exert a strong influence on the difference in seizure susceptibility between B6 and D2 mice in a variety of diverse paradigms (Buck et al., 1997, 1999; Ferraro et al., 1997, 1999, 2001). Recently, the gene(s) responsible for distal chromosome 1 seizure susceptibility was mapped to a 6.6 Mb interval between Pbx1 and D1Mit150 (Ferraro et al., 2004). The prior discovery of mutations in single ion-channel genes that are sufficient individually to cause seizures both in animals and humans (Noebels 2003) facilitates the identification of several genes in the critical interval as high-priority candidates for analysis. The most compelling of these are the potassium ion channel genes Kcnj9 and Kcnj10.
Evidence exists to support a role for both Kcnj9 and Kcnj10 in the biology of seizures. Kcnj10 encodes a pH-sensitive, inward rectifier potassium channel (KIR4.1) and is expressed throughout the brain in both neurons and glia (Bredt et al., 1995; Neusch et al., 2001; Li et al., 2001, Wu et al., 2004). Targeted knockout of Kcnj10 in mice produces a severe neurological phenotype involving motor abnormalities and postnatal lethality (∼3 weeks; Kofuji et al., 2000). Among common strains of inbred mice, Kcnj10 exhibits a polymorphism that predicts a threonine (B6) to serine (D2 and most other common inbred strains) amino acid substitution at position 261 of the parent protein (Ferraro et al., 2004). Furthermore, the gene maps to a haplotype block within the critical interval on chromosome 1 that discriminates the strains as a function of their threshold for electrically induced seizures (Ferraro et al., 2004). Kcnj9 is a member of the G-protein-activated inward rectifier potassium channel (GIRK) family that is expressed ubiquitously in the brain (Lesage et al., 1994; Dissman et al., 1996). It has been shown to be involved indirectly in seizure mechanisms in that the spontaneous seizure phenotype observed in mice with a targeted knockout of the Kcnj6 gene is exacerbated when this knockout is combined with a Kcnj9 knockout (Torrecilla et al., 2002). Kcnj9 maps within the D2 seizure susceptibility haplotype block, although no coding SNPs have been identified (Ferraro et al., 2004). Nonetheless, Kcnj9 is both a positional and biological candidate for the previously mapped seizure susceptibility locus on distal chromosome 1.
Quantitative trait loci (QTL) mapping studies have provided the chromosomal localization of genes for many complex traits but it is clear that there are limitations with respect to identifying the responsible genes and the causative sequence variation within those genes (Flint et al., 2005). As in other areas of QTL research, mapped loci for seizure-related traits are plentiful; however, identification of specific genetic variants associated with seizure susceptibility or resistance has not been readily forthcoming. Various approaches have been utilized to translate QTL into quantitative trait genes (QTGs) but there is no general formula for success and no single criterion that must be satisfied in order to document a genotype–phenotype correlation. Rather, there is support for the idea that each QTL should be assessed in ways that are most relevant to its specific biology (Abiola et al., 2003). Despite the fact that not all QTL may be amenable to a transgenic approach, the use of gene transfer to analyze the phenotypic effects of a QTL is a potentially powerful method for QTG identification (Ikeda et al., 2002; Henkel et al., 2005). Furthermore, gene transfer is useful for translational studies of complex traits where human gene variations can be isolated and studied in experimental animals (Jiang et al., 2005). Transgenic animals carrying large genomic fragments provide more comprehensive information with regard to genetic effects than animals harboring shorter constructs, and this is especially important for studying clustered genes (Gao et al., 2005). Thus, in order to test more formally the potential influence of Kcnj9 and Kcnj10 on seizure susceptibility in the B6-D2 model, we have created transgenic lines of mice in which the effect of a Kcnj9- and Kcnj10-containing BAC could be studied in relation to the distal chromosome 1 seizure susceptibility QTL Szs1 (Ferraro et al., 1997). Results of the study document BAC-mediated increase of seizure threshold and narrow the region of DNA harboring the causative gene variation to a 186-Kb interval.
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We obtained litters from all seven foster dams and in total they comprised 36 progeny. From these, we identified four tail-positive pups, three of which (TG-4, TG-12, and TG-18) were independent germ line founders of new transgenic lines. When mated with wildtype B6.D2-Mtv7a-27d congenic mice, founder mice TG-4 and TG-12 passed on BAC DNA to approximately 50% of offspring (equal numbers of males and females) whereas inheritance from founder TG-18 was much lower at approximately 15%. Transgenic progeny derived from founder TG-18 pass the BAC to approximately 50% of offspring, similar to TG-4 and TG-12 mice. This result suggests delayed integration of BAC DNA (after the first cleavage of the fertilized egg) in founder TG-18 resulting in somatic and germ cell mosaicism. BAC transgenic mice are observed to be generally indistinguishable from nontransgenic littermates. They exhibit the expected B6 coat color and their body weight is similar to same-gender littermates. They are viable into adulthood, exhibit grossly normal locomotor activity, and demonstrate typical breeding patterns.
Neither the transgenic founders nor any of their progeny have been observed to exhibit spontaneous seizures or any other major behavioral or motor dysfunction; however, assessment of seizure susceptibility documented significantly higher MEST in transgenic mice compared to nontransgenic littermates for all three lines with TG-18 exhibiting the largest magnitude of effect (13, 7, and 6 mAmp for TG-18, TG-12, and TG-4 lines, respectively). The difference in MEST between transgenic mice and nontransgenic littermates was statistically significant for all lines (Fig. 3) and this result was independent of gender (female data not shown). Estimation of copy number using the Pigm expression assay documented an amplification difference of 2 CT for TG-18 and 1 CT for TG-4 and TG-12. These results suggest that eight copies of Pigm are present (six copies of the BAC are inserted) in line TG-18 and four copies of Pigm present (two copies of the BAC inserted) in TG-4 and TG-12. Thus, the magnitude of the effect on seizure phenotype correlated with estimated copy number as TG-18 mice showed a two-fold greater MEST increase versus nontransgenic littermates compared to TG-12 and TG-4 mice. It is possible that incorporation of a longer BAC concatamer into the genome of founder TG-18 is related to the delay in integration proposed above to explain the initial low BAC transmission rate.
Figure 3. Seizure phenotype in parental (B6 and D2), congenic (B6.D2-Mtv7a-27d) and BAC-transgenic mice. Maximal electroshock seizure threshold in 8- to 9-week-old male mice is shown. Strain designations: B = B6; D = D2, C = B6.D2-Mtv7a-27d (Ferraro et al., 2004); the symbols “+” and “−“ refer to BAC-positive transgenic mice and their nontransgenic littermates, respectively. The top of each bar represents the mean MEST (+ SD). Values (given in mAmp): B = 65 ± 7, D = 25 ± 2, C = 43 ± 6, TG-4 (−) = 47 ± 3, TG-4 (+) = 54 ± 3, TG-12 (−) = 44 ± 5, TG-12 (+) = 51 ± 3, TG-18 (−) = 45 ± 4, TG-18 (+) = 58 ± 4; * p < 0.05, ** p < 0.01 vs. nontransgenic littermates; *** p < 0.001 vs. B and D (ANOVA, Newman–Keuls; n = 20–22 per group).
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We confirmed in vivo expression of genes from the BAC construct by using allele-specific RT-PCR/RFLP assays. Transgenic mice exhibited both B6 and D2 alleles for Atp1a2, Kcnj9, Kcnj10, and Pigm (data not shown), evidence that these genes are transcribed from the integrated BAC. Results of quantitative RT-PCR analysis show that relative message levels for Atp1a2, Kcnj9, Kcnj10, and Pigm are consistently higher in transgenic mice compared to nontransgenic littermates and although minor regional differences between the three lines are noted, overall gene expression is increased approximately two-fold (Table 1). We also conducted RT-PCR analysis in parental strains and show that, in general, expression levels are similar between the strains; however, it is noted that D2 mice exhibit significantly higher levels of Kcnj10 in hindbrain (1.74-fold) and significantly higher levels of Atp1a2 in all brain regions (1.9 to 2.7 fold) compared to B6 mice (Table 1).
Table 1. Regional brain gene expression1 in transgenic mice, nontransgenic littermates, and parental strains
| ||TG-4||TG-12||TG-18||Parental strains|
| FB||1.00||0.66–1.25||1.07||0.77–1.41||0.96||0.54–1.69|| 1.91*||1.61–2.29|
| MB||1.32||0.77–1.93||1.20||1.09–1.27||1.35||1.01–1.83|| 2.70**||2.52–2.88|
| HB||0.85||0.65–1.09||0.85||0.70–1.05||1.25||1.03–1.53|| 2.33*||1.68–3.24|
| MB||0.99||0.59–1.63||1.57||1.17–2.10|| 2.20*||1.87–2.60||0.83||0.82–0.93|
| MB||1.70||1.06–2.75||1.12||0.98–1.27|| 2.44*||1.92–3.12||1.00||0.85–118|
| HB||1.39||0.82–2.05||1.02||0.89–1.19|| 3.03**||2.42–3.78|| 1.74*||1.55–1.92|
| FB||1.26||0.84–1.59|| 1.99*||1.57–2.50||1.41||1.09–1.83||1.11||0.91–1.39|
| MB|| 2.05*||1.81–2.33|| 2.35*||2.06–2.73|| 2.59*||2.20–3.01||1.42||1.14–1.60|
| HB|| 1.97*||1.71–2.35||2.10||1.64–2.56|| 1.90*||1.44–2.31||1.33||1.08–1.64|
We show results of Western blot analyses in Figs. 4 and 5. With respect to Kir3.3 (KCNJ9) immunoreactivity, there is a major band visible at the level of the 45 kD marker in all samples (Fig. 4 left panel). This result is consistent with the predicted size of the Kir3.3 monomer that contains 376 amino acid residues (NCBI accession #NM_008429). Several minor bands are also noted on the blot; however, preincubation of primary antibody serum with peptide antigen (supplied by the antibody manufacturer) for 2 h at 37°C resulted in loss of the major band at 45 kD without altering the other minor bands (data not shown). Relative to actin, we demonstrate that Kir3.3 immunoreactivity is significantly higher in forebrain and midbrain of transgenic mice compared to nontransgenic littermates; differences in hindbrain did not reach levels of statistical significance (Figure 5). Western blot results also show the expected banding pattern for Kir4.1 (KCNJ10) immunoreactivity. Thus, there is a single band that migrates with the 45 kD size marker in all samples (Fig. 4) and this is consistent with the predicted size of the Kir4.1 monomer that contains 379 amino acid residues (NCBI accession #AB039879.1). Blots incubated with serum that was preincubated with the antigen peptide supplied by the primary antibody manufacturer were blank (data not shown). Relative to actin, we show that Kir4.1 immunoreactivity is significantly higher in midbrain and hindbrain of transgenic mice compared to nontransgenic littermates whereas there was no difference in forebrain levels (Fig. 5). The magnitude of the difference in both Kir3.3 and Kir4.1 immunoreactivity between transgenic and nontransgenic mice is consistent with the magnitude of differences in mRNA level. Comparison of Kir3.3 and Kir4.1 immunoreactivity in brain from parental B6 and D2 mice shows no statistically significant differences with the exception of hindbrain where D2 mice have significantly more Kir4.1 immunoreactivity compared to B6 (Fig. 5).
Figure 4. Western blot analysis. Representative experiment showing Kir3.3 (left) and Kir4.1 (right) immunoreactivity in the hindbrain from individual transgenic mice and nontransgenic littermates (n = 3 each).
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Figure 5. Quantification of Western blots. The top of each bar represents the mean (+ SD) level of Kir3.3/KCNJ9 or Kir4.1/KCNJ10 immunoreactivity in parental mice, transgenic mice, and nontransgenic littermates normalized to actin. FB, forebrain; MB, midbrain; HB, hindbrain. * p < 0.05; ** p < 0.01 (ANOVA, Neuman–Keuls; n = 3 per group for transgenic and nontransgenic littermates; n = 6 for parental B6 and D2 mice).
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In the present study, we demonstrate elevation of electrical seizure threshold through in vivo transfer of a BAC that harbors six linked genes, two of which (Kcnj9 and Kcnj10) are primary candidates for major seizure susceptibility QTL on distal chromosome 1 reported previously in B6 (seizure resistant) and D2 (seizure susceptible) mice (Ferraro et al., 1997; 1999; 2001). The experimental strategy involved random insertion transgenesis in which the BAC construct was delivered by microinjection into 1-day old fertilized oocytes that were derived from a seizure-susceptible congenic strain, B6.D2-Mtv7a/Ty-27d, produced during refinement of the chromosome 1 QTL map position (Ferraro et al., 2004). In general, phenotypes that result from random insertion methods must be interpreted with caution since it is possible that genomic integration of exogenous DNA can interrupt the sequence of an endogenous (unknown) gene(s), effectively yielding a functional knockout. In our study, demonstration of decreased seizure susceptibility (increased seizure threshold) in three independent transgenic lines (Fig. 3) minimizes this possibility and suggests that a BAC-derived gene(s) is responsible for the observed phenotype. Further support for this conclusion may be generated by comparing the magnitude of the change in seizure susceptibility in relation to transgene (BAC) copy number. Thus, the MEST increases shown for male mice in Fig. 3 represent fold-increases of 1.3 for TG-18, the line harboring six copies of the BAC, compared to 1.15 and 1.08-fold increases for lines TG-12 and TG-4, respectively, each of which harbor two copies. Overall, female mice had lower MEST values compared to males (data not shown); however, the ability of BAC genes to reverse the seizure susceptible phenotype was independent of gender and, as for the males, the magnitude of phenotypic effect was greater in TG-18 females compared to females from the other two lines again emphasizing the potential effect of copy number variation.
It is not clear which of the BAC transgenes is responsible for the observed effects on seizure threshold. Of the six known genes that are present on BAC 157J4, biological plausibility is highest for Kcnj9 and Kcnj10 with the latter gene supported by the largest body of data. We reported previously that Kcnj10 exhibits a nonsynonymous (coding) SNP that predicts an amino acid variation in the intracellular C-terminus of the channel protein at position 262 wherein the B6 sequence encodes a threonine residue and the D2 sequence encodes serine (Ferraro et al., 2004). Although this change is conservative, it is possible that it could affect the phosphorylation state of the protein since threonine and serine residues are substrates for different kinases (Light et al., 2000; Wischmeyer and Karschin, 1996). The C-terminal region of the protein is also known to be involved in channel gating as well as interaction with other channel subunits and membrane-associated proteins (Nishida and MacKinnon, 2002) and therefore subtle genetic variation may have significant functional effects. Human KCNJ10 exhibits a coding SNP, Arg271Cys, which predicts an amino acid variation at a relative position nine residues removed from the variation discovered in the mouse and which discriminates patients with common forms of epilepsy from control subjects in genetic association studies (Buono et al., 2004). Importantly, this positive result has been replicated by an independent laboratory (Lenzen et al., 2005) making it one of the few confirmed genetic association findings in common epilepsies and strengthening the hypothesis that Kncj10 is involved in differences in seizure susceptibility between B6 and D2 mice. In vitro studies aimed at examining the electrophysiology of these channel polymorphisms were unable to document significant functional differences when the variants were expressed in Xenopus oocytes (Shang et al., 2005) suggesting that the influence of the variation is subtle at the physiological level, that it requires participation of molecular elements not present in oocytes or that it is nonfunctional but is in genetic association with the causative variation. Further study is required to evaluate these possibilities.
With regard to Kcnj9, it is first important to note that there are no known coding SNPs between B6 and D2 mice. Therefore, if this gene influences seizure phenotype in our model, it must involve a strain-specific difference in expression either at the message or protein level. This hypothesis is not supported by data generated in parental B6 and D2 mice that document similar relative levels of both Kcnj9 mRNA and Kir3.3 immunoreactivity throughout the brain in the two strains. Thus, the likelihood that Kcnj9 contributes to the observed differences in seizure phenotype is reduced.
Also located on BAC 157J4 are two genes encoding alpha subunits of Na+/K+-ATPase, Atp1a4 and Atp1a2. Although both of these genes are biologically plausible candidates for influencing seizure susceptibility in terms of the critical role of their enzyme system in membrane physiology, enthusiasm for them is weak for several reasons. The α4 subunit gene exhibits testes-specific expression (Underhill et al., 1999) and is therefore not likely to be involved in a nervous system phenotype. The α2 gene is expressed at high levels in excitable tissues including brain; however, there are no coding SNPs between B6 and D2 mice. Differences in brain mRNA expression level exist (Table 1); however, mice with a targeted knockout of Atp1a2 do not experience spontaneous seizures nor do they exhibit a phenotype involving behavioral hyperexcitability (Moseley et al., 2003). With regard to studies in human epilepsy, analysis of SNPs in ATP1A2 has not uncovered genetic association with common forms of the disease (Buono et al., 2000; Lohoff et al., 2005).
Another gene found on the BAC, Igsf8, encodes a protein involved in immune function. There is a coding SNP in the gene between B6 and D2 mice, His221Arg, and it warrants further consideration since there is evidence of causal links between immune function and seizure susceptibility (McKnight et al., 2005).
The final known gene present on BAC 157J4 is Pigm. Pigm encodes a transferase involved in the transport of carbohydrate moieties used to modify proteins related to G-protein function (Maeda et al., 2001). Thus, Pigm plays a role in signal transduction mechanisms. It is expressed in the brain and contains a coding SNP, Gln206His, between B6 and D2 mice. This potentially functional variation cannot yet be excluded as a putative seizure susceptibility factor particularly in light of a recent report documenting that a Pigm promoter mutation in humans causes a severe clinical phenotype that includes seizures (Almeida et al., 2006). Finally, Ensembl reports a novel, hypothetical gene at the 3′ end of the BAC (ENSMU00000066692). It is estimated to be 99 Kb in size with two exons that contain an open reading frame of 104 bp encoding a 34-amino acid peptide with no known sequence homology.
Nonsynonymous coding SNPs are not the only gene variants of potential functional significance. Promoter and intronic SNPs can also have important effects with respect to levels of gene expression. However, despite the fact that sequence variation exists between B6 and D2 mice in the putative 5′ regulatory regions of all genes present on the BAC, analysis of relative mRNA using RT-PCR showed similar levels between strains for primary candidates Kcnj9 and Kcnj10. An exception to this pattern is observed in hindbrain where D2 mice express higher levels of Kcnj10 compared to B6 (Table 1). It is unlikely that higher expression of Kcnj10 in D2 hindbrain explains the relative seizure susceptibility of this strain since overexpression of Kcnj10 in the BAC transgenic lines results in decreased susceptibility (increased MEST). Although it is possible that coordinated regulation of transcription and turnover could mask potential strain differences in message level, our results suggest that the classic B6 and D2 difference in seizure susceptibility is not related to a difference in the expression of either of these two genes. It may be more likely that the phenotypic effect is related to the expression of a functional protein variant resulting from a coding SNP as opposed to expression of a greater amount of a protein that has an inherent level of activity similar to the endogenous protein. An important caveat relates to the anatomical resolution of the methods employed in that a brain nucleus-specific difference would likely be difficult to detect. In addition to functional effects of variation in promoter and coding sequences, it is also possible that intronic SNPs could have biological significance by altering splicing mechanisms and resulting species of message; however, in the present study, Northern blot analyses using mRNA extracted from brains of B6 and D2 mice demonstrated single bands for Atp1a2, Kcnj9, and Kcnj10 (data not shown).
It must also be considered that the mechanism responsible for seizure protective effects in the BAC-transgenic mice may be distinct from that involved in differences in seizure phenotype between the parental strains. Thus, although Kcnj10 subunits may assemble to form homomeric channel complexes, in some parts of the brain they are coexpressed and coassembled with Kcnj16 (Wu et al., 2004; Butt and Kalsi 2006) such that overexpression of Kcnj10 alone could alter local channel formation and/or properties. Kcnj9 also contributes to the formation of heteromeric protein complexes (Jelacic et al., 2000); however, this subunit may be detrimental to the assembly of functional channels (Schoots et al., 1999) possibly via a mechanism whereby channel traffic is directed to lysosomes rather than the cell membrane (Ma et al., 2002). Thus, although we did not observe Kcnj9 or Kcnj10 expression differences between B6 and D2 mice, altered expression levels of BAC-derived genes might alter the stoichiometry that defines the function of inward rectifier potassium channels.
B6 and D2 mice differ with respect to numerous phenotypic traits, and as a result, they have been the subject of many studies since being established as inbred strains in the middle of the 20th Century. Remarkable B6 and D2 strain differences with respect to seizure susceptibility were noted shortly after their introduction into biomedical research by comparing their responses to convulsive auditory stimuli (Hall 1947). Whereas the greater relative susceptibility of D2 mice to audiogenic seizures is evident only when the animals are very young (about 3 weeks of age or less), these mice retain relative seizure susceptibility through adulthood when other experimental seizure paradigms are employed including chemoconvulsant and electroshock tests (Engstrom and Woodbury, 1988). Another paradigm that exploits this strain difference involves susceptibility to ethanol withdrawal seizures (Goldstein and Kakihana, 1975). Studies designed to map the chromosomal location of genes that mediate differences in seizure susceptibility between B6 and D2 mice have documented a complex polygenic determinism with QTL detected on many different chromosomes; however, two loci in particular have been detected repeatedly. Thus, QTL on distal chromosome 1 (∼90 cM) and mid-chromosome 4 (∼40 cM) have been reported in screens using kainic acid (Ferraro et al., 1997), alcohol withdrawal (Buck et al., 1997), pentylenetetrazol (Ferraro et al., 1999), and pentobarbital withdrawal (Buck et al., 1999). It is likely that any gene influencing such diverse seizure phenotypes will be of fundamental importance in controlling neuronal excitability. Moreover, identification of such genes could be relevant to many different types of human epilepsy, particularly common, multifactorial forms that comprise the vast majority of all cases.
Over the past several years, considerable evidence has accumulated to suggest that the gene on chromosome 4 that mediates differences between B6 and D2 mice in susceptibility to depressant withdrawal seizures is Mpdz, a PDZ domain-containing protein (Fehr et al., 2002; Shirley et al., 2004). PDZ proteins are adapter proteins that serve as central organizers of protein complexes at the plasma membrane (Sarkar et al., 2004). Interestingly, Kir channels have functional PDZ binding domains (Yoo et al., 2004) and analysis of Kir4.1 and Kir3.3 sequences with the Eukaryotic Linear Motif (ELM) resource for functional sites in proteins (http://elm.eu.org/) reveals consensus PDZ1 motifs in the extreme C-terminal region of both proteins. Whereas functional analysis of PDZ domains on Kir proteins suggests that Kir3.3 does not bind PDZ sequences (Nehring et al., 2000), a number of studies have shown that Kir4.1 does (Horio et al., 1997; Kurschner et al., 1998; Tanemoto et al., 2004; Tanemoto et al., 2005). Thus, given that previous results from analysis of locus–locus interactions detected epistasis between the distal chromosome 1 kainate seizure susceptibility QTL and the kainate susceptibility QTL on mid-chromosome 4 (Ferraro et al., 1997), it is possible that a potential functional relationship exists between Mpdz and the product of the chromosome 1 QTL.
In summary, we have used an in vivo BAC transgenesis strategy involving B6 genes from the critical interval of the chromosome 1 seizure QTL to protect against classical D2 seizure susceptibility. Given the modest increases in expression noted between transgenic and nontransgenic littermates for the BAC genes studied, and the 100% identity of amino acid sequence between B6 and D2 mice for Kcnj9 and Atp1a2, the nonsynonymous (Thr262Ser) coding SNP in Kcnj10 remains the variation that is most likely to define the seizure-related QTL on distal chromosome 1. Phenotypic effects caused by functional changes in populations of inward rectifier potassium channels resulting from an alteration of the balance of subunit expression cannot be ruled out nor can the effects of closely associated gene variants. These issues will be addressed by BAC recombineering (knock-in) experiments ongoing currently in our lab in which the B6 SNP in Kcnj10 will be changed to D2. We predict that full characterization of these transgenic models will provide new insight into mechanisms of seizure susceptibility.