BACKGROUND
the megencephaly mice   <cit>  have an  <dig> base pair deletion in the shaker-like voltage-gated potassium channel subunit kv <dig>   <cit> .
this mutation causes progressive postnatal complex partial seizures and a unique pathologic brain overgrowth  <cit> .
the enlargement is not uniform but restricted to the hippocampus and ventral cortex, with 28% and 72% larger area compared to wildtype at  <dig> weeks of age  <cit> .
the enlargement is in part due to that the numbers of both neurons and glia cells are dramatically increased in the hippocampus which is caused by increased proliferation and/or reduced apoptosis .
the  <dig> base pair deletion in kv <dig>  leads to a frame shift and a premature stop codon.
the predicted truncated kv <dig>  protein  will retain only the n-terminal  domain, the first transmembrane domain  and the first extracellular loop.
voltage gated potassium channels form a diverse group of membrane proteins, regulating membrane potential, neuronal excitability and nerve signaling  <cit> .
the channels are hetero- or homotetramers, formed by a great variety of subunits, classified in  <dig> subfamilies  <cit> .
kv <dig>  belongs to the kv <dig> subfamily, consisting of eight members .
kv <dig> , kv <dig>  and kv <dig>  are the most abundant kv <dig> subunits expressed in the brain  <cit> .
although the heteromeric structure means that a very large number of k channels can be formed in theory  <cit> , the composition of the heteromultimers in the mammalian brain seems to be precisely regulated  <cit> .
a direct link between potassium channel dysfunction and apoptosis is that reduced intracellular potassium levels appear to promote critical events early in the suicide program.
treatment with potassium channel blockers have been shown to block apoptosis in various cell types  <cit> .
however, neither kv <dig>  mutations, the kv <dig>  null mouse  <cit>  nor other epileptic models have previously been associated with pathologic brain overgrowth.
therefore, we hypothesized that the truncated kv <dig>  would be expressed and dysregulate other kv <dig> subunits in the mceph/mceph mice.
truncated proteins are usually not expressed.
this is because mrnas with a premature stop codon are degraded through nonsense-mediated mrna decay   <cit> .
one exception to this is genes with no introns such as the kv <dig> genes  <cit> .
in mceph/mceph mice in situ hybridization have shown that there is no decay but instead an increased expression of kv <dig>  mrna in the hippocampus, cortex and ventral cortex  <cit> .
thus, it is possible that mceph is expressed.
in humans kv <dig>  point mutations are reported in patients with the autosomal dominant disorder episodic ataxia type <dig> ,  <cit> .
most are missense mutations but there is one report of a premature stop codon, resulting in a kv <dig>  protein that is truncated in the c-terminal .
the patient carrying this mutation suffers from a drug resistant form of ea <dig>  <cit> .
truncated kv <dig>  channels have previously been studied in cell culture and xenopus oocytes.
when the kv <dig> Δc <dig> protein is expressed in cell culture it is trapped in the endoplasmic reticulum  and degraded  <cit> .
however, full length kv <dig>  in cell culture is also retained in the er  <cit> .
another experimental kv <dig>  protein truncated in the extracellular loop between s <dig> and s <dig> is able to assemble with full length kv <dig>  subunits in cell culture and the resulting complexes are trapped in the er  <cit> .
in xenopus oocytes both the n- and c-terminal truncated kv <dig>  variants have a dominant negative effect on currents when coinjected with full length kv <dig>  and kv <dig>   <cit> .
knowing if the mceph protein is expressed and how it interacts with other kv <dig> subunits would provide further understanding of kv <dig> trafficking and clues to the downstream effects seen in the mceph/mceph mice.
in this paper we show that a truncated kv <dig>  protein, mceph is expressed in the brain of the megencephaly mouse.
the truncated protein is trapped in the er and does not reach the plasma membrane of neurons.
mceph has the ability to form multimers with kv <dig> .
it has a dominant negative effect on kv <dig>  and kv <dig>  currents in xenopus oocytes, but in the brain it does not appear to retain kv <dig>  in the er.
RESULTS
antibody characterization
a polyclonal antiserum was produced by immunization of rabbits with a synthetic peptide corresponding to amino acids  <dig> to  <dig> of kv <dig> .
the n-terminal epitope was necessary to be able to detect the truncated mceph protein.
western blot on brain lysate from wild type mice using the antiserum showed a strong band at  <dig> kda but also several weaker bands.
the  <dig> kda band had the same size as that detected by the previously characterized monoclonal antibody against a c-terminal epitope of kv <dig>   <cit> .
preimmune serum did not recognize the  <dig> kda band but showed the same weaker bands as the antiserum.
the antiserum was affinity purified against the peptide used for immunization which reduced the number of unspecific bands significantly .
preincubation of the affinity purified antibody with the peptide used for immunization absorbed the signal .
the purified kv <dig>  n-terminal antibody was used for all further experiments.
in immunblotting of lysate from kv <dig>  null mice an  <dig> kda band was present but at a lower intensity than in wild type .
since the null mouse does not have any kv <dig>  protein this means that the antibody cross reacts with another protein.
kv <dig> subunits have a high degree of similarity so it is likely that the antibody recognizes another kv <dig> subunit.
the peptide used for immunization share a stretch of six amino acids with kv <dig> .
both kv <dig>  and kv <dig>  are expressed in the brain as mature glycoproteins that are reported to be  <dig> and  <dig> kda, respectively  <cit> .
the shared amino acids and the size of the band makes kv <dig>  the likely candidate for the cross reactivity.
acetone post fixed wild type and kv <dig> -null brains were used for immunohistochemistry.
in wild type the staining resembled kv <dig>  but the background was high.
the pattern in kv <dig>  null brain was very similar to wild type, indicating cross reactivity .
immunohistochemistry on formalin fixed wild type brain sections showed the same staining pattern that previously has been reported for kv <dig>   <cit> .
in the hippocampus the immunoreactivity was mainly localized in the fiber networks in the middle part of the molecular layer, in the hilus and the pyramidal cell layer of ca <dig> .
when formalin fixed tissue was used the pattern in kv <dig>  null was changed and clearly distinguishable from wild type .
the conclusion is that the antibody recognizes kv <dig> .
it is cross reactive when used for immunoblotting but this is greatly reduced when used for immunohistochemistry on formalin fixed tissue.
mceph expression in the brain
the  <dig> bp deletion in kv <dig>  in mceph/mceph mice leads to a premature stop codon.
the predicted truncated protein, mceph, has a calculated weight of  <dig> kda.
to determine if mceph is expressed in the brain the polyclonal kv <dig>  n-terminal antibody was used for immunoblotting and immunohistochemistry.
immunoblotting showed a band at approximately  <dig> kda in brain lysate from mceph/mceph mice .
the intensity of the band was much lower than that of the kv <dig>  protein in wild type mice suggesting a low expression of the truncated protein.
the mceph/mceph mice do not have any full length kv <dig>  but, like the kv <dig>  null mice, they have the  <dig> kda cross reactivity-caused band.
however, the  <dig> kda band was only present in mceph/mceph mice.
no other band specific for mceph/mceph was detected.
immunohistochemistry was performed on formalin fixed mceph/mceph mice brain sections.
interestingly, in mceph/mceph there was staining only around the nuclei of cells, and no staining in fibers .
there was some background staining resembling that of the kv <dig>  null mouse but the overall pattern was significantly different.
the main areas with mceph immunoreactivity were the hippocampus and ventral cortex.
in the other areas examined there were only a few stained cell bodies.
in the hippocampus mceph was expressed in neurons in the dentate gyrus, ca <dig> and in ca <dig>  this pattern is in agreement with the previously reported mrna expression  <cit> .
taken together, the immunoblotting and immunohistochemistry results show that mceph is expressed in the brain of the megencephaly mouse.
trafficking of mceph
kv <dig>  has a single n-glycosylation site on the extracellular loop between s <dig> and s <dig> and is expressed on the plasma membrane as a mature glycoprotein.
this glycosylation site is preserved in mceph which makes it possible to study the trafficking of mceph by analyzing its glycosylation pattern.
glycosylation starts in the er where a high mannose carbohydrate is transferred to an asparagine residue.
this carbohydrate can be cleaved off by the glycosidases endoh and pngasef.
next, the protein is transported to the golgi where the carbohydrate is modified and becomes resistant to endoh but not to pngasef.
this is used as a marker for er to golgi transport.
to investigate the glycosylation status, brain lysates from wild type and mceph/mceph mice were treated with the enzymes endoh and pngasef and analyzed with immunoblotting.
for kv <dig>  in wild type mice endoh did not affect the size of the  <dig> kda band but pngasef reduced it with approximately  <dig> kda.
this is in agreement with other studies that have shown that the  <dig> kda band is the mature glycosylated form of kv <dig>  expressed in the plasma membrane  <cit> .
both endoh and pngasef reduced the apparent molecular weight of mceph with approximately  <dig> kda .
this strongly indicates that mceph is core glycosylated in the er but not transported to the golgi.
neither mature nor unglycosylated mceph was detected in the brain.
interactions of mceph
kv <dig> channels are tetramers and the assembly of the kv <dig> subunits takes place in the er  <cit> .
mceph contains the n-terminal tetramerization domain and might thus bind other kv <dig> subunits.
interactions with kv <dig>  in the brain
kv <dig>  and kv <dig>  are often associated in the same channels in the brain  <cit> .
we have previously reported a disturbance in the expression of kv <dig>  in the mceph/mceph hippocampus  <cit> .
to investigate if this disturbance could be caused by an interaction between mceph and kv <dig> , immunoprecipitation followed by immunoblotting was performed on brain lysate from wild type, kv <dig>  null and mceph/mceph mice.
the monoclonal kv <dig>  and kv <dig>  antibodies each precipitated both kv <dig>  and kv <dig>  in wild type brain, verifying that correct experimental procedures were used.
as a control the same antibodies were used for immunoprecipitation of lysate from kv <dig>  null brain.
as expected, in the kv <dig>  null lysate no kv <dig>  coprecipitated with kv <dig> .
also, when the kv <dig>  antibody was used for immunoprecipitation neither kv <dig>  nor kv <dig>  was detected .
the kv <dig> -n-terminal antibody precipitated kv <dig>  and kv <dig>  in wild type but also kv <dig>  in kv <dig>  null lysate.
this means that it is not specific when used for immunoprecipitation.
for mceph/mceph, immunoprecipitation was performed with anti-kv <dig>  whereas the kv <dig> -n-terminal antibody was used for immunoblotting.
with this approach it was not possible to detect any mceph co-precipitating with kv <dig>  .
this suggests that there is no persistent interaction between kv <dig>  and mceph in the brain.
on western blots, kv <dig>  in brain appears as two bands at  <dig> and  <dig> kda representing core and mature glycosylated form, respectively  <cit> .
if mceph binds and traps kv <dig>  in the er, the amount of core glycosylated kv <dig>  would be expected to increase and the mature glycosylated form decrease.
immunoblotting was performed on lysates from both whole brains and isolated hippocampi.
the hippocampus was investigated since mceph is expressed primarily in this brain region.
most of the kv <dig>  protein in the hippocampus had mature glycosylation and there was only a very small amount of core glycosylated protein.
no difference in kv <dig>  core glycosylation was seen between wild type and mceph/mceph in neither whole brain nor hippocampus lysate .
when the immunoprecipitation and glycosylation data is combined it appears that mceph does not retain kv <dig>  in the er in brain.
interactions with kv <dig>  in hek <dig> cells
to test if mceph can form multimers its ability to interact with wild type kv <dig>  was investigated by overexpression in cell culture.
this system was chosen to allow higher expression levels and easier detection.
for this experiment kv <dig>  and mceph were cloned into the vectors pzsgreen and pdsred <dig> to construct fluorescent fusion proteins.
hek293a cells were transfected with the mceph-zsgreen and the kv <dig> -dsred constructs together or separately and analyzed  <dig> hours after transfection.
the green and the red fluorescent signals were colocalized around the cell nucleus .
no staining was observed on the plasma membrane.
western blot with the polyclonal kv <dig>  n-terminal antibody on lysate from cotransfected hek <dig> cells showed bands at  <dig> and  <dig> kda corresponding to the mceph and kv <dig>  fusion proteins .
corresponding bands were found in cells transfected with single constructs which confirmed the identity of the bands.
to investigate the interaction between mceph and kv <dig> , immunoprecipitation was performed on cell lysate from cotransfected cells using the monoclonal kv <dig>  c-terminal antibody.
immunoblotting with the polyclonal kv <dig>  n-terminal antibody detected the mceph-zsgreen fusion protein as well as the kv <dig> -dsred fusion protein .
since the c-terminal kv <dig>  antibody is specific for wild type kv <dig>  the detected mceph protein was associated with the kv <dig>  protein.
interactions with kv <dig>  and kv <dig>  in xenopus oocytes
to further investigate the ability of mceph to interact with kv <dig> subunits we chose to analyze the effects of expressing mceph in combination with kv <dig>  or kv <dig>  in xenopus laevis oocytes.
figure 5a shows the kv <dig>  and kv <dig>  currents associated with a series of voltage steps, followed by a step to + <dig> mv.
both channels show delayed rectifier behaviour and lack fast inactivation.
kv <dig> , however, shows a marked slow inactivation.
mceph was found to reduce the expression of both kv <dig>  and kv <dig>  in the plasma membrane.
figure 5b shows the mean results of injecting mceph mrna together with kv <dig>  or kv <dig>  mrna  given as the effect on peak conductance versus voltage curves .
as seen, the curves for kv <dig>  and kv <dig>  are down-scaled by about  <dig> and 60%, respectively, without marked changes in slope or midpoint value.
the mceph shift of the kv <dig>  activation curve was slightly bigger than that of kv <dig>  .
the activation time course of the currents associated with voltage steps was negligibly affected by mceph, reflected in the unaffected time to peak current at + <dig> mv for both kv <dig>  and kv <dig>  .
similar effects were observed for the mceph effect on the slow inactivation kinetics.
mean inactivation versus voltage curves, measured from the peak current associated with the second pulse  are shown in figure 5c.
the mceph shift of the kv <dig>  inactivation curve was slightly bigger than that of kv <dig>  .
in summary, the measurements show that mceph has a dominant negative effect on both kv <dig>  and kv <dig>  channel expression in xenopus oocytes.
furthermore, the small effects on activation and inactivation kinetics suggest that mceph does not form functional channel complexes with kv <dig>  or kv <dig>  subunits in the plasma membrane.
heteromeric complexes involving mceph subunits are expected to drastically affect channel kinetics.
discussion
in this study we show that mceph/mceph mice express a truncated kv <dig> , mceph.
the mceph protein is expressed in neurons throughout the brain but is trapped in the er.
mceph is able to bind full length kv <dig>  in cell culture and has a dominant negative effect when coexpressed with kv <dig>  and kv <dig>  in xenopus oocytes.
in contrast, in the brain mceph does not appear to retain kv <dig>  in the er.
to be able to detect the truncated mceph protein an antibody against the n-terminal part of kv <dig>  was generated.
immunoblotting on brain lysate from mceph/mceph mice showed a band that has the same size as the predicted mceph protein, approximately  <dig> kda.
this band is present in all mceph/mceph brains studied but not in any wild type or kv <dig>  null mice.
based on this we conclude that mceph is expressed in the brain of the megencephaly mouse.
kv <dig>  in the brain is a mature glycoprotein that is mostly localized to axons and nerve terminals  <cit> .
truncated kv <dig>  variants have previously been shown to be retained in the er in cell culture  <cit> .
the draw back of these studies is that full length kv <dig>  homomers in cell culture is also largely retained in the er since they are undetectable at the cell surface by immunohistochemical methods  <cit>  but detectable at low levels with electrophysiological methods  <cit> .
lack of surface expression was seen by us both in hek <dig> cells and in the neuron-like cell line pc <dig> .
the reason is a strong er retention motif in the pore region  <cit> .
the mceph/mceph mouse provides an opportunity to study the trafficking of a truncated kv <dig>  in neurons.
both endoh and pngasef treatment reduced the molecular weight of mceph by approximately  <dig> kda, suggesting that the protein is core glycosylated  <cit> .
we detected neither maturely glycosylated nor unglycosylated mceph.
the glycosylation data suggest that mceph is trapped in the er and not further transported.
this is supported by immunohistochemistry experiments were mceph immunoreactivity surrounds the nuclei in an er-like pattern.
there is no immunoreactivity in fibers consistent with the lack of mature glycosylated mceph.
the low amount of mceph protein detected with immunoblotting compared to the high levels of mrna suggests that the mceph protein is rapidly degraded.
this is most likely due to er-associated degradation , a quality control mechanism in the er that recognizes misfolded proteins  <cit> .
these proteins are retro-translocated to the cytosol and degraded by proteasomes.
we have previously seen disturbances of staining pattern of kv <dig>  and kv <dig>  in the mceph/mceph hippocampus, which was not due to transcriptional regulation  <cit> .
previous studies of other truncated k <dig>  variants have shown that they have a dominant negative effect in oocytes and form oligomers with full length kv <dig> subunits in cell culture.
we hypothesized that mceph interacts with kv <dig> subunits and causes the expression disturbance possibly by retaining these subunits in the er.
kv <dig>  often forms heterotetramers with kv <dig>  subunits in the brain  <cit> .
nevertheless, we found no evidence for a persistent interaction between mceph and kv <dig>  in the brain, suggesting that there is no retention of kv <dig>  in the er.
however, we found mceph to be able to oligomerize when expressed in hek <dig> cells.
moreover, electrophysiological measurements on xenopus oocytes showed that mceph reduces both kv <dig>  and kv <dig>  currents, suggesting interaction between mceph and kv <dig> subunits in this cell type.
this indicates that the lack of er retention seen for kv <dig>  in the brain is not caused by an inability of mceph to form multimers
it can be assumed that lack of kv <dig>  could lead to defect trafficking of kv <dig> channels.
this is not the case since the kv <dig>  null mouse has normal expression of kv <dig>  in the hippocampus  and also the cerebellum and sciatic nerve  <cit> .
one possibility is that the kv <dig>  and kv <dig>  expression disturbances in mceph/mceph hippocampus are related to the growth.
the mceph/mceph hippocampus is significantly enlarged and has an increased amount of neurons .
this could lead to disturbed organization of the dendritic trees and give rise to the abnormal expression patterns.
also, since mceph contains the n-terminal t <dig> domain, which has been shown to mediate beta subunit association  <cit> , it is possible that mceph sequesters these auxiliary subunits within the er and prevents them from forming complexes with kv <dig>  and kv <dig>  in mceph/mceph mice.
beta subunit association has been shown to promote kv <dig> trafficking to the cell surface as well as to cause axonal targeting of kv <dig>   <cit> .
the megencephaly mouse is not a model for any known human disease.
still, it provides a unique tool to study kv <dig> channels in the brain.
the ea <dig> mutation r417stop gives rise to a kv <dig>  protein that lacks the final  <dig> amino acids in the c-terminal domain .
the kv <dig> Δc <dig> protein and mceph have some similarities but there are major differences.
both truncated proteins have a dominant negative effect in oocytes and can assemble with full length subunits in cell culture.
for the kv <dig> Δc <dig>  change in current kinetics when coinjected with kv <dig>  in xenopus oocytes indicates that some channels containing truncated subunits can reach the plasma membrane  <cit> .
this is not seen for mceph which is most likely due to the difference in length.
kv <dig> Δc <dig> might evade the rapid degradation by cellular control mechanisms since most of the protein remains.
the truncated protein can then cause more damage by retaining subunits or by forming defect channels.
this is supported by the inheritance patterns.
the r417stop mutation is dominant which suggests a new function for the truncated protein, while mceph is recessive, which indicates a loss of function.
the mechanism behind the brain growth in mceph/mceph is still unknown.
we now know that mceph is expressed in the brain, but we do not know if it is mceph or lack of functional kv <dig>  that is responsible for the overgrowth.
the kv <dig>  null mouse has not been reported to have an enlarged brain which suggests that mceph has an effect.
however, the low levels of protein and recessive inheritance indicates that the mceph mutation causes a loss of function of kv <dig> .
in line with both scenarios, it was previously shown that potassium channel dysfunction appears to reduce apoptosis  <cit> , which might contribute to the overgrowth of the mceph/mceph brain.
CONCLUSIONS
for the first time we report the expression of a truncated kv <dig>  protein in brain.
this protein is trapped within er and has the ability to interact with kv <dig> subunits.
the megencephaly mice, where the protein is expressed, constitute a tool to study kv <dig>  trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.

 the megencephaly mice have an base pair deletion in the shaker-like voltage-gated potassium channel subunit kv. this mutation causes progressive postnatal complex partial seizures and a unique pathologic brain overgrowth. the enlargement is not uniform but restricted to the hippocampus and ventral cortex, with 28% and 72% larger area compared to wildtype at weeks of age. the enlargement is in part due to that the numbers of both neurons and glia cells are dramatically increased in the hippocampus which is caused by increased proliferation and/or reduced apoptosis. the base pair deletion in kv leads to a frame shift and a premature stop codon. the predicted truncated kv protein will retain only the n-terminal domain, the first transmembrane domain and the first extracellular loop. voltage gated potassium channels form a diverse group of membrane proteins, regulating membrane potential, neuronal excitability and nerve signaling. the channels are hetero- or homotetramers, formed by a great variety of subunits, classified in subfamilies. kv belongs to the kv subfamily, consisting of eight members. kv, kv and kv are the most abundant kv subunits expressed in the brain. although the heteromeric structure means that a very large number of k channels can be formed in theory, the composition of the heteromultimers in the mammalian brain seems to be precisely regulated. a direct link between potassium channel dysfunction and apoptosis is that reduced intracellular potassium levels appear to promote critical events early in the suicide program. treatment with potassium channel blockers have been shown to block apoptosis in various cell types. however, neither kv mutations, the kv null mouse nor other epileptic models have previously been associated with pathologic brain overgrowth. therefore, we hypothesized that the truncated kv would be expressed and dysregulate other kv subunits in the mceph/mceph mice. truncated proteins are usually not expressed. this is because mrnas with a premature stop codon are degraded through nonsense-mediated mrna decay. one exception to this is genes with no introns such as the kv genes. in mceph/mceph mice in situ hybridization have shown that there is no decay but instead an increased expression of kv mrna in the hippocampus, cortex and ventral cortex. thus, it is possible that mceph is expressed. in humans kv point mutations are reported in patients with the autosomal dominant disorder episodic ataxia type,. most are missense mutations but there is one report of a premature stop codon, resulting in a kv protein that is truncated in the c-terminal. the patient carrying this mutation suffers from a drug resistant form of ea. truncated kv channels have previously been studied in cell culture and xenopus oocytes. when the kv Δc protein is expressed in cell culture it is trapped in the endoplasmic reticulum and degraded. however, full length kv in cell culture is also retained in the er. another experimental kv protein truncated in the extracellular loop between s and s is able to assemble with full length kv subunits in cell culture and the resulting complexes are trapped in the er. in xenopus oocytes both the n- and c-terminal truncated kv variants have a dominant negative effect on currents when coinjected with full length kv and kv. knowing if the mceph protein is expressed and how it interacts with other kv subunits would provide further understanding of kv trafficking and clues to the downstream effects seen in the mceph/mceph mice. in this paper we show that a truncated kv protein, mceph is expressed in the brain of the megencephaly mouse. the truncated protein is trapped in the er and does not reach the plasma membrane of neurons. mceph has the ability to form multimers with kv. it has a dominant negative effect on kv and kv currents in xenopus oocytes, but in the brain it does not appear to retain kv in the er.  antibody characterization a polyclonal antiserum was produced by immunization of rabbits with a synthetic peptide corresponding to amino acids to of kv. the n-terminal epitope was necessary to be able to detect the truncated mceph protein. western blot on brain lysate from wild type mice using the antiserum showed a strong band at kda but also several weaker bands. the kda band had the same size as that detected by the previously characterized monoclonal antibody against a c-terminal epitope of kv. preimmune serum did not recognize the kda band but showed the same weaker bands as the antiserum. the antiserum was affinity purified against the peptide used for immunization which reduced the number of unspecific bands significantly. preincubation of the affinity purified antibody with the peptide used for immunization absorbed the signal. the purified kv n-terminal antibody was used for all further experiments. in immunblotting of lysate from kv null mice an kda band was present but at a lower intensity than in wild type. since the null mouse does not have any kv protein this means that the antibody cross reacts with another protein. kv subunits have a high degree of similarity so it is likely that the antibody recognizes another kv subunit. the peptide used for immunization share a stretch of six amino acids with kv. both kv and kv are expressed in the brain as mature glycoproteins that are reported to be and kda, respectively. the shared amino acids and the size of the band makes kv the likely candidate for the cross reactivity. acetone post fixed wild type and kv -null brains were used for immunohistochemistry. in wild type the staining resembled kv but the background was high. the pattern in kv null brain was very similar to wild type, indicating cross reactivity. immunohistochemistry on formalin fixed wild type brain sections showed the same staining pattern that previously has been reported for kv. in the hippocampus the immunoreactivity was mainly localized in the fiber networks in the middle part of the molecular layer, in the hilus and the pyramidal cell layer of ca. when formalin fixed tissue was used the pattern in kv null was changed and clearly distinguishable from wild type. the conclusion is that the antibody recognizes kv. it is cross reactive when used for immunoblotting but this is greatly reduced when used for immunohistochemistry on formalin fixed tissue. mceph expression in the brain the bp deletion in kv in mceph/mceph mice leads to a premature stop codon. the predicted truncated protein, mceph, has a calculated weight of kda. to determine if mceph is expressed in the brain the polyclonal kv n-terminal antibody was used for immunoblotting and immunohistochemistry. immunoblotting showed a band at approximately kda in brain lysate from mceph/mceph mice. the intensity of the band was much lower than that of the kv protein in wild type mice suggesting a low expression of the truncated protein. the mceph/mceph mice do not have any full length kv but, like the kv null mice, they have the kda cross reactivity-caused band. however, the kda band was only present in mceph/mceph mice. no other band specific for mceph/mceph was detected. immunohistochemistry was performed on formalin fixed mceph/mceph mice brain sections. interestingly, in mceph/mceph there was staining only around the nuclei of cells, and no staining in fibers. there was some background staining resembling that of the kv null mouse but the overall pattern was significantly different. the main areas with mceph immunoreactivity were the hippocampus and ventral cortex. in the other areas examined there were only a few stained cell bodies. in the hippocampus mceph was expressed in neurons in the dentate gyrus, ca and in ca this pattern is in agreement with the previously reported mrna expression. taken together, the immunoblotting and immunohistochemistry results show that mceph is expressed in the brain of the megencephaly mouse. trafficking of mceph kv has a single n-glycosylation site on the extracellular loop between s and s and is expressed on the plasma membrane as a mature glycoprotein. this glycosylation site is preserved in mceph which makes it possible to study the trafficking of mceph by analyzing its glycosylation pattern. glycosylation starts in the er where a high mannose carbohydrate is transferred to an asparagine residue. this carbohydrate can be cleaved off by the glycosidases endoh and pngasef. next, the protein is transported to the golgi where the carbohydrate is modified and becomes resistant to endoh but not to pngasef. this is used as a marker for er to golgi transport. to investigate the glycosylation status, brain lysates from wild type and mceph/mceph mice were treated with the enzymes endoh and pngasef and analyzed with immunoblotting. for kv in wild type mice endoh did not affect the size of the kda band but pngasef reduced it with approximately kda. this is in agreement with other studies that have shown that the kda band is the mature glycosylated form of kv expressed in the plasma membrane. both endoh and pngasef reduced the apparent molecular weight of mceph with approximately kda. this strongly indicates that mceph is core glycosylated in the er but not transported to the golgi. neither mature nor unglycosylated mceph was detected in the brain. interactions of mceph kv channels are tetramers and the assembly of the kv subunits takes place in the er. mceph contains the n-terminal tetramerization domain and might thus bind other kv subunits. interactions with kv in the brain kv and kv are often associated in the same channels in the brain. we have previously reported a disturbance in the expression of kv in the mceph/mceph hippocampus. to investigate if this disturbance could be caused by an interaction between mceph and kv, immunoprecipitation followed by immunoblotting was performed on brain lysate from wild type, kv null and mceph/mceph mice. the monoclonal kv and kv antibodies each precipitated both kv and kv in wild type brain, verifying that correct experimental procedures were used. as a control the same antibodies were used for immunoprecipitation of lysate from kv null brain. as expected, in the kv null lysate no kv coprecipitated with kv. also, when the kv antibody was used for immunoprecipitation neither kv nor kv was detected. the kv -n-terminal antibody precipitated kv and kv in wild type but also kv in kv null lysate. this means that it is not specific when used for immunoprecipitation. for mceph/mceph, immunoprecipitation was performed with anti-kv whereas the kv -n-terminal antibody was used for immunoblotting. with this approach it was not possible to detect any mceph co-precipitating with kv. this suggests that there is no persistent interaction between kv and mceph in the brain. on western blots, kv in brain appears as two bands at and kda representing core and mature glycosylated form, respectively. if mceph binds and traps kv in the er, the amount of core glycosylated kv would be expected to increase and the mature glycosylated form decrease. immunoblotting was performed on lysates from both whole brains and isolated hippocampi. the hippocampus was investigated since mceph is expressed primarily in this brain region. most of the kv protein in the hippocampus had mature glycosylation and there was only a very small amount of core glycosylated protein. no difference in kv core glycosylation was seen between wild type and mceph/mceph in neither whole brain nor hippocampus lysate. when the immunoprecipitation and glycosylation data is combined it appears that mceph does not retain kv in the er in brain. interactions with kv in hek cells to test if mceph can form multimers its ability to interact with wild type kv was investigated by overexpression in cell culture. this system was chosen to allow higher expression levels and easier detection. for this experiment kv and mceph were cloned into the vectors pzsgreen and pdsred to construct fluorescent fusion proteins. hek293a cells were transfected with the mceph-zsgreen and the kv -dsred constructs together or separately and analyzed hours after transfection. the green and the red fluorescent signals were colocalized around the cell nucleus. no staining was observed on the plasma membrane. western blot with the polyclonal kv n-terminal antibody on lysate from cotransfected hek cells showed bands at and kda corresponding to the mceph and kv fusion proteins. corresponding bands were found in cells transfected with single constructs which confirmed the identity of the bands. to investigate the interaction between mceph and kv, immunoprecipitation was performed on cell lysate from cotransfected cells using the monoclonal kv c-terminal antibody. immunoblotting with the polyclonal kv n-terminal antibody detected the mceph-zsgreen fusion protein as well as the kv -dsred fusion protein. since the c-terminal kv antibody is specific for wild type kv the detected mceph protein was associated with the kv protein. interactions with kv and kv in xenopus oocytes to further investigate the ability of mceph to interact with kv subunits we chose to analyze the effects of expressing mceph in combination with kv or kv in xenopus laevis oocytes. figure 5a shows the kv and kv currents associated with a series of voltage steps, followed by a step to + mv. both channels show delayed rectifier behaviour and lack fast inactivation. kv, however, shows a marked slow inactivation. mceph was found to reduce the expression of both kv and kv in the plasma membrane. figure 5b shows the mean results of injecting mceph mrna together with kv or kv mrna given as the effect on peak conductance versus voltage curves. as seen, the curves for kv and kv are down-scaled by about and 60%, respectively, without marked changes in slope or midpoint value. the mceph shift of the kv activation curve was slightly bigger than that of kv. the activation time course of the currents associated with voltage steps was negligibly affected by mceph, reflected in the unaffected time to peak current at + mv for both kv and kv. similar effects were observed for the mceph effect on the slow inactivation kinetics. mean inactivation versus voltage curves, measured from the peak current associated with the second pulse are shown in figure 5c. the mceph shift of the kv inactivation curve was slightly bigger than that of kv. in summary, the measurements show that mceph has a dominant negative effect on both kv and kv channel expression in xenopus oocytes. furthermore, the small effects on activation and inactivation kinetics suggest that mceph does not form functional channel complexes with kv or kv subunits in the plasma membrane. heteromeric complexes involving mceph subunits are expected to drastically affect channel kinetics. discussion in this study we show that mceph/mceph mice express a truncated kv, mceph. the mceph protein is expressed in neurons throughout the brain but is trapped in the er. mceph is able to bind full length kv in cell culture and has a dominant negative effect when coexpressed with kv and kv in xenopus oocytes. in contrast, in the brain mceph does not appear to retain kv in the er. to be able to detect the truncated mceph protein an antibody against the n-terminal part of kv was generated. immunoblotting on brain lysate from mceph/mceph mice showed a band that has the same size as the predicted mceph protein, approximately kda. this band is present in all mceph/mceph brains studied but not in any wild type or kv null mice. based on this we conclude that mceph is expressed in the brain of the megencephaly mouse. kv in the brain is a mature glycoprotein that is mostly localized to axons and nerve terminals. truncated kv variants have previously been shown to be retained in the er in cell culture. the draw back of these studies is that full length kv homomers in cell culture is also largely retained in the er since they are undetectable at the cell surface by immunohistochemical methods but detectable at low levels with electrophysiological methods. lack of surface expression was seen by us both in hek cells and in the neuron-like cell line pc. the reason is a strong er retention motif in the pore region. the mceph/mceph mouse provides an opportunity to study the trafficking of a truncated kv in neurons. both endoh and pngasef treatment reduced the molecular weight of mceph by approximately kda, suggesting that the protein is core glycosylated. we detected neither maturely glycosylated nor unglycosylated mceph. the glycosylation data suggest that mceph is trapped in the er and not further transported. this is supported by immunohistochemistry experiments were mceph immunoreactivity surrounds the nuclei in an er-like pattern. there is no immunoreactivity in fibers consistent with the lack of mature glycosylated mceph. the low amount of mceph protein detected with immunoblotting compared to the high levels of mrna suggests that the mceph protein is rapidly degraded. this is most likely due to er-associated degradation, a quality control mechanism in the er that recognizes misfolded proteins. these proteins are retro-translocated to the cytosol and degraded by proteasomes. we have previously seen disturbances of staining pattern of kv and kv in the mceph/mceph hippocampus, which was not due to transcriptional regulation. previous studies of other truncated k variants have shown that they have a dominant negative effect in oocytes and form oligomers with full length kv subunits in cell culture. we hypothesized that mceph interacts with kv subunits and causes the expression disturbance possibly by retaining these subunits in the er. kv often forms heterotetramers with kv subunits in the brain. nevertheless, we found no evidence for a persistent interaction between mceph and kv in the brain, suggesting that there is no retention of kv in the er. however, we found mceph to be able to oligomerize when expressed in hek cells. moreover, electrophysiological measurements on xenopus oocytes showed that mceph reduces both kv and kv currents, suggesting interaction between mceph and kv subunits in this cell type. this indicates that the lack of er retention seen for kv in the brain is not caused by an inability of mceph to form multimers it can be assumed that lack of kv could lead to defect trafficking of kv channels. this is not the case since the kv null mouse has normal expression of kv in the hippocampus and also the cerebellum and sciatic nerve. one possibility is that the kv and kv expression disturbances in mceph/mceph hippocampus are related to the growth. the mceph/mceph hippocampus is significantly enlarged and has an increased amount of neurons. this could lead to disturbed organization of the dendritic trees and give rise to the abnormal expression patterns. also, since mceph contains the n-terminal t domain, which has been shown to mediate beta subunit association, it is possible that mceph sequesters these auxiliary subunits within the er and prevents them from forming complexes with kv and kv in mceph/mceph mice. beta subunit association has been shown to promote kv trafficking to the cell surface as well as to cause axonal targeting of kv. the megencephaly mouse is not a model for any known human disease. still, it provides a unique tool to study kv channels in the brain. the ea mutation r417stop gives rise to a kv protein that lacks the final amino acids in the c-terminal domain. the kv Δc protein and mceph have some similarities but there are major differences. both truncated proteins have a dominant negative effect in oocytes and can assemble with full length subunits in cell culture. for the kv Δc change in current kinetics when coinjected with kv in xenopus oocytes indicates that some channels containing truncated subunits can reach the plasma membrane. this is not seen for mceph which is most likely due to the difference in length. kv Δc might evade the rapid degradation by cellular control mechanisms since most of the protein remains. the truncated protein can then cause more damage by retaining subunits or by forming defect channels. this is supported by the inheritance patterns. the r417stop mutation is dominant which suggests a new function for the truncated protein, while mceph is recessive, which indicates a loss of function. the mechanism behind the brain growth in mceph/mceph is still unknown. we now know that mceph is expressed in the brain, but we do not know if it is mceph or lack of functional kv that is responsible for the overgrowth. the kv null mouse has not been reported to have an enlarged brain which suggests that mceph has an effect. however, the low levels of protein and recessive inheritance indicates that the mceph mutation causes a loss of function of kv. in line with both scenarios, it was previously shown that potassium channel dysfunction appears to reduce apoptosis, which might contribute to the overgrowth of the mceph/mceph brain.  for the first time we report the expression of a truncated kv protein in brain. this protein is trapped within er and has the ability to interact with kv subunits. the megencephaly mice, where the protein is expressed, constitute a tool to study kv trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth. 
interactions between mceph and other kv subunits were studied in cell culture, xenopus oocytes and the brain. mceph can form tetramers with kv in cell culture and has a dominant negative effect on kv and kv currents in oocytes. however, high kv mrna levels in mceph/mceph brain indicated that it escaped this control mechanism. mceph was found to lack mature glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum. therefore, we hypothesized that the truncated kv would be expressed and dysregulate other kv subunits in the mceph/mceph mice. we found that the mceph protein is expressed in the brain of mceph/mceph mice. the predicted mceph protein is truncated at amino acid out of truncated proteins are usually not expressed since nonsense mrnas are most often degraded. the megencephaly mice express a truncated kv in the brain, and constitute a unique tool to study kv trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.
 the megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. the responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel kv. the predicted mceph protein is truncated at amino acid out of truncated proteins are usually not expressed since nonsense mrnas are most often degraded. however, high kv mrna levels in mceph/mceph brain indicated that it escaped this control mechanism. therefore, we hypothesized that the truncated kv would be expressed and dysregulate other kv subunits in the mceph/mceph mice.  we found that the mceph protein is expressed in the brain of mceph/mceph mice. mceph was found to lack mature glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum. interactions between mceph and other kv subunits were studied in cell culture, xenopus oocytes and the brain. mceph can form tetramers with kv in cell culture and has a dominant negative effect on kv and kv currents in oocytes. however, it does not retain kv in the er of neurons.  the megencephaly mice express a truncated kv in the brain, and constitute a unique tool to study kv trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth. 