The Role of Presenilin and its Interacting Proteins in the Biogenesis of Alzheimer 's Beta Amyloid The biogenesis and accumulation of the beta amyloid protein -LRB- Aβ -RRB- is a key event in the cascade of oxidative and inflammatory processes that characterises Alzheimer 's disease . The presenilins and its interacting proteins play a pivotal role in the generation of Aβ from the amyloid precursor protein -LRB- APP -RRB- . In particular , three proteins -LRB- nicastrin , aph-1 and pen-2 -RRB- interact with presenilins to form a large multi-subunit enzymatic complex -LRB- γ-secretase -RRB- that cleaves APP to generate Aβ . Reconstitution studies in yeast and insect cells have provided strong evidence that these four proteins are the major components of the γ-secretase enzyme . Current research is directed at elucidating the roles that each of these protein play in the function of this enzyme . In addition , a number of presenilin interacting proteins that are not components of γ-secretase play important roles in modulating Aβ production . This review will discuss the components of the γ-secretase complex and the role of presenilin interacting proteins on γ-secretase activity . Introduction : the generation of beta amyloid by presenilin and its binding proteins Alzheimer 's disease -LRB- AD -RRB- is a complex , progressive neurodegenerative disorder that is neuropathologically characterised by extensive neuronal loss and the presence of neurofibrillary tangles and senile plaques . While the majority of AD cases are sporadic , ∼ 5 % of AD cases are familial -LRB- FAD -RRB- with mutations in three genes , amyloid precursor protein , presenilin 1 -LRB- PS1 -RRB- and presenilin 2 -LRB- PS2 -RRB- accounting for the majority of cases . A major feature of both sporadic and familial forms of AD , is the accumulation and deposition of a small peptide referred to as beta amyloid -LRB- Aβ -RRB- within brain tissue of AD sufferers . The mechanisms that underlie the disease processes are poorly understood . However , the accumulation of Aβ is thought to play a pivotal role in neuronal loss or dysfunction through a cascade of events that include the generation of free radicals , mitochondrial oxidative damage and inflammatory processes -LRB- reviewed in Refs . -LSB- 1 , 2 -RSB- -RRB- . The primary event that results in the abnormal accumulation of Aβ is thought to be the dysregulated proteolytic processing of its parent molecule , the amyloid precursor protein -LRB- APP -RRB- . The APP molecule is a transmembrane glycoprotein that is proteolytically processed by two competing pathways , the non-amyloidogenic and amyloidogenic -LRB- Aβ forming -RRB- pathways . How these pathways are regulated remains unclear . However , there are many factors including diet , hormonal status , and genetic mutations that influence the processing of APP to generate Aβ -LRB- reviewed in Refs . -LSB- 1 , 3 -RSB- -RRB- . Three major secretases are postulated to be involved in the proteolytic cleavage of APP . These include α-secretase -LRB- of which the metalloproteases ADAM17/TACE and ADAM 10 are likely candidates -RRB- , beta APP cleaving enzyme -LRB- BACE , formally known as β-secretase -RRB- and the γ-secretase . The α-secretase cleaves within the Aβ domain of APP thus precluding the formation Aβ and generating non-amyloidogenic fragments and a secreted form of APP -LRB- α-APPs -RRB- . In the amyloidogenic pathway -LRB- Fig. 1 -RRB- , BACE cleaves near the N-terminus of the Aβ domain on the APP molecule , liberating another soluble form of APP , β-APPs and a C-terminal fragment -LRB- C99 -RRB- containing the whole Aβ domain . The final step in the amyloidogenic pathway is the intramembranous cleavage of the C99 fragment by γ-secretase , to liberate the Aβ peptide -LRB- reviewed in Ref . -LSB- 1 -RSB- -RRB- . Identification of multiple cleavage sites within the APP molecule has suggested the presence of other secretases , namely ε - and ζ-secretase ; that generate the APP intracellular domain -LRB- AICD -RRB- and a longer fragment of Aβ -LRB- Aβ46 -RRB- , respectively -LSB- 4 -- 7 -RSB- . Although different enzymes may exists , the same γ-secretase complex that generates Aβ may also generate AICD and Aβ46 as these products are inhibited by γ-secretase inhibitors -LSB- 4 -- 7 -RSB- indicating that this enzyme has multiple cleavage sites . However , over-expression and mutagenesis studies have shown that the production of Aβ and AICD may be independent catalytic events -LSB- 8 , 9 -RSB- . Fig 1Formation of the γ-secretase complex and Aβ generation . A schematic representation of the proposed formation of the γ-secretase complex and the enzymatic activity resulting in Aβ generation . Evidence has been provided to show that PS , nicastrin -LRB- NCT -RRB- , aph-1 , and pen-2 are the only components of the γ-secretase complex . Nicastrin and aph-1 are involved in the early formation of the complex , interacting with each other and forming a dimeric sub-complex . The presenilin holoprotein is incorporated into the aph-1 : NCT sub-complex , nicastrin undergoes post-translational modifications and the complex is transported to the cell surface -LRB- or other Aβ generating compartments , i.e. TGN -RRB- as a trimeric complex . Pen-2 is incorporated into the complex and presenilin is cleaved forming the active γ-secretase complex . Following cleavage of APP by BACE to liberate secreted APP , the APP-C99 fragment left embedded within the membrane undergoes regulated intramembranous processing -LRB- RIP -RRB- by the γ-secretase complex , liberating Aβ1 -- 40 or Aβ1 -- 42 -LRB- Modified from Ref . -LSB- 165 -RSB- -RRB- The γ-secretase enzyme is thought to be an aspartyl protease that has the unusual ability to regulate intramembrane proteolysis -LRB- RIP -RRB- for a growing list of type 1 integral membrane proteins which include , APP , APP like proteins -LRB- APLPs -RRB- , E-Cadherin , CD44 , lipoprotein receptor related protein -LRB- LRP -RRB- , Notch , sterol regulatory element-binding protein -LRB- SREBP -RRB- , interferon response element -LRB- IRE1 -RRB- and activated transcription factor 6 -LRB- ATF-6 -RRB- -LRB- for recent review see -LSB- 10 -RSB- -RRB- . The mechanism of RIP and γ-secretase activity is unknown . However a transient hydrophilic environment for catalysis within the lipid membrane must be created . Furthermore , the enzyme -LRB- or an enzyme domain -RRB- must have the ability to bend and unwind the α-helical substrates , exposing their amide bonds to hydrolysis . It is therefore conceivable that the γ-secretase enzyme is an integral protein of the lipid bi-layer and contains a number of proteins that may have different functions within an enzyme complex . The exact conformation or molecular architecture of the γ-secretase enzyme remains unknown . However , large molecular mass complexes of ∼ 250 kDa -LSB- 11 , 12 -RSB- , ∼ 500 kDa -LSB- 13 -- 17 -RSB- and ∼ 2,000 kDa -LSB- 13 , 18 -RSB- have been identified . Over the last few years biochemical and genetic approaches have identified four components of the γ-secretase complex , presenilins , nicastrin , anterior pharynx defective -LRB- aph-1 -RRB- and presenilin enhancer 2 -LRB- pen-2 -RRB- . Over-expression and expression knockdown studies have provided strong evidence that these proteins are essential for γ-secretase activity -LSB- 15 , 19 -- 24 -RSB- . Subsequent reconstitution studies in non-mammalian cells have provided evidence that these are the only components responsible for γ-secretase catalytic activity -LSB- 25 -- 27 -RSB- . However the active component and the active catalytic site of this enzymatic complex are yet to be identified . The roles of presenilin and its binding partners within the γ-secretase complex in Aβ generation Presenilin 1 : the catalytic component of the γ-secretase enzyme Presenilin 1 -LRB- PS1 -RRB- is thought to be the most critical component of the γ-secretase complex as evidence indicates that it is the catalytic component of this proteolytic enzyme . The finding that mutations in PS1 account for the majority of inherited early onset forms of AD and result in the overproduction of the highly amyloidogenic Aβ42 -LSB- 28 -- 30 -RSB- provided the initial evidence that PS1 facilitates γ-secretase activity . Subsequent studies showed that PS1 ablation or mutagenesis of two highly conserved aspartate residues within transmembrane domains 6 and 7 of PS1 results in a reduction in Aβ levels -LSB- 31 -- 34 -RSB- . Furthermore , aspartyl protease inhibitors and transition state analogue inhibitors which are designed to target the active site of the protease , all reduce Aβ40 and Aβ42 levels and have been shown to affinity label and bind to PS1 -LSB- 35 -- 37 -RSB- . In addition , physical interactions between PS1 and the APP-C99 fragment have been identified -LSB- 38 -- 40 -RSB- . The studies outlined above provide substantial evidence to indicate that PS1 is the catalytic subunit of γ-secretase . However , this transmembrane protein is not typical of a conventional aspartyl protease , and lacks the typical D -LRB- T/S -RRB- G motif for an aspartyl protease active site -LRB- recently reviewed in -LSB- 41 -RSB- . Aspartyl proteases require the presence of two aspartate residues for the hydrolysis of the peptide bond . Although PS1 lacks the typical D -LRB- T/S -RRB- G aspartyl protease active site it does contain the two aspartyl residues -LRB- D257 and D385 -RRB- which are either critical for the active site on the γ-secretase complex or constitute the active site -LSB- 42 -RSB- . Other proteases that contain atypical aspartyl protease motifs and have weak homology with PS1 include the bacterial membrane embedded aspartyl protease , type 4 prepilin protease and the signal peptidases , which are all transmembrane proteins . These proteins share the unusual aspartyl motif GxGD and perform intramembranous cleavage of type I membrane proteins -LRB- reviewed in -LSB- 43 -RSB- -RRB- . The topology and atypical characteristics of these proteins make them an unprecedented class of protease and as is the case for PS1 , would require interaction with of other proteins within the complex . The interaction between the components of the γ-secretase complex is not sufficient to create a macromolecular enzyme complex such as γ-secretase -LRB- i.e. total mass of all the components is ∼ 200 kDa -RRB- . Thus , dimer or trimer formation must occur within the complex . To date the only member of the complex that is known to undergo this process is PS1 . The full length PS1 protein is rapidly endoproteolytically cleaved within its characteristic large hydrophilic loop into amino - and carboxy-terminal fragments -LRB- NTF/CTF -RRB- of ∼ 27 and ∼ 17 kDa , respectively -LSB- 44 , 45 -RSB- . These fragments are thought to interact with each other to form the catalytic component of the γ-secretase enzyme since in the absence of full length PS1 , co-expressed NTF and CTF were able to generate γ-secretase activity in presenilin deficient mammalian cells -LSB- 46 , 47 -RSB- . The stoichiometry and the nature of the interaction between these fragments remain unclear . It has been shown by many studies that the NTF : CTF form a heterodimer in mammalian cells -LSB- 12 , 48 -- 51 -RSB- leading to suggestions that this heterodimer is the active γ-secretase -LSB- 52 -RSB- . However , Cervantes and colleagues -LSB- 53 -RSB- provided evidence that the presenilin fragments can form a tetramer by identifying heterodimers as well as NTF and CTF homodimers in yeast . Evidence for heterodimer and NTF homodimer -LRB- but not CTF homodimer -RRB- formation has been provided by photo-affinity labelled cross-linking studies -LSB- 54 -RSB- . This formation provides a core of aspartyl residues required for aspartyl protease activity . However , it has yet to be established whether the hypothetical `` core '' is formed between fragments from one PS1 molecule or multiple molecules within the complex . Further , if PS1 is the `` catalytic core '' of the γ-secretase complex the roles of the other interacting proteins need to be determined . Nicastrin : the substrate docking site and scaffolding for building the γ-secretase complex Nicastrin was discovered to be part of the γ-secretase complex when it was found to co-immunoprecipitate with PS1 -LSB- 55 -RSB- . Nicastrin is a type I transmembrane protein with a 670 amino acid long hydrophilic N-terminal domain , a transmembrane domain , and a relatively short cytoplasmic C-terminus of twenty amino acid residues -LSB- 55 -RSB- . The protein undergoes glycosylation and sialylation within the secretory pathway to yield a mature ∼ 150 kDa protein , the largest component of the γ-secretase complex -LSB- 14 -RSB- , -LSB- 56 -- 58 -RSB- . Nicastrin has been shown to be a critical component of the γ-secretase complex as inhibition of nicastrin function in vitro and in vivo results in a complete loss of APP and Notch cleavage -LSB- 14 , 55 , 59 -- 61 -RSB- . Within this complex , nicastrin appears to be involved in the early formation of the complex , particularly in the formation of the first sub-complex between itself and aph-1 -LRB- Fig. 1 -RRB- . Recent evidence suggests that nicastrin is critical for the correct assembly of the γ-secretase complex within the endoplasmic reticulum and the intracellular trafficking of the complex to the cell surface -LSB- 62 , 63 -RSB- . Furthermore , nicastrin is essential for the interaction between the complex and APP-C99 and thus may act as a receptor for γ-secretase substrates -LSB- 63 , 64 -RSB- . Therefore , although nicastrin is a critical component , evidence to date suggests that it is unlikely to be a catalytic component of the γ-secretase complex . Instead , nicastrin acts as a scaffold for the building of the active complex , and a possible substrate docking site . Anterior pharynx defective 1 -LRB- aph-1 -RRB- : scaffolding partner for nicastrin and may have possible catalytic activity The aph-1 protein is a ∼ 30 kDa , 308 amino acid long 7-transmembrane protein -LSB- 65 -RSB- existing in two homologous forms , located on chromosome 1 and chromosome 15 -LRB- aph-1a & aph-1b respectively ; -LSB- 19 -RSB- -RRB- . In addition , as the result of alternate splicing , a long and short isoform of aph-1a have also been reported , with the short isoform more abundantly expressed in most tissues -LSB- 21 -RSB- . Although the exact function of these isoforms remains unknown , multiple γ-secretase complexes containing aph-1 isoforms are thought to exist -LSB- 66 , 67 -RSB- . The aph-1a isoform appears to be incorporated into active γ-secretase complexes . Deletion of aph-1a causes a reduction in Aβ generation -LSB- 66 -RSB- and is the major isoform present in γ-secretase complexes during embryonic development -LSB- 67 -RSB- . The γ-secretase complexes incorporating aph-1b are thought to have a redundant function as aph-1b has been found not to be involved in the regulation of other γ-secretase components or γ-secretase activity -LSB- 66 -RSB- . The aph-1 protein forms a stable and intermediate ∼ 140 kDa complex with Nicastrin , before binding PS1 . Aph-1 has been shown to interact with immature and mature forms of presenilins and nicastrin -LSB- 21 -RSB- . The GXXXG motif located within transmembrane 4 of aph-1 has been shown to be important in this interaction -LSB- 68 -RSB- , -LSB- 69 -RSB- . The aph-1 : nicastrin sub-complex appears to play a role in the stabilisation and assembly of PS1 in the γ-secretase complex -LRB- Fig 1 ; -LSB- 24 , 70 , 68 -RSB- -RRB- . However , the exact role of aph-1 in the γ-secretase complex remains unknown . Expression studies have shown that removal of aph-1 destabilises the presenilin holoprotein and the addition of aph-1 leads to the accumulation of the holoprotein -LSB- 11 , 19 -- 24 , 71 -RSB- . Thus together with nicastrin , aph-1 is thought to contribute the initial formation and trafficking of the γ-secretase complex and thereby provide a scaffold for the proteolytic processing of PS1 and the formation of the active site . Aph-1 is critical for the activity of the fully constructed γ-secretase complex . Evidence has been provided that as well as binding to immature components of γ-secretase in early stages of complex formation , aph-1 also interacts with the mature forms of PS1 , nicastrin and pen-2 -LSB- 68 , 72 -RSB- . Furthermore , recent evidence has been provided to suggest that this interaction occurs on the cell surface where it also binds the γ-secretase substrate , Notch and facilitates its cleavage -LSB- 72 -RSB- . These studies suggest that apart from the scaffolding function it shares with nicastrin , aph-1 has another function within the fully constructed , active γ-secretase complex . Structural and functional similarities between aph-1 and other proteases that possess the ability for intramembranous cleavage -LRB- such as rhomboid , -LSB- 68 , 73 -RSB- suggests that this transmembrane protein may have an enzymatic function within the complex . This postulated novel function for aph-1 needs to be validated by future studies . Presenilin Enhancer 2 -LRB- pen-2 -RRB- : the elusive presenilinase or `` molecular clamp '' that holds the complex together ? The pen-2 protein is a ∼ 12 kDa , 101 amino acid long protein containing two transmembrane domains and its N - and C-terminals facing the lumen of the endoplasmic reticulum -LSB- 74 , 75 -RSB- . Pen-2 has been shown to interact with Nicastrin , aph-1 , and also PS1 . Without this interaction pen-2 has been shown to be degraded , possibly by the proteasome -LSB- 76 , 77 -RSB- . Ablation of pen-2 results in significantly reduced PS1 endoproteolysis and Aβ production , suggesting that pen-2 is essential for both presenilinase and γ-secretase activity -LSB- 15 , 19 , 22 -- 24 -RSB- . Likewise , combinational expression studies have shown that when pen-2 is co-expressed with the other components of the γ-secretase complex in a step-wise manner , endoproteolysis of the PS1 holoprotein was enhanced -LSB- 11 , 22 , 24 -- 27 , 71 -RSB- . A trimeric sub-complex between presenilin , nicastrin and aph-1 has been identified in cells lacking pen-2 -LSB- 24 , 70 -RSB- suggesting that pen-2 may be the final protein incorporated into the γ-secretase complex -LRB- Fig. 1 -RRB- . Recent evidence has suggested that pen-2 is incorporated into the γ-secretase complex through an interaction with presenilins . The `` DYLSF '' domain of pen-2 and a `` NF '' motif on transmembrane 4 of PS1 have been shown to be critical for the interaction between these proteins -LSB- 78 -- 80 -RSB- . The cleavage of PS1 occurs within the hydrophilic loop and is distal from the interaction site on transmembrane 4 , suggesting that another domain of pen-2 is responsible for the cleavage of PS1 or pen-2 may present PS1 to the elusive `` presenilinase '' which may be another member of the γ-secretase complex . The C-terminal end of pen-2 has been shown to be important for γ-secretase activity since altering the length of this region by addition or deletion of residues has been shown to reduce Aβ40 and 42 generation without altering the binding of pen-2 to the complex -LSB- 78 -RSB- suggesting that pen-2 may have an alternative function within the complex . One suggestion put forward by Hasegawa and colleagues -LSB- 78 -RSB- is that the C-terminus of pen-2 may be a linker/space molecule that maintains spatial interactions between proteins within the complex . However , recent evidence using pen-2 C-terminal loss of function mutations suggest that the C-terminus acts as a `` molecular clamp '' holding together the presenilin fragments and the whole γ-secretase complex -LSB- 81 -RSB- . If this is the case then PEN-2 is an integral part of the catalytic process holding the complex together whilst the γ-secretase products are generated . The newest member of the complex : TMP21 , a modulator of γ-secretase activity Recently another member of the γ-secretase complex was immuno-purified from wild-type PS1 and PS2 blastocyte derived cells -LSB- 82 -RSB- . The type 1 transmembrane protein termed TMP21 is a member of the p24 cargo-family which may have a signalling role in the sorting and transport of proteins from the endoplasmic reticulum to the Golgi -LSB- 83 , 84 -RSB- . It was shown by Chen and colleagues -LSB- 82 -RSB- that TMP21 is a member of the complex as it was isolated in a high molecular weight presenilin complex , interact with all of the known components of the γ-secretase complex , co-localised with the complex components in the ER , Golgi and cell surface and destabilised from the complex in the absence of the presenilins and pen-2 . Although the over-expression of TMP21 did not alter γ-secretase activity , its suppression resulted in and increase in Aβ40 and Aβ42 . However , suppression of TMP21 did not alter the production of AICD , notch cleavage to generate NICD or cleavage of E-cadherin to generate CICD . These results are consistent with the notion that γ - and ε-secretase cleavage activities are independently regulated and indicate a role for TMP21 in modulating γ-secretase activity to generate Aβ . This role for TMP21 appears to be independent of its role in protein transport since the suppression of both TMP21 and p24a -LRB- a member of the p24 cargo family that interacts with TMP21 -RRB- does not result in additional increases in Aβ production to that observed following the suppression of TMP21 only -LSB- 82 -RSB- . This finding led the authors to postulate that there are two pools of TMP21 , a major pool that is stabilised by , p24a and has no role in Aβ production and a minor pool that modulates Aβ production , independent of p24a . Although , further investigation is required to determine the precise mechanism of action , it appears that TMP21 may function to regulate intramembrane proteolysis controlling γ-secretase activity and thus preventing the over-production of Aβ . This modulator role for TMP21 is important to consider when elucidating mechanisms on how γ-secretase activity is altered -LRB- for example by presenilin mutations -RRB- resulting in enhanced Aβ40 and Aβ42 production . Presenilin 2 -LRB- PS2 -RRB- : capable of forming a fully functional γ-secretase complex The presenilin proteins share an overall homology of 67 % with the highest similarities in the predicted transmembrane domains . Fewer similarities are found at the N-terminus and in the central region of the hydrophilic loop -LSB- 85 , 86 -RSB- . Although there are some structural similarities between PS1 and PS2 several lines of evidence suggest that these proteins may have distinct functions . Mice lacking PS1 die before birth and have severe skeletal and brain deformities , whilst those lacking PS2 only develop a mild pulmonary fibrosis and haemorrhage with age -LSB- 33 , 87 -- 90 -RSB- . Compared to neuronal cultures isolated from PS1 ablated mice , those isolated from PS2 knockout mice exhibit higher Aβ production and are less sensitive to γ-secretase inhibitors -LSB- 89 , 91 -RSB- . Lai and colleagues -LSB- 91 -RSB- also provided evidence to suggest that distinct PS1 containing and PS2 containing complexes exist -LSB- 91 -RSB- . If this is the case , evidence to date suggests that PS2 containing complexes have different functions and have less γ-secretase processing power than PS1 containing complexes . However , recent in vitro evidence has suggested that chimeras of PS1 and PS2 fragments retain γ-secretase activity -LSB- 92 -RSB- suggesting that active complexes containing both PS1 and PS2 could occur . However , this is unlikely to occur in vivo as PS2 and PS1 transgenic mice have differential effects on γ-secretase activity -LSB- 93 -RSB- . In this study mice lacking PS1 were crossbred with transgenic mice containing wild type or mutant PS2/PS1 . Compared to mice harbouring wild-type PS1 , those harbouring wild-type PS2 had a ∼ 4-fold reduction in Aβ40 and ∼ 2-fold reduction Aβ42 brain levels . In addition , the authors reported that although mutations in PS1 lead to higher brain levels of Aβ40 and Aβ42 than PS2 mutations , those mice harbouring PS2 mutations had higher brain levels of Aβ42 , whilst Aβ40 brain levels remain unchanged -LSB- 93 -RSB- . Taken together these studies suggest that PS1 containing complexes predominate in generating Aβ40 and Aβ42 . However , the finding that PS2 is involved in Aβ42 -LRB- but not Aβ40 -RRB- generation suggests that two Aβ generating γ-secretase complexes -LRB- γ-40 secretase and γ-42 secretase -RRB- exist , with γ-42 secretase complexes containing both PS1 and PS2 . The roles of presenilin binding proteins not incorporated into the active γ-secretase complex In addition to Aβ generation , the presenilins have been implicated in a variety of intracellular processes including membrane trafficking , Notch signalling , neuronal plasticity , cell adhesion , regulation of calcium homeostasis , the unfolded protein response , and apoptosis . Therefore it is not surprising that the presenilins have a growing list of binding partners -LRB- see Table 1 -RRB- . Though the presenilin binding proteins that are incorporated into the γ-secretase complex are critical for the activity of this enzyme , those that are not integral components have equally important roles in modulating Aβ production . A number of non-complex presenilin binding proteins have been shown to alter γ-secretase activity -LRB- see Table 1 -RRB- . However , only a few have had supporting evidence that their direct interaction with the presenilins modulate γ-secretase activity . These are described below and shown in Fig. 2 . Table 1Presenilin interacting proteinsInteracting proteinProposed functionInteraction with presenilins shown to modulate Aβ production ReferenceComplex componentsAPH-1Component of the γ-secretase complex ; role for initial formation of the complex , or a proteolytic roleYes -LSB- 19 , 65 , 67 , 123 -RSB- . NicastrinComponent of γ-secretase complex ; possible role as a receptor for APP-C99Yes -LSB- 14 , 55 -- 58 -RSB- . PEN-2Component of the γ-secretase complex , role in proteolytic cleavage of the presenilins Yes -LSB- 19 , 24 , 70 , 76 , 77 -RSB- . TMP21Recently identified component of the γ-secretase complex , role in modulating Aβ productionYes -LSB- 82 -RSB- γ-secretase substratesAPP-C99Substrate for the γ-secretase complex : precursor to Aβ generationYes -LSB- 38 -- 40 -RSB- E/N cadherinγ-secretase substrate ; role in Cell-Cell AdhesionNo -LSB- 124 -RSB- IRE1γ-secretase substrate ; role in unfolded protein responseNo -LSB- 125 , 126 -RSB- LRPγ-secretase substrate ; Lipid metabolismYes -LSB- 119 -RSB- Notch1 γ-secretase substrate ; precursor to NICD generationYes -LSB- 112 -RSB- Interacting binding proteinsABP-280 , Filamin homolog1Actin binding proteinNo -LSB- 127 -RSB- Bcl-2 Anti-apoptotic moleculeNo -LSB- 128 -RSB- Bcl-XLAnti-apoptotic moleculeNo -LSB- 129 -RSB- CalmyrinPossible calcium-myrstol switchNo -LSB- 130 -RSB- CALP - calsenilin like proteinNovel member of the calsenilin/KChIp protein familyNo -LSB- 131 -RSB- CalsenilinCalcium binding proteinYes -LSB- 94 , 96 -- 99 , 101 , 102 -RSB- CLIP-170Linking membrane organelles to microtubules No -LSB- 132 -RSB- DRALLIM-domain containing proteinNo -LSB- 133 -RSB- FHL2Role in PI3K/Akt activationNo -LSB- 134 -RSB- FKBP38Role in mitochondria mediated apoptosis No -LSB- 135 -RSB- GoSignalling moleculeNo -LSB- 136 -RSB- GSK3βWnt signalling , serine threonine protein kinase No -LSB- 137 -RSB- HC5/ZETASubunits of the catalytic 20S proteasomeYes -LSB- 138 -RSB- Met1Putative methyltransferaseNo -LSB- 139 -RSB- Modifier of cell adhesion protein -LRB- MOCA -RRB- Regulates proteasomal activity on APPYes -LSB- 140 -RSB- Omi/HtrA2Serine protease , proapoptoticNo -LSB- 141 -RSB- PAGNeuronal proliferation proteinNo -LSB- 142 -RSB- PAMP and PARLMetalloproteases No -LSB- 143 -RSB- PKASerine/Theronine protein kinase ; β-catenin phosphorylationNo -LSB- 144 -RSB- PlakoglobinRole in b-catenin/Tcf -4 activityNo -LSB- 145 -RSB- PLD1Phospholipid-modifying enzymeYes -LSB- 146 -RSB- PSAPPDZ like proteinNo -LSB- 147 -RSB- QM/Jif1Negative regulator of c-JunNo -LSB- 148 -RSB- Rab proteinsVesicle mediated protein traffickingYes -LSB- 149 , 150 -RSB- RyR2Cardiac ryanodine receptor No -LSB- 151 -RSB- SEL-10Ubiquitination of proteinsYes -LSB- 152 -RSB- SorcinCalcium binding regulator of ryanodine receptorNo -LSB- 153 -RSB- Syntaxin 1ASynaptic plasma-membrane proteinNo -LSB- 154 -RSB- Syntaxin 5ER-Golgi vesicular transportYes -LSB- 155 , 156 -RSB- TauMicrotubule binding proteinNo -LSB- 137 -RSB- TelencephalinNeuron specific adhesion moleculeNo -LSB- 40 -RSB- TPIPTetratricopeptide repeat-containing proteinNo -LSB- 157 -RSB- UbiquilinUbiquitin domain-containing proteinNo -LSB- 158 -- 160 -RSB- X11 family of proteinsCytoplasmic adaptor proteinsYes -LSB- 110 , 111 -RSB- β-catenin/δ-catenin -LRB- NPARP -RRB- Wnt signalling . Cell adhesionNo -LSB- 161 -- 163 -RSB- μ-CalpainCalcium-dependent thiol proteaseNo -LSB- 164 -RSB- Fig. 2The postulated roles for presenilin interacting proteins not incorporated into the γ-secretase complex in Aβ generation . A schematic representation of the postulated roles of presenilin interacting proteins in -LRB- A -RRB- promoting or -LRB- B -RRB- attenuating γ-secretase activity and Aβ generation . -LRB- A -RRB- Together with the formation of the γ-secretase complex , calsenilin have been shown to interact with the presenilins and promote Aβ generation when over-expressed in mammalian cells . The role of calsenilin in Aβ generation remains unclear but maybe involved in the presentation of the substrate to the γ-secretase complex . -LRB- B -RRB- The presenilin interacting protein , X11 and other γ-secretase substrates that compete for enzyme activity such as Notch fragments -LRB- i.e. NEXT -RRB- have all been shown to reduce Aβ generation when over-expressed in mammalian cells . X11 may reduce Aβ production by binding to PS and APP preventing PS mediated cleavage of APP or its presentation to the γ-secretase complex Calsenilin Calsenilin -LRB- also called DREAM or kChIP3 -RRB- is a calcium binding protein , that was first identified through yeast two hybrid screening using the PS2 C-terminus as the bait protein -LSB- 94 -RSB- . The interaction between calsenilin and the presenilins appear to have a role in APP processing . It was initially demonstrated that calsenilin can form a complex with PS1 and PS2 and regulate presenilin fragment formation in mammalian cells -LSB- 95 -RSB- . This study also showed that calsenilin may facilitate presenilin mediated apoptosis , since calsenilin was shown to preferentially interact with the C-terminal fragment of PS2 that results from caspase cleavage of the presenilin holoprotein -LSB- 96 -RSB- . Expression studies in vivo -LRB- Xenopus -RRB- and in vitro -LRB- mammalian cells -RRB- have shown that the interaction between presenilin and calsenilin may be involved in ER calcium release and apoptosis -LSB- 97 , 98 -RSB- . Over-expressing calsenilin in neuronal cells resulted in cell death which was enhanced by the co-expression of wild-type or PS1 mutations -LSB- 99 -RSB- . These studies have suggested that the interaction between calsenilin and presenilins may function in apoptotic pathways . However , several lines of evidence suggest that calsenilin may also be implicated in APP processing and Aβ generation . Initial in vitro studies showed that over-expression of calsenilin in mouse neuroblastoma cells resulted in a gene dose dependent accumulation of Aβ42 -LSB- 100 -RSB- . Using a γ-secretase luciferase based assay and a cell free system established by Li and colleagues -LSB- 18 -RSB- to characterise the γ-secretase complex in purified cell membranes , Jo and colleagues -LSB- 101 -RSB- recently reported that the over-expression of calsenilin resulted in an increase in γ-secretase activity . This calsenilin/presenilin , mediated activity was markedly reduced using a well-characterised γ-secretase inhibitor -LRB- L-685 , 458 -RRB- . Furthermore , compared to wild-type mice , brain Aβ40 and Aβ42 levels were reduced in calsenilin ablated mice -LSB- 102 -RSB- . Together these studies would suggest that calsenilin has a second function , to facilitate γ-secretase activity . X11/Mint proteins A family of cytoplasmic adaptor proteins called X11 -LRB- also known as munc-18-interacting proteins -- mints -RRB- have also shown to have a direct role in APP processing . In particular , several studies have reported that X11α and X11β stabilise APP and inhibit the secretion of Aβ and APP from neuronal cells -LSB- 103 -- 106 -RSB- . The molecular mechanisms by which the X11s influence APP processing and Aβ production are not clear . However , this effect appears to be modulated by a direct interaction between X11 and APP . The interaction between the phosphotyrosine binding -LRB- PTB -RRB- domain within X-11 and the YENPTY motif within APP appears to modulate Aβ generation , since introduction of point mutations within these domains enhanced Aβ generation -LSB- 103 , 106 -- 109 -RSB- . The binding of X11 appears to stabilise APP , leading to its intracellular accumulation -LSB- 103 , 106 -- 109 -RSB- . It is conceivable that the X11 -- APP interaction may inhibit secretase accessibility to APP resulting in impaired Aβ secretion . The X11 proteins have multiple binding domains and have been shown to interact with the PS1 C-terminal fragment through their PDZ domain -LSB- 110 -RSB- . The authors also showed an interaction between PS1 and APP in the presence of X11 that was otherwise ablated in the absence of X11 . This suggests that an interaction between APP and PS1 is mediated through X11 . A subsequent study has reported that X11β and alcadein -LRB- and X11β-binding protein -RRB- expression prevents the binding of PS1 to APP -LSB- 111 -RSB- . The significance of the results from these interaction studies remains unclear . However , they do suggest that X11 proteins may act as inhibitory molecules preventing PS1 mediated proteolytic processing of APP and thus attenuating Aβ production . Competing substrates Some presenilin binding proteins reduce the likelihood of Aβ generation by competing for the catalytic activity offered by γ-secretase . One example is Notch1 which undergoes cleavage by γ-secretase to liberate an intracellular domain -LRB- NICD -RRB- that translocates to the nucleus where it co-activates transcription of genes involved in several developmental pathways -LSB- 112 -- 115 -RSB- . The first evidence of substrate competition came from one study that treated neurons with the Noch1 ligand , delta , and found a dose dependent reduction in Aβ levels -LSB- 116 -RSB- . In contrast , the over-expression of APP in these neurons resulted in a reduction in Notch signalling -LSB- 116 -RSB- . Further evidence for substrate competition was provided by Kimberley and colleagues -LSB- 117 -RSB- where APP and Notch based substrates directly competed for γ-secretase activity in a cell free assay . Similar results were achieved in a subsequent study where cells expressing Notch showed a reduction in Aβ production -LSB- 118 -RSB- . Additional evidence for substrate competition has been provided recently for another substrate of γ-secretase , the cytoplasmic tail of the low-density lipoprotein receptor -- related protein -LRB- LRP -RRB- , which has also been shown to interact with PS1 -LSB- 119 -RSB- . The co - expression of the LRP C-terminal fragment and APP in CHO cells has been shown to result in a reduction in secreted levels of Aβ40 and Aβ42 -LSB- 119 -RSB- . Taken together , these studies not only suggest substrate competition but also the presence of a single γ-secretase . However , the presence of multiple γ-secretase complexes can not be ruled out . As discussed above there is evidence in the literature for the formation of γ-secretase complexes with distinct functions . All of the studies described above are undertaken in conditions where there is a large amount of substrate available which could down-regulate the activity of one γ-secretase for another . Whether substrate competition occurs under physiological conditions and if there are certain conditions that promote a higher expression of one substrate over another remains to be determined . Concluding comments It is well established that together with presenilin , its binding proteins , nicastrin , aph-1 and pen-2 are the only proteins that constitute the γ-secretase complex responsible for generating Aβ from APP . The function -LRB- s -RRB- of each γ-secretase component within the complex is gradually becoming apparent . However , there are many facets of γ-secretase activity that are yet to be identified such as the domain responsible for actual catalytic activity ; the mechanisms of enzymatic activity within the lipid bi-layer ; and the existence of single or multiple γ-secretase complexes . Further investigation using appropriate protein expression models and reconstitution studies such as those used to validate the protein components of γ-secretase complex is required to provide some insight into the processes that underlie the catalytic activity of this enzyme . Indeed , reconstitution of the γ-secretase complex in insect cells and purification of this complex from the cells have provided researchers with the first 3D images of the structure of this intricate enzyme -LSB- 120 , 121 -RSB- . Identification of the specific catalytic domain -LRB- s -RRB- within the complex would provide a site that could be a potential target to develop agents that specifically modulate Aβ levels without altering the other known -LRB- and unknown -RRB- activities of the γ-secretase enzyme . Recent insight into this notion was provided by the finding that certain compounds selectively block the cleavage of APP but not Notch -LSB- 122 -RSB- . However , it is important to note that other presenilin interacting proteins not incorporated into the complex and the pathways they are involved in play important roles in APP processing and Aβ generation . These interactions assist in many facets of γ-secretase activity , including modulating substrate/enzyme availability , and thus are important to consider when developing appropriate therapeutic strategies .

O O O B O O B I O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

amyloid precursor protein
beta amyloid
presenilin
interacting proteins


Functional analysis of a novel potassium channel -LRB- KCNA1 -RRB- mutation in hereditary myokymia Myokymia is characterized by spontaneous , involuntary muscle fiber group contraction visible as vermiform movement of the overlying skin . Myokymia with episodic ataxia is a rare , autosomal dominant trait caused by mutations in KCNA1 , encoding a voltage-gated potassium channel . In the present study , we report a family with four members affected with myokymia . Additional clinical features included motor delay initially diagnosed as cerebral palsy , worsening with febrile illness , persistent extensor plantar reflex , and absence of epilepsy or episodic ataxia . Mutation analysis revealed a novel c. 676C > A substitution in the potassium channel gene KCNA1 , resulting in a T226K nonconservative missense mutation in the Kv1 .1 subunit in all affected individuals . Electrophysiological studies of the mutant channel expressed in Xenopus oocytes indicated a loss of function . Co-expression of WT and mutant cRNAs significantly reduced whole-oocyte current compared to expression of WT Kv1 .1 alone . Introduction Myokymia is defined as spontaneous involuntary muscle fiber group contraction , is visible as vermiform movement of the overlying skin , and may be hereditary or acquired . Electromyography -LRB- EMG -RRB- shows continuous motor unit activity with spontaneous discharges of high intraburst frequency . Myokymia occurs in autoimmune disease with antibodies to voltage-gated potassium channel subunits in Isaacs -- Merton syndrome and Morvan fibrillary chorea -LSB- 1 -RSB- . Hereditary myokymia with episodic ataxia type 1 -LRB- OMIM # 160120 -RRB- arises from mutations in the voltage-gated potassium channel KCNA1 -LSB- 2 -RSB- . The ataxia is characterized by profound attacks of cerebellar ataxia lasting seconds to minutes . Partial epilepsy has been reported in some families with KCNA1-associated disease -LSB- 3 , 4 -RSB- . Another rare cause of myokymia with neonatal epilepsy but lacking episodic ataxia results from a KCNQ2 potassium channel mutation -LSB- 5 -RSB- . Reduced frequency and severity of myokymia and episodic ataxia have been reported after treatment with acetazolamide or carbamazepine -LSB- 3 , 6 -RSB- . Approximately a dozen missense mutations and one truncating mutation in KCNA1 have been reported -LSB- 3 , 4 , 7 -- 10 -RSB- . The functional consequences of human KCNA1 mutations causing typical myokymia and EA1 have been reported -LSB- 11 -RSB- , much less is known about the functional consequences of mutations causing unusual KCNA1-associated phenotypes . In the present study , we describe the clinical and genetic features of a new family with hereditary myokymia lacking epilepsy or episodic ataxia . Molecular studies of KCNA1 in the proband revealed a c. 676C > A transversion resulting in the substitution of Lys for Thr at codon 226 -LRB- T226K -RRB- and a c. 1355A > C transversion resulting in Y452S . Further genetic studies showed that all affected family members carried the T226K mutation , which was absent in unaffected family members and ethnically matched normal controls , whereas the Y452S substitution was present in both affected and unaffected family members and normal controls . In transfected mammalian cells , electrophysiologic studies demonstrated functional effects of the c. 676C > A transversion with no currents above background levels recorded from oocytes injected with the mutant subunit . Co-injection of the wild-type and mutant cRNAs produced significantly smaller whole-oocyte currents than injection with the WT cRNA alone . Materials and methods Clinical data The family pedigree is shown in Fig. 1 . Six family members -LRB- two unaffected and four affected -RRB- were available for clinical and genetic studies approved by the Yale Human Investigations Committee . Fig. 1Family pedigree . Squares indicate males , circles indicate females . Blackened symbols denote individuals with myokymia . Alleles at codon 226 are indicated -LRB- Thr = wild-type , Lys = mutation -RRB- Subject II-1 The proband , a 13-year-old boy -LRB- II-1 -RRB- , was the product of an unremarkable pregnancy and delivery . He was diagnosed with cerebral palsy because of leg stiffness and delayed walking at 18 months old . Cognitive development was normal ; his medical history was significant for esotropia . At 4 years old , he was hospitalized with increasing leg pain , stiffness , and inability to walk during the course of a flu-like illness . Creatine kinase -LRB- CK -RRB- was elevated at 520 U/l acutely -LRB- normal < 195 U/l -RRB- , but was normal when repeated interictally . Examination showed periorbital myokymia , mild abdominal and leg muscle hypertrophy , leg stiffness , spastic gait , hyperreflexia , and bilateral Babinski sign . Magnetic resonance imaging -LRB- MRI -RRB- of the brain and spine were normal . Metabolic studies including electrolytes , amino and organic acids , carnitine , ammonia , lactate , and thyroid functions were normal . Routine studies of cerebral spinal fluid and an electroencephalogram were normal . Needle EMG of deltoid and iliopsoas muscles showed irregular , polyphasic continuous motor unit discharges with normal interference pattern . Additional family members Six family members were evaluated . Four affected members -LRB- mother and three sons -RRB- had myokymia on clinical examination , EMG examination , or both . Nerve conduction velocities were normal . There was no history of seizures or episodic ataxia in any of them . The mother -LRB- I-2 , Fig. 1 -RRB- is a 40-year-old woman who had had muscle twitching since childhood , hyperreflexia , bilateral Babinski sign , and tendency to run on her toes . Her husband is unaffected and had a normal neurological exam . A 12-year-old son -LRB- II-2 -RRB- also had delayed walking , leg stiffness , and was diagnosed with cerebral palsy . At age 3 years , he had an episode of prolonged generalized paralysis with no muscle response to tetanic stimulation after general anesthesia for esotropia surgery . He recovered within 1 day . MRI of the brain and spine were normal . An 8-year-old daughter -LRB- II-3 -RRB- is unaffected . A 5-year-old son -LRB- II-4 -RRB- had symptoms identical to his brothers and was hospitalized twice for episodes of increased muscle stiffness with presumed viral gastroenteritis , elevated CK during both episodes -LRB- 606 U/l and 997 U/l -RRB- but had normal CKs measured while well . Carbamazepine treatment resulted in marked improvement of muscle symptoms in three of the brothers who remain asymptomatic . The mother , with milder symptoms , declined treatment . Three family members with myokymia had esotropia ; one patient required three eye surgeries . Molecular studies Genomic DNA was extracted from blood samples or buccal swabs . The coding region of the single-exon 1,448-bp gene KCNA1 -LRB- GenBank sequence NM_000217 -RRB- was amplified . Polymerase chain reactions -LRB- PCRs -RRB- were performed in a 25-μl reaction volume containing 50 ng genomic DNA , 50 ng of each primer , 1.5 mM MgCl2 , 200 μM dNTPs , 1 × PCR buffer -LRB- Finnzyme -RRB- , and 2.5 U of Finnzyme using a GeneAmp PCR System 9700 -LRB- Applied Biosystems , Foster City , CA -RRB- . Cycling parameters consisted of a modified touchdown -LRB- stepdown -RRB- protocol as follows : an initial hot start denaturation at 94 °C for 5 min -LRB- hot start -RRB- ; 94 °C for 45 s , annealing temperature -LRB- three cycles each at 70 , 67 , 64 , 61 , and 58 °C -RRB- for 45 s , and extension at 72 °C for 2 min for a total of 15 cycles followed by 25 cycles of 94 °C for 45 s , 55 °C for 45 s , 72 °C for 1 min ; a final extension step of 72 °C for 10 min followed by a 4 °C hold . PCR products were separated by electrophoresis on a 1 % agarose gel and visualized by ethidium bromide staining . PCR products were purified by spin column -LRB- Qiagen , Valencia , CA -RRB- or by enzymatic method -LRB- ExoSAP-IT , USB , Cleveland , OH -RRB- and analyzed for mutations by automated sequencing -LRB- Applied Biosystems , Foster City , CA -RRB- . The c. 1355A > C transversion resulted in the creation of a DdeI restriction site , so the presence of the sequence variant was tested by sequencing and confirmed with restriction digest in all subjects . Restriction digests were performed with DdeI -LRB- New England Biolabs , Beverly , MA -RRB- at 37 °C for 2 h. Oocyte expression and electrophysiological studies Human Kv1 .1 cDNA was subcloned into Kpn1 and HindIII sites of psGEM . The Kv1.1-T226K mutation was introduced by site-directed mutagenesis and confirmed by automated sequencing . cRNAs were synthesized using an mMessage mMachine kit -LRB- Ambion , Austin , TX -RRB- and quantified by spectroscopy . Oocytes were isolated from Xenopus laevis and defolliculated by collagenase treatment . Each oocyte was injected with either 5 ng of human Kv1.1-WT , 5 ng Kv1.1-T226K , or 2.5 ng WT plus 2.5 ng T226K cRNA . Whole-oocyte currents were measured with two electrode voltage clamp techniques -LRB- Oocyte Clamp , Warner Instruments , Hamden , CT -RRB- with constant perfusion -LRB- 1 ml/min , solution exchange \ 3 s -RRB- after 16 h. Data were sampled at 1 kHz and filtered at 0.25 kHz . Standard bath solution was ND-96 -LRB- in mM -RRB- : 96 NaCl , 2 KCl , 1 MgCl2 , 1.8 CaCl2 , and 5 HEPES/NaOH , pH 7.5 . Results Mutation analysis of KCNA1 Mutation analysis of KCNA1 identified two sequence variants : a c. 676C > A transversion resulting in the substitution of Lys for Thr at codon 226 -LRB- T226K -RRB- and a c. 1355A > C transversion resulting in Y452S and creating a DdeI restriction site . Either a Tyr -LRB- NM_000217 -RRB- or a Ser -LRB- AAI01734 -RRB- had been reported at codon 452 , but the population frequency and association with disease were unknown . Therefore , we tested all six family members -LRB- four affected , two unaffected -RRB- and 10 normal controls for the SNP and found that all 16 subjects were homozygous for the Y452S substitution , compatible with a common polymorphism . In contrast , all -LRB- 4/4 -RRB- affected subjects were heterozygous for the previously unreported T226K missense mutation , which was absent in both unaffected family members and absent in 190 chromosomes from ethnically matched normal controls -LRB- Fig. 2 -RRB- . Fig. 2Sequence chromatograms demonstrating c. 676C > A resulting in T226K substitution in KCNA1 . A DNA sequence from an affected patient -LRB- I-2 -RRB- heterozygous for the mutation -LRB- top -RRB- and from a normal control -LRB- bottom -RRB- are presented Electrophysiological studies of the human Kv1 .1 mutant The genetic studies of the segregation of the T226K mutation favored it as a pathological mutation , but given that two missense mutations were identified , we sought additional supporting evidence . We tested whether the missense mutation alters human Kv1 .1 channel function by expressing Kv1.1-T226K in Xenopus oocytes . No currents above background levels were recorded from Kv1.1-T226K cRNA-injected oocytes , whereas Kv1 .1 wild-type channels produced typical delayed rectifier currents -LRB- Fig. 3 -RRB- . We then mimicked the heterozygous situation by co-injecting equal amounts of Kv1 .1 WT and T226K mutant cRNAs . Co-injection produced significantly smaller whole-oocyte currents than injection with Kv1 .1 WT cRNA alone -LRB- Fig. 3c and d -RRB- . These results indicate that the T226K mutation significantly reduced efflux of potassium ions during depolarization and is therefore likely to result in increased muscle cell excitability . Fig. 3Missense mutation T226K induces loss of function of human Kv1 .1 channels expressed in Xenopus oocytes . a -- c Representative family of whole-oocyte currents recorded from oocytes expressing human Kv1.1-WT -LRB- a -RRB- , Kv1.1-T226K -LRB- b -RRB- , and both Kv1 .1 WT and T226K -LRB- c -RRB- . Currents were evoked by a series of 350-ms test pulses from − 80 to +60 mV in 10-mV increments from a hold potential of − 90 mV . Tail currents were recoded at − 40 mV . Scale bar , 100 ms and 1 μA . d I -- V curves for three experimental conditions in a -- c. Data were collected for 10 -- 15 oocytes in each group . The peak currents in a and c are significantly different at test voltages between 0 and +60 mV -LRB- P < 0.05 -RRB- Discussion Mutations in KCNA1 are typically associated with episodic ataxia type 1 -LRB- EA1 -RRB- , characterized by profound cerebellar ataxia lasting seconds to minutes and interictal myokymia -LSB- 6 -RSB- . The expanded phenotypic spectrum associated with proven KCNA1 mutation includes a family with prolonged episodes of episodic ataxia and epilepsy , but without myokymia -LSB- 12 -RSB- . Other families have been reported with myokymia and adducted thumbs , congenital contractures , kyphoscoliosis , or epilepsy with myokymia -LSB- 4 , 7 -- 10 -RSB- . We report a family with autosomal dominant myokymia lacking either ataxia or epilepsy . The mother had self-described lifelong `` muscle twitchiness '' but never sought medical attention for her condition until the hospitalizations of her sons . Myokymia in the periorbital region and on EMG were the features that led to genetic studies of KCNA1 . Although the stiffness , toe walking , and esotropia could be caused by continuous muscle contraction , the extensor plantar reflex -LSB- observed on multiple exams by experienced two neurologists -LRB- LRM and FMH -RRB- -RSB- is a new finding for KCNA1-associated disease and suggests involvement of the corticospinal tract . The KCNA1 knockout mouse has been reported to exhibit altered inhibition of layer V pyramidal neocortical neurons -LSB- 13 -RSB- . KCNA1 on chromosome 12p13 encodes Kv1 .1 , a potassium channel subunit . Delayed-rectifier voltage-gated-type potassium channels are important in the rapid neuronal repolarization after an action potential and are multimeric proteins containing four subunits around a central pore . The Kv1 .1 potassium channel subunit is expressed during development in neuronal and glial cells in the central and peripheral nervous systems -LSB- 14 -RSB- . Kv1 .1 plays an important role in regulating neuronal excitability in the central nervous system , as shown by studies of the KCNA1 knockout mouse with an increased susceptibility to seizures -LSB- 15 -RSB- . The present family has generalized myokymia due to a T226K mutation in the voltage-gated potassium channel KCNA1 . Unusual clinical features include pyramidal dysfunction and worsening with febrile illness or anaesthesia . One previously reported 3-year-old boy presented in a similar fashion to our patients with a 12-h episode of inability to walk , increased tone , and elevated CK during a febrile illness -LSB- 3 -RSB- . No anesthesia exposure was reported . That child had a P244H mutation in the intracellular loop between transmembrane segments 2 and 3 . The whole-cell current amplitudes elicited by injection of P244H did not differ from wild-type amplitudes . Co-injection of P244H plus an equal amount of wild-type cRNA yielded a peak current amplitude that was 200 % of wild-type alone . We identified a novel , nonconservative T226K missense mutation that is located in the second transmembrane domain of Kv1 .1 and is invariant from Drosophila melanogaster to mice and humans -LSB- 6 -RSB- . In contrast to the functional effects reported for the P244H mutation , heterologous expression of the T226K mutation identified in the present family showed that the mutant potassium channel expressed in oocytes demonstrated no current above background levels , and its co-expression with the wild-type channel significantly reduced whole-oocyte current , compatible with a severe effect of the mutation . The T226K mutation is likely to reduce efflux of potassium ions during depolarization , thereby increasing cellular excitability . The phenotypic similarity between the P244H and the T226K mutations contrasts with their biophysical effects . Therefore , additional factors must contribute to the functional effects of these mutations . Kv1 .1 forms heteromeric channels with other subunits and interacts with other accessory subunits -LSB- 16 -RSB- . In addition to their well-known role as ion channels that regulate neuronal firing , potassium channels have also been shown to activate enzymes linked to intracellular signaling pathways -LSB- 16 , 17 -RSB- . One of these novel interactions may contribute to differences in the manifestation of disease-causing mutations .

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