expression constructs  mouse asic1a was cloned into pmt3 
  ( 44 ) for heterologous 
  expression . human -actinin-1 was a gift of c. otey , university of north 
  carolina , and -actinin-2 , -3 , and -4 were gifts of m. sheng , 
  massachusetts institute of technology . full - length human 
  -actinin-14 were cloned into pegfp vectors ( clontech 
  laboratories ) to generate enhanced green fluorescent protein 
  ( egfp)--actinin fusion proteins ; previous studies showed that the 
  localization and function of -actinins were unaffected by gfp tags 
  ( 4547 ) . 
  a dominant - negative n - terminal - truncated ( missing amino acid 3249 ) 
  human -actinin-1 ( rod - actinin ) 
  ( 48 ) was a gift of a. 
  huttenlocher , university of wisconsin . rod - actinin contains the central 
  -actinin spectrin domain including the c terminus but lacks the 
  n - terminal head domain . dimers containing rodactinin can bind other proteins , 
  but the absence of the head domain interrupts cross - linking to the filamentous 
  actin cytoskeleton ( 48 , 
  49 ) . rod - actinin would be 
  expected to interfere with all -actinins , and consistent with that 
  conclusion , it disrupted the effect of -actinins in human embryonic 
  kidney cells , which express both -actinin-1 and 
  -4.3 chinese hamster 
  ovary ( cho ) cells also express -actinin-1 and -actinin-4 
  ( 50 ) . ha - tagged asic1a was 
  made by adding the influenza hemagglutinin epitope ( ypydvpdyagv ) to the n 
  terminus of asic1a . cell culture and transfection  for patch clamp studies cho 
  cells were transfected with 16 g of dna using the transfast 
  lipid reagent ( promega madison , wi ) and cultured on glass coverslips in 
  35-mm petri dishes . to identify transfected cells using epifluorescence , we 
  used egfp at an asic1a : egfp ratio of 6:1 . empty vector dna was used to 
  maintain a constant final dna concentration for all transfections . hippocampal neuronal culture and transfection  rat e18 
  hippocampal neurons ( brain bits , springfield , il ) were transfected after 
  78 days in culture using calcium phosphate transfection . conditioned 
  medium was collected and replaced by freshly prepared neurobasal medium with 
  b-27 supplement ( invitrogen ) 30 min before transfection . a total of 5 g of 
  vector dna was added in a transfection mix consisting of 50 ml of water , 5 ml 
  of cacl2 ( 2.5 m ) , and 60 ml of 2 pbs per dish . after 3 h of incubation at 37 c and after an 8-min hanks ' balanced salt 
  solution washing step , conditioned media was reapplied to cultures . at day 10 , 
  neurons were used for whole - cell , patch clamp experiments . immunoprecipitation and immunoblotting  whole mouse brains 
  from adult animals were homogenized in ice - cold buffer ( 5 ml / brain ) containing 
  300 mm sucrose , 10 mm tris ( ph 7.5 ) , 20 mm 
  nacl plus protease inhibitors ( complete mini , edta - free , roche applied 
  science ) using a glass teflon homogenizer . samples were spun for 2 min in a 
  tabletop centrifuge at 2000  g to remove large debris . supernatants were centrifuged for 30 min at 135,000  g in a 
  beckman tla100.3 rotor at 4 c . the membrane pellet was washed once on ice 
  and then solubilized in 6 ml of 50 mm tris ( ph 7.5 ) , 150 
  mm nacl , protease inhibitors ( complete mini ) with 1% triton x-100 . 
  for immunoprecipitation , samples were centrifuged ( 10 min , 700  
  g ) to remove particulate debris and precleared by the addition of 
  protein g - agarose ( roche applied science , 50 l , 15 mg / ml ) . either 
  undiluted affinity - purified asic1 antibody ( raised in rabbits against the 
  22-amino acid peptide from the c terminus of asic1 and purified as described 
  previously ( 16 ) ) or a mouse 
  monoclonal -actinin antibody bm-75.2 that binds to all four actinin 
  isoforms ( 1 l , sigma - aldrich ) was added to 750 l of protein extract 
  and incubated for 3 h while rotating at 4 c . for precipitation , protein 
  g - agarose beads ( roche applied science , 50 l 15 mg / ml ) were added , and 
  samples were rotated overnight at 4 c . after centrifugation , 3 wash steps 
  were performed to clear samples ( buffer 1 : 50 mm tris ( ph 7.5 ) , 150 
  mm nacl , 1% nonidet p-40 , 0.5% sodium deoxycolate , protease 
  inhibitors ( complete mini ) ; buffer 2 : 50 mm tris ( ph 7.5 ) , 500 
  mm nacl , 0.1% nonidet p-40 , 0.05% sodium deoxycolate ; buffer 3 : 10 
  mm tris ( ph 7.5 ) , 0.1% nonidet p-40 , 0.05% sodium deoxycolate ) . bound proteins were extracted at 95 c in sds sample buffer ( 0.125 
  mm tris ( ph 7.5 ) , 3.4% sds , 17% glycerol , 67 mm 
  dithiothreitol , 0.008% bromphenol blue ) for 10 min and were resolved by 
  sds - page and immunoblotted with the indicated antibodies . co - immunoprecipitation from cos-7 cells was performed 48 h after 
  electroporation ( 10 cells , 10 or 15 g of dna at 1:1 
  asic1a:-actinin ratio per sample ) . electroporation was performed with 
  400 l of prechilled cell suspension ( 120 mm kcl , 25 
  mm hepes , 0.15 mm nacl , 10 mm 
  kpo4 , 2 mm egta , 5 mm mgcl2 with 2 
  mm atp and 5 mm glutathione using a single 25-s pulse of 
  0.320 v and 975 microfarads . cells in 100-mm dishes were washed twice with 
  ice - cold pbs and harvested in lysis buffer ( 50 mm tris ( ph 7.5 ) , 
  150 mm nacl , 1% nonidet p-40 , and protease inhibitors ( complete 
  mini ) . supernatants from cell extracts were pre - cleared with protein 
  a - sepharose ( pierce ) gently agitated for 1 h at 4 c . either asic1 
  antibody ( 16 ) or rabbit 
  polyclonal anti - gfp antibody ( clontech , living colors full - length a.v . polyclonal antibody ) were added to 250500 l of protein extract 
  after 1 h of rotation at 4 c . protein a - sepharose was added followed by 
  incubation overnight with rotation at 4 c . immunoprecipitates absorbed to 
  protein a - sepharose were washed three times as described above . the 
  immunoprecipitated proteins were resuspended in sds sample buffer and 
  electrophoresed . for immunoblotting , primary antibodies were detected with 
  horseradish peroxidase - conjugated goat - anti rabbit or anti - mouse igg from ecl 
  ( ge healthcare ) . cho cells were transfected with ha - asic1a:-actinin : gfp at 1:1:1 
  ratio . for control , -actinin was replaced with gfp so the ratio was 1:2 
  for ha - asic1a : gfp . cells were washed 3 with ice - cold 
  pbs+/+ , and 3 ml of 0.5 mg / ml nhs - biotin in pbs+/+ was added to each 10-cm 
  dish followed by incubation at 4 c for 30 min with gentle rocking . cells 
  were washed once with pbs+/+ , and 0.1 m glycine in pbs+/+ was added 
  to quench the reaction followed by 2 washes with pbs+/+ . cells were lysed in 
  pbs with 1% nonidet p-40 , 0.5% deoxycholate , 0.5% sds , and freshly added 
  proteinase inhibitors ( roche applied science ) . protein concentration was quantified 
  using a modified bradford assay kit ( bio - rad ) . for neutravidin pulldown , 60 
  l of a 50% slurry of neutravidin beads were added to 200 l of cell 
  lysate and incubated at 4 c overnight with gentle rotation . beads were 
  washed 3 with wash buffer ( tris 50 mm ( ph 7.4 ) , 1% triton 
  x-100 ) . beads were then boiled in 80 l of sds sample buffer with or 
  without reducing agent . rt - pcr  rat hippocampal neurons and rat muscle tissue were 
  lysed in cell lysis buffer , and first - strand cdna was synthesized using the 
  cells - to - cdna ii kit ( ambion ) . for pcr , the primer pairs for detection of 
  -actinin-1 were 5-gatgcagacaaggagcgcct-3 ( bp 
  18871906 ) and 5-gggacccaacgtgccggag-3 ( bp 
  24952513 ) ; for -actinin-2 they were 
  5-agaatgaggtggagaaggtga-3 ( bp 17871807 ) and 
  5-tggaaggtgaccgtgccttg-3 ( bp 24672486 ) ; for 
  -actinin-3 they were 5-ctgcagctggagtttgctcg-3 ( bp 
  16071625 ) and 5-tgtgctccatgctgtagacc-3 ( bp 
  21962215 ) , and for -actinin-4 they were 
  5-agcaatcacatcaagctgtcg-3 ( bp 19061927 ) and 
  5-ccacactcatgatccggttg-3 ( bp 25422561 ) . we generated small interfering rnas 
  ( sirnas ) against each of the four rat -actinin isoforms using 
  oligoengine to locate 19 nucleotides within exons of the target and 
  immediately downstream of aa dinucleotides and to exclude identity to other 
  sequences in the ncbi data base . sequences with potential polymerase iii 
  termination sites and mrna splice sites were avoided . the 19-nucleotide sense sequence and the inverted antisense sequence at the 
  3 end , a penta - thymidine motif provided a polymerase iii termination 
  site . sirnas were cloned into psilencer vector ( ambion ) for expression 
  of the respective sequences under the u6 promotor . dsred and its 
  cytomegalovirus promoter from pdsred2-n1 were cloned into the kpn site 
  upstream of the u6 promoter within the psilencer vector ; this vector 
  was used for identification of transfected neurons . to test for efficacy and 
  specificity of sirna , cho cells were co - transfected with either one of the 
  sirnas and egfp - tagged -actinin at a 10:1 cdna ratio ( total 11 g 
  dna ) using lipofectamine 2000 in opti - mem i media according to the 
  manufacturer 's recommendation ( invitrogen ) . rnai1 targeted at 476494 
  from the start sequence of rat -actinin-1 ( the sense chain was 
  5-gatctccaacgtcaacaagttcaagagacttgttgacgttggagatctttttt-3. rnai2 
  targeted at 224242 from the start sequence of rat -actinin-2 ; 
  the sense chain was 
  5-tggcctgatggatcatgagttcaagagactcatgatccatcaggccatttttt-3. rnai3 
  targeted at 455473 from the start sequence of rat -actinin-3 ; 
  the sense chain was 
  5-aacgagaagctgatggaggttcaagagacctccatcagcttctcgtttttttt-3. rnai4 
  targeted at 493511 from the start sequence of rat -actinin-4 ; 
  the sense chain was 
  5-gactatccaggagatgcagttcaagagactgcatctcctggatagtctttttt-3. electrophysiology  whole - cell , patch clamp recordings were 
  performed on cho cells on 10-mm glass coverslips continuously superfused with 
  a bath solution containing : 128 mm nacl , 5.4 mm kcl , 2 
  mm cacl2 , 1 mm mgcl2 , 10 
  mm hepes , 10 mm mes , 5.55 mm glucose , 
  adjusted to ph 7.4 with tmaoh . the standard pipette solution contained 10 
  mm nacl , 121 mm kcl , 5 mm egta , 2 
  mm mgcl2 , 2 mm na2atp , 10 
  mm hepes adjusted to ph 7.25 with tmaoh . for whole - cell , patch 
  clamp experiments with hippocampal neurons the bath solution contained 100 
  mm nacl , 5.4 mm kcl , 2 mm cacl2 , 1 
  mm mgcl2 , 10 mm hepes , 10 mm mes , 
  10 mm glucose adjusted to ph 7.4 with tmaoh . the pipette solution 
  contained 10 mm nacl , 70 mm potassium gluconate , 10 
  mm kcl , 10 mm egta , 1 mm mgcl2 , 3 
  mm na2atp , 25 mm hepes adjusted to ph 7.25 
  with tmaoh . bath solutions with different ph values were adjusted with tmaoh 
  and applied with a rapid solution exchanger ( rsc-200 & evh-9 , biologic 
  science instruments ) . recording pipettes were pulled from capillary glass with 
  a micropipette puller ( sutter instruments ) and polished ( mf830 , narishige , 
  japan ) . recordings were made at room temperature using an axo - patch 200b amplifier 
  with pclampex 8.1 software ( axon instruments ) . amplitude was determined by subtracting the 
  base - line current at ph 7.4 from the peak current amplitude . the  of 
  desensitization ( des ) was calculated by fitting the data to a 
  single exponential equation with clampfit ( axon instruments ) . asic1a contains a putative binding site for 
  -actinin  in the c terminus of asic1a , we identified an 
  amino acid motif that resembled sequences in the cytosolic portions of the 
  nmda receptor subunit 1 and the p2x7 purinoceptor , two ion channels known to 
  physically interact with -actinin 
  ( fig . the potential 
  -actinin - binding site in asic1a lies in the cytosolic portion of the 
  subunit and would , therefore , be accessible for interaction with other 
  cytoskeletal proteins . the sequence was not present in asic2 or asic3 , 
  suggesting that -actinin might specifically interact with asic1 . 
  because asic1a and -1b splice variants share the same c terminus , interactions asic1a associates with -actinin-1 and 
  -actinin-4to test the hypothesis that asic1a and 
  -actinin interact , we expressed asic1a with each of the four 
  -actinin isoforms in cos-7 cells . 2 ) . conversely , 
  precipitating -actinin-1 or -actinin-4 co - precipitated asic1a . asic1a weakly co - precipitated -actinin-3 , but not the converse , and we 
  did not detect an interaction with -actinin-2 . these data suggest that 
  asic1a resides in close proximity with at least two of the actinins , 
  -actinin-1 and -actinin-4 . figure 1.the cytosolic c terminus of asic1a contains a putative 
  -actinin - binding site . analysis of the asic1 c terminus revealed an 
  amino acid motif that is present in -actinin binding cation channels . the gray box highlights the conserved motif in the primary sequence 
  of the mouse asic1a cytosolic c terminus . analysis of the asic1 c terminus revealed an 
  amino acid motif that is present in -actinin binding cation channels . the gray box highlights the conserved motif in the primary sequence 
  of the mouse asic1a cytosolic c terminus . because the localization and function of 
  -actinin is not affected by gfp tags 
  ( 4547 ) , 
  we co - expressed egfp - tagged isoforms of -actinin-1 , -2 , -3 , or -4 with 
  asic1a in cos-7 cells . for immunoprecipitation ( ip ) , either 
  anti - asic1a ( top panels ) or anti - gfp ( middle panels ) 
  antibodies were used . western blots were probed with anti - gfp or anti - asic1a 
  antibodies as indicated . note that anti - asic1a antibody co - precipitated 
  egfp - tagged-actinin-1 and-actinin-4 , and conversely , anti - gfp 
  antibody co - immunoprecipitated asic1a with -actinin-1 and 
  -actinin-4 . the lower panel shows western blots of total cell 
  lysates ( 2% of total lysate ) used for co - immunoprecipitation experiments . shown are western blots showing isoform - specific interactions 
  between -actinins and asic1a . because the localization and function of 
  -actinin is not affected by gfp tags 
  ( 4547 ) , we co - expressed egfp - tagged isoforms of -actinin-1 , -2 , -3 , or -4 with 
  asic1a in cos-7 cells . for immunoprecipitation ( ip ) , either 
  anti - asic1a ( top panels ) or anti - gfp ( middle panels ) 
  antibodies were used . western blots were probed with anti - gfp or anti - asic1a 
  antibodies as indicated . note that anti - asic1a antibody co - precipitated 
  egfp - tagged-actinin-1 and-actinin-4 , and conversely , anti - gfp 
  antibody co - immunoprecipitated asic1a with -actinin-1 and 
  -actinin-4 . the lower panel shows western blots of total cell 
  lysates ( 2% of total lysate ) used for co - immunoprecipitation experiments . cos-7 
  cells were transfected with asic1a or asic1amut alone or together 
  with egfp - labeled -actinin-4 . for immunoprecipitation ( ip ) , 
  either anti - asic1a ( upper panels ) or anti - gfp ( middle 
  panels ) antibodies were used ( igg indicates nonspecific mouse or rabbit 
  igg used as a control ) . western blots were probed with either anti - asic1a or 
  anti - gfp antibody . note that wild - type asic1a , but not asic1amut , 
  co - immunoprecipitated with -actinin - gfp . conversely , 
  -actinin - gfp co - immunoprecipitated with wild - type asic1a but not 
  asic1amut . the lower panels show total cell lysates ( 2% of 
  total lysate ) used for co - immunoprecipitation experiments . cos-7 
  cells were transfected with asic1a or asic1amut alone or together 
  with egfp - labeled -actinin-4 . for immunoprecipitation ( ip ) , 
  either anti - asic1a ( upper panels ) or anti - gfp ( middle 
  panels ) antibodies were used ( igg indicates nonspecific mouse or rabbit 
  igg used as a control ) . western blots were probed with either anti - asic1a or 
  anti - gfp antibody . note that wild - type asic1a , but not asic1amut , 
  co - immunoprecipitated with -actinin - gfp . conversely , 
  -actinin - gfp co - immunoprecipitated with wild - type asic1a but not 
  asic1amut . the lower panels show total cell lysates ( 2% of 
  total lysate ) used for co - immunoprecipitation experiments . to learn whether the potential -actinin binding motif in the c 
  terminus of asic1a was involved in the interaction between asic1a and 
  -actinin , we expressed -actinin-4 with an asic1a variant that 
  contained mutations in the potential -actinin binding motif 
  ( asic1amut residues 
  lslddvk mutated 
  to lsadava ) . 
  mutating the c - terminal motif prevented asic1a from co - precipitating 
  -actinin-4 ( fig . these results suggest a direct association between -actinin-4 and 
  asic1a that depends on the -actinin binding motif . for most of the 
  remainder of the studies , we focused on -actinin-4 . -actinin did not affect cell surface expression of 
  asic1a  to determine whether -actinin might regulate surface 
  expression of asic1a , we biotinylated cell surface proteins in cho cells 
  transfected with asic1a . co - expressing asic1a with -actinin-4 , 
  -actinin-1 , or rod - actinin did not alter the fraction of asic1a on the 
  cell surface ( fig . 4 , a and 
  b ) . figure 4.-actinin did not alter cell surface expression of asic1a . a , cho cells were transfected with ha - asic1a and either gfp ( as a 
  control ) , -actinin-4 , -actinin-1 , or rod - actinin . after 48 h lanes with total protein 
  were loaded with 20% of the cell lysate used for neutravidin binding . b , quantification of asic1a protein surface : total ratio ( n = 
  6 from 3 separate experiments ) . there were no significant differences between 
  experimental groups . a , cho cells were transfected with ha - asic1a and either gfp ( as a 
  control ) , -actinin-4 , -actinin-1 , or rod - actinin . after 48 h lanes with total protein 
  were loaded with 20% of the cell lysate used for neutravidin binding . b , quantification of asic1a protein surface : total ratio ( n = 
  6 from 3 separate experiments ) . there were no significant differences between 
  experimental groups . -actinin influences asic1a current density  to learn 
  whether -actinin also regulates the function of asic1a , we co - expressed 
  them and measured currents . -actinin-4 reduced asic1a current density , 
  whereas -actinin-1 had no effect 
  ( fig . 5 , a and 
  b ) . these results suggest that even though both 
  -actinins interact with asic1a , they have selective functional effects . 
  as an additional test of the effect of -actinin , we co - expressed asic1a 
  with rod - actinin , a dominant - negative construct 
  ( 48 , 
  49 ) . we found that the 
  dominant - negative rod - actinin fragment had the opposite effect of 
  -actinin-4 and increased current density 
  ( fig . 5 , a and 
  b ) . finding that rod - actinin increased current amplitude suggested that it 
  disrupted an interaction between asic1a and an endogenous -actinin . such an interaction predicts that asic1amut , which did not interact 
  with -actinin , would have a greater current density than asic1a . these data also 
  suggest that the effect of -actinin-4 on current results from a direct 
  interaction with asic1a . -actinin alters the properties of asic1a currents  the 
  findings that -actinin-4 did not change the amount of asic1a on the 
  cell surface whereas it decreased current density suggested that 
  -actinin-4 must have also altered the properties of the current 
  generated by asic1a or increased the proportion of silent channels . to test 
  this possibility , we examined several characteristics of asic1a current , 
  including the ph sensitivity , the rate of desensitization , and the time course 
  of recovery from desensitization . figure 5.-actinin regulated asic1a acid - evoked current in cho cells . 
 a , representative currents evoked by application of ph 5.0 to cho 
  cells expressing asic1a or asic1amut alone or co - expressing asic1a 
  with -actinin-1 , -actinin-4 , or rod - actinin . b , mean 
  current densities evoked by ph 5.0 superfusion of cho cells expressing the 
  indicated constructs . data are normalized to currents obtained in cho cells 
  expressing wild - type asic1a alone . mean 
  h - gated current densities in cho cells expressing 
  asic1amut were not affected by co - expression with-actinin-1 , 
  -actinin-4 , or rod - actinin ( n = 717 ) . * , p 
  < 0.02 compared with asic1a ( left ) or asic1amut 
  ( right ) alone . * * , p < 0.05 compared with asic1a . -actinin regulated asic1a acid - evoked current in cho cells . 
 a , representative currents evoked by application of ph 5.0 to cho 
  cells expressing asic1a or asic1amut alone or co - expressing asic1a 
  with -actinin-1 , -actinin-4 , or rod - actinin . b , mean 
  current densities evoked by ph 5.0 superfusion of cho cells expressing the 
  indicated constructs . data are normalized to currents obtained in cho cells 
  expressing wild - type asic1a alone . mean 
  h - gated current densities in cho cells expressing 
  asic1amut were not affected by co - expression with-actinin-1 , 
  -actinin-4 , or rod - actinin ( n = 717 ) . * , p 
  < 0.02 compared with asic1a ( left ) or asic1amut 
  ( right ) alone . * * , p < 0.05 compared with asic1a . conversely , the 
  dominant - negative rod - actinin had the opposite effect , reducing ph 
  sensitivity . consistent with these data , when we disrupted the asic1a 
  -actinin - binding site ( asic1amut ) , ph sensitivity fell , and 
  -actinin-4 and rod - actinin failed to alter the ph sensitivity of this 
  variant ( fig . these results 
  indicate that the interaction with -actinin influenced the sensitivity 
  of asic1a to extracellular protons . table 1-actinin and ph sensitivity of asic1a in cho cellsdata are ph values evoking 50% of current compared to current induced by ph 
  5.0.transfected dnahalf - maximal activation , ph50 asic1a 
   6.46  0.09 
   asic1a + -actinin-4 
   6.67  0.14 
   asic1a + rod - actinin 
   6.30  0.07 
   asic1amut 6.28  0.05 
   asic1amut + -actinin-4 
   6.22  0.06 
 
  asic1amut + rod - actinin 
 
  6.29  0.06 
   -actinin and ph sensitivity of asic1a in cho cells data are ph values evoking 50% of current compared to current induced by ph 
  5.0 . after acid - evoked activation , asic1a currents desensitize in the continued 
  presence of acid ( fig . we found that neither -actinin-1 nor -4 
  coexpression altered the time constant of asic1a current desensitization 
  ( des ) ( fig . however , disrupting the -actinin - binding site in asic1amut 
  or co - expressing rod - actinin with asic1a increased des . 
  moreover , disrupting the -actinin binding motif prevented the effect of 
  rod - actinin . these data suggest that -actinin can alter the 
  desensitization rate of asic1a channels , an effect that is mediated in part by 
  interactions between asic1a and ubiquitously expressed -actinins . asic1a currents also show a characteristic time - dependent recovery from 
  desensitization in acidic solution 
  ( 8 , 
  10 ) . we activated and 
  desensitized asic1a currents with a 60-s exposure to ph 6.0 solution 
  ( fig . then , after 
  varying time intervals at ph 7.4 , we re - exposed the channels to ph 6.0 . 
  co - conversely , 
  co - expressing a dominant - negative rod - actinin with asic1a or disrupting the 
  -actinin binding motif with asic1amut slowed the rate of 
  recovery . a , ph sensitivity of asic1a expressed alone in cho cells 
  (  ) or asic1a co - expressed with either -actinin-4 (  ) or 
  rod - actinin (  ) . -actinin-4 increased ph sensitivity of asic1a , 
  whereas rod - actinin decreased ph sensitivity ( * . i indicates peak 
  current amplitude at given ph , and iph5.0 indicates peak 
  current amplitude at ph 5.0 . b , asic1amut (  , 
  n = 613 ) had a reduced ph sensitivity compared with wild - type 
  asic1a (  , n = 6 ) ( * , p < 0.05 ) . n = 
  613 ) nor coexpressing -actinin-4 with asic1amut 
  (  , n = 413 ) altered the ph sensitivity of 
  asic1amut . -actinin modulated ph sensitivity of asic1a current in cho 
  cells . a , ph sensitivity of asic1a expressed alone in cho cells 
  (  ) or asic1a co - expressed with either -actinin-4 (  ) or 
  rod - actinin (  ) . -actinin-4 increased ph sensitivity of asic1a , 
  whereas rod - actinin decreased ph sensitivity ( * . i indicates peak 
  current amplitude at given ph , and iph5.0 indicates peak 
  current amplitude at ph 5.0 . b , asic1amut (  , 
  n = 613 ) had a reduced ph sensitivity compared with wild - type 
  asic1a (  , n = 6 ) ( * , p < 0.05 ) . note that neither 
  co - expressing rod - actinin with asic1amut (  , n = 
  613 ) nor coexpressing -actinin-4 with asic1amut 
  (  , n = 413 ) altered the ph sensitivity of 
  asic1amut . these results indicate that an association with -actinin-4 altered 
  several characteristics of asic1a channel function . the effects of the 
  dominant - negative rod domain and the asic1amut variant suggest that 
  asic1a interacts with -actinins that are endogenous in the heterologous 
  cells we used to express asic1a . therefore , in the studies described below we 
  tested for an effect of endogenous -actinins . figure 7.effect of -actinin on desensitization of asic1a and 
  asic1amut currents . data are time constants of desensitization 
  ( des ) of currents evoked by ph 5.0 . des of 
  acid - activated current in cho cells expressing asic1amut were not 
  affected by co - expression of -actinin-1 , -actinin-4 , or 
  rod - actinin . n = 1643 for currents with asic1a and the 
  -actinin variants , and n = 615 for currents with 
  asic1amut and the -actinin variants . * , p < 0.01 
  compared with asic1a alone ( left ) or asic1amut 
  ( right ) . * * , p < 0.05 compared with asic1a . data are time constants of desensitization 
  ( des ) of currents evoked by ph 5.0 . des of 
  acid - activated current in cho cells expressing asic1amut were not 
  affected by co - expression of -actinin-1 , -actinin-4 , or 
  rod - actinin . n = 1643 for currents with asic1a and the 
  -actinin variants , and n = 615 for currents with 
  asic1amut and the -actinin variants . * , p < 0.01 
  compared with asic1a alone ( left ) or asic1amut 
  ( right ) . * * , p < 0.05 compared with asic1a . acid - activated currents were desensitized by ph 6.0 application 
  for 60 s followed by application of ph 7.4 for the indicated length of time 
  ( 540 s ) . then , a second ph 6.0 application was used to assess recovery 
  of asic1a current . a , representative tracings showing recovery from 
  desensitization during 5 s and 20 s at ph 7.4 in cho cells expressing asic1a 
  alone . b , percentage recovery of current from desensitization in 
  response to ph 6.0 application for 60 s. data were compared with currents of 
  the first ph 6.0 application . , asic1a , n = 49 ;  , 
  asic1a + rod - actinin , n = 510 ;  , asic1a + -actinin-4 , n = 46 ;  , asic1amut , 
  n = 45 . * , acid - activated currents were desensitized by ph 6.0 application 
  for 60 s followed by application of ph 7.4 for the indicated length of time 
  ( 540 s ) . then , a second ph 6.0 application was used to assess recovery 
  of asic1a current . a , representative tracings showing recovery from 
  desensitization during 5 s and 20 s at ph 7.4 in cho cells expressing asic1a 
  alone . b , percentage recovery of current from desensitization in 
  response to ph 6.0 application for 60 s. data were compared with currents of 
  the first ph 6.0 application . , asic1a , n = 49 ;  , 
  asic1a + rod - actinin , n = 510 ;  , asic1a + -actinin-4 , n = 46 ;  , asic1amut , 
  n = 45 . * , figure 9.-actinin associated with asic1a in the brain . a , 
  western blot of -actinin and asic1a co - immunoprecipitated ( ip ) 
  from brain lysates . for immunoprecipitation anti - asic1a or 
  anti--actinin antibodies ( bm-75.2 , which recognizes all four 
  actinin - isoforms ) were used . western blots were probed with 
  anti--actinin antibody ( upper panels ) or anti - asic1a antibody 
  ( lower panels ) . note that anti - asic1a antibody co - precipitated 
  -actinin , and conversely , anti--actinin antibody co - precipitated 
  asic1a . the right panels show blots of total cell lysates used for 
  co - immunoprecipitation experiments and were probed with anti - asic1a and 
  anti--actinin antibody . b , brain lysates from wild - type and 
  asic1a mice 
  ( 5 ) were immunoprecipitated 
  with anti - asic1a or anti--actinin antibody as indicated . the 
  immunoprecipitates were probed with asic1a antibody . -actinin associated with asic1a in the brain . a , 
  western blot of -actinin and asic1a co - immunoprecipitated ( ip ) 
  from brain lysates . for immunoprecipitation anti - asic1a or 
  anti--actinin antibodies ( bm-75.2 , which recognizes all four 
  actinin - isoforms ) were used . western blots were probed with 
  anti--actinin antibody ( upper panels ) or anti - asic1a antibody 
  ( lower panels ) . note that anti - asic1a antibody co - precipitated 
  -actinin , and conversely , anti--actinin antibody co - precipitated 
  asic1a . the right panels show blots of total cell lysates used for 
  co - immunoprecipitation experiments and were probed with anti - asic1a and 
  anti--actinin antibody . b , brain lysates from wild - type and 
  asic1a mice 
  ( 5 ) were immunoprecipitated 
  with anti - asic1a or anti--actinin antibody as indicated . the 
  immunoprecipitates were probed with asic1a antibody . asic1a and -actinin associate in brain  to 
  test the hypothesis that asic1a and -actinins associate in neurons , we 
  used immunoprecipitation from mouse brain . we found that asic1a 
  co - precipitated -actinin , and -actinin co - precipitated asic1a 
  ( fig . -actinin failed to co - immunoprecipitate asic1a from 
  asic1a mouse brain 
  ( fig . these 
  results suggest that asic1a and -actinin reside in close proximity in 
  brain . -actinin regulates hippocampal acid - activated 
  currents  in the brain asic1a has been implicated in learning and 
  memory , and an association of asic1a with the cytoskeletal linker protein 
  -actinin could be one mechanism underlying synaptic plasticity . our 
  data suggested that in heterologous cells endogenous -actinins regulate 
  asic currents ; therefore , we asked if this was the case in neurons . we used 
  rt - pcr to learn which -actinin genes are expressed in the hippocampus 
  and found -actinin-1 , -2 , and -4 but not -actinin-3 
  ( fig . to reduce 
  their expression , we constructed vectors encoding sirnas directed against each 
  -actinin isoform . the efficacy and specificity of the rna interference 10b ) . with this information , for subsequent studies we 
  reduced -actinin expression by co - transfecting primary cultures of 
  hippocampal neurons with a mix of the sirnas against -actinin-1 , -2 , 
  and -4 ( rnai to -actinin-1 , -2 , and -4 ) . acid - evoked currents in hippocampal neurons are produced by a mixture of 
  asic1a , -2a , and -2b subunits 
  ( 7 , 
  53 , 
  54 ) . rnai against 
  -actinin-1 , -2 , and -4 increased endogenous asic current density in 
  hippocampal neurons ( fig . in addition , it reduced the ph sensitivity of 
  hippocampal acid - activated currents ( fig . although the effects were small , it also slowed the 
  rate of recovery from desensitization within the first 2 s 
  ( fig . 10e ) . these 
  results are consistent with our findings in a heterologous system expressing 
  asic1a alone . in contrast to our results with asic1a homomultimers studied in 
  cho cells , des of hippocampal asic currents were not affected 
  by reducing endogenous -actinins 
  ( fig . previous studies localized -actinin to dendritic spines and the 
  postsynaptic density 
  ( 2935 ) . 
  likewise , asic1a localized to dendritic spines and was enriched in the 
  postsynaptic membrane ( 5 , 
  14 , 
  15 ) . those earlier studies 
  positioned -actinin-4 and asic1a where they could interact . in this 
  study , we now show physical and functional links between these two proteins 
  both in heterologous cells and in neurons . the data also indicate that the 
  asic1a--actinin interaction occurred through a motif in the asic1a c 
  terminus . this interaction appears to be specific , as the -actinin 
  binding motif is not conserved in asic2 or -3 . in addition , asic1a associated 
  with -actinin-1 and -4 but not with -actinin-2 or -3 . these 
  results suggest specificity in the interaction between these two families of 
  proteins . -actinins bind actin and 
  may thereby link asic1a to the predominant cytoskeletal element of dendritic 
  spines ( 55 , 
  56 ) . the c termini of 
  -actinins also contain pdz binding motifs that attach to several 
  scaffolding proteins , including densin-180 
  ( 21 , 
  33 , 
  57 ) . thus , -actinin may 
  tie asic1a to multiple other proteins to form a signaling complex in the 
  postsynaptic membrane ( 21 ) . a 
  potential example ( 25 ) is the 
  functional coupling between asic1a and nmda receptors ; nmda receptors are also 
  regulated by -actinin 
  ( 38 , 
  58 ) . ( 25 ) suggested that during 
  acidosis , ca / calmodulin - dependent protein kinase ii is recruited 
  to nmda receptors where it phosphorylates asic1a , thereby increasing its ph 
  sensitivity . interestingly , asic1a 
  ( 5 ) , -actinin 
  ( 37 ) , nmda receptors 
  ( 58 ) , actin 
  ( 59 ) , and many of their 
  associated components contribute to synaptic plasticity . the mechanism by which -actinin-4 reduced current density appears to 
  be a direct effect on channel function . this conclusion is supported by the 
  finding that -actinin-4 altered multiple characteristics of the current 
  without changing the amount of asic1a on the cell surface . in cho cells , overexpressing -actinin-4 increased h - evoked current 
  density and increased ph sensitivity , whereas we saw the opposite effect when 
  we expressed the dominant - negative rod - actinin or mutated the -actinin 
  interaction motif in asic1a . likewise , in neurons , knockdown of 
  -actinin increased current density and reduced ph sensitivity . however , 
  there were some differences between h - gated currents in cho cells 
  expressing asic1a and h - gated currents in neurons . in cho cells -actinin-4 accelerated and rod - actinin and asic1amut slowed 
  recovery from desensitization , whereas -actinin sirna had minor effects 
  on recovery from desensitization in neurons . moreover , the interaction with 
  -actinin altered the desensitization rate of asic1a currents in cho 
  cells , but -actinin sirna had no effect on h - activated 
  current in neurons . we suspect these differences result from the fact that in 
  cho cells we studied asic1a homomeric channels , whereas in neurons much of the 
  asic1a exists in heteromultimers 
  ( 7 ) . the conclusion that 
  -actinin-4 increased ph sensitivity in both cases is interesting , 
  because ph sensitivity is a property usually associated with the asic 
  extracellular domain ( 4 , 
  6062 ) . therefore , we speculate that the -actinin binding motif in the asic1a 
  intracellular c terminus somehow links asic1a to structures that control 
  gating and ph sensitivity . rt - pcr with isoform - specific primer 
  pairs revealed expression of -actinin-1 , -2 , and -4 , whereas 
  transcription of -actinin-3 was not detected . muscle from the same 
  donor contained transcripts for all four -actinin isoforms including 
  -actinin-3 . in negative control experiments ( rt ) reverse transcriptase was omitted , but the specific primers pairs for all four 
  -actinin isoforms were present . b , to assess the efficacy and 
  specificity of sirna , cho cells were co - transfected with egfp - tagged 
  -actinin-1,-2,-3 , or -4 and isoform - specific sirnas . western blot was 
  probed with either anti - gfp or anti - tubulin antibody . c  f , reduction of endogenous -actinin affected 
  properties of acid - activated current in hippocampal neurons . for controls , 
  neurons were transfected either with vector encoding dsred or sirna directed 
  against -actinin-3 . c , current density was increased by 
  simultaneous knockdown of -actinin-1 , -2 , and -4 ( n = 
  2347 , * , p < 0.02 ) . d , ph sensitivity was 
  decreased by knockdown of endogenous -actinin-1 , -2 , and -4 (  , 
  ph50 = 6.16 , n = 936 ; * , p < 0.05 ) 
  compared with controls ( dsred (  ) , ph50 = 6.30 , n = 
  721 and rnai to -actinin-3 (  ) , ph50 = 6.36 , 
  n = 1728 ) . i indicates peak current amplitude at 
  given ph , and iph5.0 indicates peak current amplitude at 
  ph 5.0 . * , e , time - dependent recovery of 
  desensitized current evoked by ph 6.0 ( rnai to -actinin-1 , -2 , and -4 
  (  ) , n = 511 and rnai to -actinin-3 (  ) , 
  n = 311 ; * , p < 0.05 ) . f , time constants 
  of desensitization of currents evoked by ph 6.0 ( n = 
  1843 ) . rt - pcr with isoform - specific primer 
  pairs revealed expression of -actinin-1 , -2 , and -4 , whereas 
  transcription of -actinin-3 was not detected . muscle from the same 
  donor contained transcripts for all four -actinin isoforms including 
  -actinin-3 . in negative control experiments ( rt ) reverse transcriptase was omitted , but the specific primers pairs for all four 
  -actinin isoforms were present . b , to assess the efficacy and 
  specificity of sirna , cho cells were co - transfected with egfp - tagged 
  -actinin-1,-2,-3 , or -4 and isoform - specific sirnas . western blot was 
  probed with either anti - gfp or anti - tubulin antibody . c  f , reduction of endogenous -actinin affected 
  properties of acid - activated current in hippocampal neurons . for controls , 
  neurons were transfected either with vector encoding dsred or sirna directed 
  against -actinin-3 . c , current density was increased by 
  simultaneous knockdown of -actinin-1 , -2 , and -4 ( n = 
  2347 , * , p < 0.02 ) . d , ph sensitivity was 
  decreased by knockdown of endogenous -actinin-1 , -2 , and -4 (  , 
  ph50 = 6.16 , n = 936 ; * , p < 0.05 ) 
  compared with controls ( dsred (  ) , ph50 = 6.30 , n = 
  721 and rnai to -actinin-3 (  ) , ph50 = 6.36 , 
  n = 1728 ) . i indicates peak current amplitude at 
  given ph , and iph5.0 indicates peak current amplitude at 
  ph 5.0 . * , e , time - dependent recovery of 
  desensitized current evoked by ph 6.0 ( rnai to -actinin-1 , -2 , and -4 
  (  ) , n = 511 and rnai to -actinin-3 (  ) , 
  n = 311 ; * , p < 0.05 ) . f , time constants 
  of desensitization of currents evoked by ph 6.0 ( n = 
  1843 ) . -actinin is not the only protein that interacts with asic1a ; pick1 
  ( protein interacting with c kinase ) , stomatin , and annexin ii light chain p11 
  also bind the channel . in pick1 , a pdz domain interacts with the asic1a c 
  terminus , and the two proteins resided in dendrites , probably in postsynaptic 
  membranes ( 15 , 
  22 ) . stomatin associated with 
  asic1a in heterologous systems , although the site of interaction , remains 
  unknown ( 63 ) . neither pick1 
  nor stomatin altered the amplitude or properties of asic1a currents . the 
  annexin ii light chain p11 associated with an undefined site in the asic1a n 
  terminus ( 24 ) . in heterologous 
  cells , it increased cell surface expression of asic1a and increased asic1a 
  current amplitude without altering the characteristics of the current . thus , 
  of the proteins known to associate with asic1a , only -actinin affected 
  its channel function . the connections between -actinin and asic1a and their previously 
  established distribution in the postsynaptic membrane position these proteins 
  at a key site for influencing neuronal activity . together , these observations 
  suggest that -actinins may have an important impact on asic1a function 
  at this location .
the acid - sensing ion channel 1a ( asic1a ) is widely expressed in central and 
  peripheral neurons where it generates transient cation currents when 
  extracellular ph falls . 
 asic1a confers ph - dependent modulation on postsynaptic 
  dendritic spines and has critical effects in neurological diseases associated 
  with a reduced ph . 
 however , knowledge of the proteins that interact with 
  asic1a and influence its function is limited . here 
 , we show that 
  -actinin , which links membrane proteins to the actin cytoskeleton , 
  associates with asic1a in brain and in cultured cells . 
 the interaction 
  depended on an -actinin - binding site in the asic1a c terminus that was 
  specific for asic1a versus other asics and for -actinin-1 and 
  -4 . 
 co - expressing -actinin-4 altered asic1a current density , ph 
  sensitivity , desensitization rate , and recovery from desensitization . 
  moreover , 
 reducing -actinin expression altered acid - activated currents 
  in hippocampal neurons . 
 these findings suggest that -actinins may link 
  asic1a to a macromolecular complex in the postsynaptic membrane where it 
  regulates asic1a activity .