glutamate functions as the major excitatory neurotransmitter by binding to n - methyl - d - aspartate receptors ( nmdars ) that are widespread in the central nervous system .
the nmdars constitute a major class of ionotropic glutamate receptors and play an essential role in synaptic transmission , plasticity , and memory .
activation of nmdars results in cell membrane depolarization with an equilibrium potential near 0 mv , producing the excitatory postsynaptic potential ( epsp ) and leading to an increase of ca influx into the cell .
the intracellular ca can in turn function as a second messenger , mediating a variety of signaling cascades .
excessive activation of nmdars by glutamate mediates neuronal damage in many neurological disorders including ischemia and neurodegenerative diseases ( choi et al .
the nmdars have long been considered the main target for the treatment of excitotoxicity - related neuronal injury , and a variety of antagonists or blockers of nmdars have been developed .
unfortunately , the results of clinical trials have been disappointing because of the obvious side effects associated with blocking the physiological roles of nmdars ( chen and lipton , 2006 ) .
therefore , a better understanding of the mechanism of how nmdars can be modulated by regulatory proteins should help in the development of new therapeutic agents to counteract overactive nmda receptor function , and may represent an alternative to treating nmdar - mediated excitotoxic injury .
this short review focuses on the specific negative modulation of nmdars by a neuronal calcium sensor ( ncs ) protein , dream / calsenilin / kchip3 .
nmdars are believed to be heterotetrameric complexes composed of combinations of the obligatory nr1 subunit and nr2 and/or nr3 subunits ( chazot and stephenson , 1997 ; laube et al .
, 1998 ; schorge and colquhoun , 2003 ; furukawa et al . , 2005 ) .
the nr1 subunit is encoded by a single gene but exists as eight functional splice variants , while the nr2 ( nr2a - b ) and nr3 ( nr3a - b ) subunits are encoded by four and two different genes , respectively .
the nmdar subunits form a central ion conductance pathway selective for cations such as na , k , and ca , and share a common membrane topology , with each subunit consisting of four transmembrane ( tm ) domains ( m1m4 ) .
the long extracellular n - terminal regions of nmdar subunits are organized as a tandem of two domains .
the first domain , called the n - terminal domain ( ntd ) that includes the first 380 amino acids , is involved in tetrameric assembly ( mayer , 2006 ; paoletti and neyton , 2007 ; stroebel et al . ,
the second domain of about 300 amino acids is known as the agonist - binding domain ( abd ) that precedes the tm1 domain .
the abd binds glycine ( or d - serine ) in the nr1 and nr3 subunits , whereas the nr2 abd binds glutamate ( furukawa et al .
, 2005 ; yao and mayer , 2006 ) . the pore loop ( p loop ) , or the m2 region , forms the narrowest constriction of the channel ion conductance pathway and determines the permeation properties of nmdars .
the nmdars feature an intracellular c - terminal tail of about 400600 residues that has a strong diversity in its amino acid sequence .
the c - terminal tails of nmdar subunits contain a series of short motifs that interact with intracellular factors or binding partners involved in receptor trafficking , anchoring and signaling ( skeberdis et al .
activation of nmdars requires a simultaneous binding of two co - agonists , glutamate , and glycine with different biophysical properties of ion permeation .
the typical nmdars contain nr2 subunits with properties of high permeability to ca and extracellular mg block at hyperpolarized membrane potentials ( wrighton et al .
different from conventional nr1/nr2 heterotetramers , nr3-containing nmdars have unique properties with a five to tenfold decrease of ca permeability , insensitivity to mg block , and reduced single - channel conductance and open probability , functioning as a negative modulator for nmda receptor channel function ( das et al . , 1998 ;
2009 ; cavara et al . , 2010 ; henson et al . , 2010 ) .
nmda receptors are also regulated by other intracellular signals and proteins , including calcium , protein kinases , protein phosphatase calcineurin , and calcium - sensitive proteins such as calmodulin ( legendre et al . , 1993 ; vyklicky , 1993 ; lieberman and mody , 1994 ; tong et al . , 1995 ; ehlers et al . , 1996 ) .
calcium - dependent nmda receptor desensitization and inactivation provides a feedback mechanism capable of regulating subsequent ca entry into the postsynaptic cell through nmda channels ( figure 1 ) .
schematic representation for inhibitory effect of dream / calsenilin / kchip3 on nmdars in a ca - sensitive manner . upon activation of nmdars by glutamate
binding , ca influx through nmdars increases the association between dream and nr1 subunits , resulting in reduced surface expression of nmdars , and subsequent inhibition of nmdars - mediated ca influx and excitotoxicity .
dream functions as a ca - sensitive modulator for the negative feedback control of nmdar function .
so far , a number of nr1 or nr2 subunit binding partners have been identified in the postsynaptic density .
the nr1 binding proteins include calmodulin ( cam ) ( ehlers et al . , 1996 ; akyol et al . , 2004
) , ca / cam - dependent protein kinase ii ( camkii ) ( leonard et al . , 2002 ) , -actinin ( wyszynski et al . , 1997 ; merrill et al . , 2007 ) ,
tubulin ( van rossum et al . , 1999 ) , spectrin ( wechsler and teichberg , 1998 ) , neurofilament ( ehlers et al . ,
calmodulin binding to the nr1 subunit is ca dependent and occurs with homomeric nr1 complexes , heteromeric nr1/nr2 subunit complexes from expression systems , and nmda receptors from the brain .
calmodulin binding to nr1 causes a fourfold reduction in nmda channel open probability , mediating the negative modulation of nmdar function ( ehlers et al . , 1998 ) .
the downstream regulatory element antagonist modulator ( dream ) protein , first identified in the nucleus as a ca - regulated transcriptional repressor through its binding to dna at specific regulatory elements , contains four ca - binding ef - hand domains and belongs to the ncs family ( carrion et al . , 1999 ; burgoyne , 2007 ) .
dream was named for its ability to block gene expression in its ca - free form via direct binding with the downstream regulatory element ( dre ) sequence in target genes such as preprodynorphin ( ppd ) , c - fos , hrk , na , and ca exchanger ncx3 ( carrion et al .
, 1999 ; sanz et al . , 2001 ; gomez - villafuertes et al . ,
dream was also named calsenilin or kv channel interacting protein 3 ( kchip3 ) ( buxbaum et al . , 1998 ; an et al . , 2000
) , indicating that dream / calsenilin / kchip3 has multifunctional properties . in the nucleus
the dream protein functions as a dimer , whereas outside the nucleus kchip3 is a monomer and regulates the surface expression and gating kinetics of kv4 channels ( an et al . , 2000 ;
kim and sheng , 2004 ; scannevin et al . , 2004 ; pioletti et al .
dream / calsenilin / kchip3 is preferentially expressed in the central nervous system , as well as in non - neuronal tissues ( link et al .
dream / calsenilin / kchip3 knock - out mice display a hypoalgesic phenotype , suggesting a critical role of dream / calsenilin / kchip3 in pain modulation ( cheng et al . ,
in addition , emerging evidence reveals the role of dream / calsenilin / kchip3 in long - term potentiation ( ltp ) ( lilliehook et al . , 2003 ) and learning and memory ( alexander et al . , 2009 ; fontan - lozano et al . , 2009 ) , suggesting a possible connection between dream and nmda function .
kchip13 were initially identified from a rat brain library in yeast two - hybrid ( yth ) screens using the cytoplasmic n - terminal domain ( amino acids 1180 ) of rat kv4.3 as a bait ( an et al . , 2000 ) .
similarly , kchip4 from mouse and human was accidentally cloned using the c - terminal 43 amino acid residues of presenilin 2 ( ps2 , amino acids 406448 ) as a bait in the yth system ( morohashi et al . , 2002 ) .
kchip4 , also known as calsenilin - like protein ( calp ) , binds to ps2 which is known to facilitate intramembranous -cleavage of -amyloid protein precursor ( app ) ( morohashi et al . , 2002 ) .
kchip14 ( 216  256 amino acids ) can co - immunoprecipitate and co - localize with either kv4 from co - transfected cells or kv4 -subunits from tissues , and thus constitute integral components of native kv4 channel complexes ( wang , 2008 ) . kchip14
all share a conserved carboxy - terminal core region that contains four ef - hand - like calcium binding motifs , but have a variable amino - terminal region that causes diverse modulation of kv4 trafficking and channel function ( an et al .
, 2000 ; holmqvist et al . , 2002 ; scannevin et al . , 2004 ;
findings from co - immunoprecipitation experiments show that dream antibody can immunoprecipitate endogenous nr1 subunit and dream protein from rat hippocampal tissue ( zhang et al . , 2010 ) .
in the reciprocal co - ip studies in hek 293 cells expressing dream and nr1 - 1a ( nr1a ) proteins , nr1 antibody can also immunoprecipitate dream along with the nr1 subunit .
gst pull - down assays reveal that the n - terminus of dream directly interacts with the nr1a c - terminus , and that the dream - nr1 interaction is sensitive to ca and depends on the ef hand domains of dream ( zhang et al . , 2010 ) .
psd-95 is a major scaffolding protein in the postsynaptic density , tethering nmdars to signaling proteins , and is critical for nmda receptor function ( kim and sheng , 2004 ) .
wu et al . generated a line of transgenic mice ( tgdream ) overexpressing a dominant active dream mutant , and compared nmda receptor - mediated epscs in tgdream and wild - type mice under conditions of various stimulation intensities ( wu et al .
they found that the amplitude of nmda receptor - mediated epscs in tgdream mice is significantly reduced compared to that in wild - type mice ( wu et al . , 2010 ) .
in addition , ltd is significantly reduced in tgdream mice whereas ltp is not affected by dream , demonstrating that dream interacts with psd-95 , and that the interaction is negatively regulated by calcium ( wu et al . ,
2010 ) . in xenopus oocytes expressing nr2b - containing nmdars alone or together with dream ,
two - electrode voltage clamp recordings show that , in the absence of dream , the peak currents of nmda channels activated by glutamate ( plus glycine ) are suppressed by dream , and the current decrease is caused by a reduction in the density of nmdars at the cell surface ( figure 1 ; zhang et al . , 2010 ) .
fontan - lozano et al . recently provided another piece of evidence that dream negatively regulates the function of nmda receptors ( fontan - lozano et al . , 2011 ) .
by taking advantage of mice lacking the dream protein , they demonstrated that the facilitated learning induced by decreased expression of kv4.2 in dream mice requires the activation of nmda receptors containing the nr2b subunit ( fontan - lozano et al . , 2011 ) .
this study not only indicates the significance of the balance between kv4 channel function and nmdar activity , but also suggests the formation of a functional complex between dream / kv4.2/nmdars that regulates the synaptic efficacy mediating synaptic plasticity and learning .
excitotoxicity is caused by overactivation of nmda receptor function , and inhibition of nmdars can reverse the neuronal toxicity .
the data available so far support both the pro - apoptotic and anti - apoptotic roles of dream .
in general , the pro - apoptotic role of dream closely correlates with its interaction with presenilins , the production of amyloid beta ( a ) and the modulation in ca signaling , whereas the anti - apoptotic role of dream is conferred by its transcriptional repressor activity on the apoptotic protein hrk .
jo et al . reported that hela cells transiently transfected with dream exhibit the morphological and biochemical features of apoptosis and that expression of presenilin potentiates dream - induced apoptosis ( jo et al . , 2001 ) .
jo et al . also reported that dream expression increases in either human neuroblastoma sk - n - be2(c ) cells or rat neuroblastoma b103 cells after exposure to a , but no other apoptotic inducers such as staurosporine , thapsigargin , and calcium ionophore a23187 .
the pro - apoptotic role of dream is selectively induced during ab toxicity . because of the involvement of presenilins/-secretase in a formation and neuronal death , dream coordinates with presenilin activity to play a crucial role in these processes through binding with the c - terminus of presenilins ( jo et al . , 2003 , 2004 ) .
stably expressed dream in h4 neuroglioma cells which showed no initiation of apoptosis in the absence of apoptosis triggers , and the apoptosis - associated caspase and calpain activities were not affected by dream ( lilliehook et al .
on the other hand , binding of the transcriptional repressor dream to the hrk gene avoids inappropriate hrk expression and apoptosis in hematopoietic progenitor cell lines ( sanz et al . , 2001 , 2002 ) .
nevertheless , the precise function of dream in pro - apoptosis and anti - apoptosis remains to be explored . from our previous observations
, we noticed that cell viability is affected by the amount of exogenous dream gene and the method of transfection .
however , we could not observe any obvious morphological changes associated with cell death after the transfection of dream . to prove the cytoprotective role of dream , we utilized cell lines and primary cultured hippocampal neurons with dream overexpression or sirna - mediated knockdown , and evaluated lactate dehydrogenase ( ldh ) leakage and propidium iodide ( pi ) uptake both in nmda and oxygen - glucose deprivation ( ogd ) -induced excitotoxic injury models ( zhang et al . , 2010 ) .
administration of nmda markedly increases the number of pi - positive cells ( dead cells ) in nmdar - transfected cho cells , whereas co - expression of dream greatly reduces pi - positive cells ( zhang et al . , 2010 ) .
ogd is commonly used in vitro to mimic ischemia - reperfusion insult to the brain , and ogd treatment induces a significant increase of ldh release ( dawson et al . , 1994 ) .
with over - expression of dream , however , ogd treatment induces a smaller increase in ldh release .
these results indicate that the over - expression of dream attenuates nmdar - mediated excitotoxicity ( zhang et al . , 2010 ) . we have previously tested the effect of dream sirna on nmda - induced current and excitotoxic injury in hippocampal neurons ( zhang et al . , 2010 ) .
knockdown of endogenous dream with sirna results in an increased amplitude of nmda current recorded by whole - cell patch - clamp assays .
after nmda exposure , pi - positive cells in the dream sirna group increase compared with control sirna , indicating the inhibitory effect of dream on nmdar - mediated current and excitotoxic injury ( zhang et al . , 2010 ) .
the ncs protein dream / calsenilin / kchip3 acts as an auxiliary subunit and suppresses nmda receptor channel function .
this negative modulation of nmda receptor function by dream likely provides a feedback mechanism by which overactive nmda receptors are inhibited .
therefore , targeting regulatory proteins of nmdars may represent an alternative approach to treating nmdar - mediated excitotoxic damage and providing neuroprotection .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 n - methyl - d - aspartate receptors ( nmdars ) are glutamate - gated ion channels highly permeable to calcium and essential to excitatory neurotransmission . 
 the nmdars have attracted much attention because of their role in synaptic plasticity and excitotoxicity . 
 evidence has recently accumulated that nmdars are negatively regulated by intracellular calcium binding proteins . 
 the calcium - dependent suppression of nmdar function serves as a feedback mechanism capable of regulating subsequent ca2 + entry into the postsynaptic cell , and may offer an alternative approach to treating nmdar - mediated excitotoxic injury . 
 this short review summarizes the recent progress made in understanding the negative modulation of nmdar function by dream / calsenilin / kchip3 , a neuronal calcium sensor ( ncs ) protein . 