BACKGROUND
n-methyl-d-aspartate receptors  are inotropic glutamate receptors  that contribute to multiple neuronal functions such as .
however, excessive nmdar activity can lead to neuronal dysfunction and neuronal death  <cit> .
functional nmdar require two glun <dig> and two glun <dig> subunits .
the glun <dig> subunit can be replaced by a glun <dig> subunit, but when present the glun <dig> subunit decreases the nmdar activity  <cit> .
there are four glun <dig> subunits   <cit> .
an increase in the expression of glun <dig>  glun2a, and glun2b subunits in the hippocampus contribute to neurological problems that develop following traumatic brain injury  <cit> .
increase in the activity/expression of glun2b has been postulated to contribute to neuronal damage following stroke, as well as to the development of diseases such as parkinson, huntington, alzheimer and chronic neuropathic pain  <cit> .
nmdar are similar to other iglurs in that each subunit has four domains, which include the amino-terminal domain , an agonist/ligand binding domain , a trans-membrane domain  and a carboxyl terminal domain  .
in glun <dig> subunits, the atd domain contains binding sites for allosteric inhibitors such as zinc  <cit>  and ifenprodil  <cit> .
the abd domain in glun <dig> and glun <dig> binds the coagonist glycine, whereas the abd domain in glun <dig> binds the agonist glutamate .
some peptides isolated from marine cone snails have shown specificity for glun <dig> subunits of the nmdar  <cit> .
one member of the conantokin family, conantokin-g is a 17-residue peptide that shows selectivity for nmdar containing the glun2b subunit  <cit> .
although, conantokin-g shows high selectivity for glun2b, its reversibility appears to be very slow and incomplete on neuronal cells  <cit> .
we designed a number of peptides with potential glun2b–nmdar antagonistic activity, this was done by using point mutations on the conantokin-g sequence and evaluating their structure as well as their binding capacity to the ligand binding domain of the glun2b subunit by using various in silico evaluations.
here we tested this prediction for two of these peptides, ear <dig> and ear <dig>  and found that both of these peptides blocked in a highly reversible manner nmda-evoked currents in hippocampal neurons, and that ear <dig> was more selective for nmdars containing the glun2b than for those containing the glun2a subunit.
the high reversibility of both of these peptides and the higher selectivity of ear <dig> for the nmdars containing the glun2b increase their potential as pharmacological agents.
methods
peptides
these values were provided by the company
apurity determine by high-performance liquid chromatograph
bmw: molecular weight, theoretical
cms : hydrogen cation mass spectrometry analysis
dms : sodium ion mass spectrometry analysis
peptide three-dimensional structure
for each peptide a three-dimensional structure model was generated using ucsf  chimera software , and a refined three-dimensional structure was obtained in an hydrophilic environment using pepstr  as previously described  <cit> .
structural model of the glun2b
details of this model are described in a separate publication  <cit> .
briefly, this model was obtained by using the amino acids 1- <dig> of the reported sequence of the glun2b subunit .
this amino acid sequence includes the amino terminal  and the ligand binding  domains.
the glun2b structural model was predicted using i-tasser , this software used several templates to determine the best model.
the best model  was obtained when the template used was the inotropic glutamate ampa  receptor   <cit> .
this template has been previously used to elucidate other functions of glutamate receptors  <cit> .
the c-score usually ranges between − <dig> and  <dig>  the most positive the value the better the model topology, the glun2b model selected has a c-core of  <dig>   <cit> .
the tm-score provides a value of the structural correspondence between two structures , values higher than  <dig>  indicate that the model topology is likely to be correct  <cit> , the selected glun2b model has a tm-score of  <dig> .
the selected glun2b model  was further evaluated using pdbsum , that provides various parameters including hydrophobicity profile, solvent accessibility, prediction of ligand binding sites.
the secondary structure of the model was also evaluated using ramachandran plots , which provides the percentage of structural regions that are either “favorable”, “allowed” or “not-allowed”.
a model with good stereo-chemistry should have close to 90% of the residues in structurally favorable regions  <cit> .
the selected model has  <dig> % of the residues located in structurally “favorable” regions, and  <dig> % of the residues in “allowed” regions  <cit> .
the model has structural topology comparable to that described for other inotropic glutamate receptors  <cit> , including the atd, ldb and the transmembrane  domains.
the atd and lbd structural configurations are composed of mostly α helices and allows the binding of modulators an agonists  <cit> .
molecular docking
molecular docking was performed with the obtained three-dimensional structures of the peptides and of the glun2b subunit by using “autodock vina” .
a grid box was generated at the center of the lbd of the glun2b model to which the corresponding hydrogen atoms and chargers were assigned.
the grid box was  <dig> Å, in the software “completeness” was set to  <dig> and the number of results per docking was set to  <dig>  for each peptide.
the peptides corresponded to the ligands.
the best conformations and orientations, which displayed the lowest values for binding energy , were visualized using autodock tools .
the docking results were then analyzed with the rosetta flexpepdock web server , which refines the peptide-receptor interaction by optimizing the peptide orientation within the binding site using the monte-carlo method and focusing on energy minimization of the ligand with the receptor.
with the starting structure flexpepdock realized  <dig> independent simulations,  <dig> simulations were done under high resolution.
the other  <dig> simulation included a step of pre-optimization at low resolution, followed by a method of refinement of high resolution.
this resulted in  <dig> models for each peptide, we selected the best one for each peptide based on the score provided by the punctuation function of rosetta   <cit> .
the docking images were generated using ucsf chimera .interactions between peptide-receptor were determine using discovery studio visualizer v <dig>  software .
hippocampal cultures
pregnant rats were maintained and used following the guidelines approved by the new york university langone medical center institutional animal care and use committee.
pregnant rats were killed using co <dig> followed by cervical dislocation.
day  <dig> embryos  were removed by caesarian section.
dissociated hippocampal cultures were prepared as previously described  <cit> .
cells were plated on 12 mm coverslips  with neurobasal medium supplemented with b <dig>  or with 2% bovine fetal serum and used between  <dig> and 37 days in vitro; the data was collected from  <dig> cells that had on average  <dig>  days in vitro .
coverslips were pre-treated by placing 50 µl of poly-d-lysine  and laminin  in the middle of the coverslip for 5 min at room temperature .
transfection of hek- <dig> cells
the plasmids pegfp-nr1a  , pegfp-nr2b  , and pegfp-nr2a   were a gift from stefano vicini  <cit> .
hek- <dig> cells  were grown on 12-mm coverslips in  <dig> well plates.
hek- <dig> cells  were transfected using lipofectamine  <dig> according to the product instructions , with a mixture of glun1a and glun2a subunits or a mixture of glun1a and glun2b subunits.
the amount of cdna for each plasmid was  <dig>  µg/well.
cells were used 2–4 days following transfection.
gfp signal was used to identify positively transfected cells.
electrophysiology
electrophysiological recordings were performed under voltage-clamp by using the whole cell conformation  <cit> .
intracellular solution :  <dig> cs-gluconate,  <dig> cscl,  <dig> hepes,  <dig> egta, 4 mg-atp,  <dig>  na-gtp, ph  <dig>    <cit> .
extracellular solution :  <dig> nacl,  <dig>  kcl,  <dig>  cacl <dig>   <dig> hepes,  <dig> d-glucose, ph  <dig> .
mg2+ was omitted to prevent the voltage-dependent block of the nmdar ion currents  <cit> .
the following blockers were added to extracellular solution:  <dig>  μm tetrodotoxin  to block na+ channels  <cit> ; 20 μm cnqx  to block ampa and kainate receptors, 50 μm bicuculine  and 100 μm picrotoxin  to block gaba receptors, and 1 μm estricnin to block glycine channels.
currents were measured using borosilicate pipettes  with resistances of 1–3 mΩ.
experiments were conducted at room temperature.
currents were measured using a 2-electrode voltage clamp amplifier axopatch 200b .
output from the voltage clamp amplifier was sent to a microcomputer using a data acquisition interface .
currents were filtered at  <dig>  or 5 khz  and sampled at 10–50 khz.
for analysis the current traces were filtered at 1 khz.
pclamp  <dig>   was used for data acquisition and data analyses.
cells were held at −60 mv and the nmdar-evoked currents were obtained by applying a 5 s pulse of nmda  followed by a 10 s pulse of bath solution through a puffer  located directly on top of the cells .
successive stimulations with nmda were done every 4 min, which under control conditions resulted in nmda-evoked ca2+ currents of similar magnitude over time .
when the effects of mk- <dig> a general nmdar blocker, ro 25- <dig>  a specific glun2b/nmdar blocker), and peptides  were evaluated, the cells were first exposed to two or three consecutive nmda pulses  to assure that the cell recording was stable, then the blocker  was applied 10 s prior and during the following nmda pulse.
in all cases the nmda pulses were followed by a 10 s pulse of bath solution through the puffer.
the chamber was continuously perfused with the bath solution , except when the puffer was on.
peptides were added at a final concentration of  <dig>   <dig> and 500 μm.
the area of the nmda-evoked inward currents was measured over a period of 5 s, from the onset of the nmda-evoked inward current.
the areas were normalized using the nmda-evoked inward current measured prior to the addition of the blocker or peptide .
figures were done using graphpad prism v <dig>  results are expressed as mean and standard error of mean .fig.
<dig> evoked nmda-currents in cultured hippocampal neurons are blocked by -mk <dig> and ro 25- <dig>  a whole-cell nmda-evoked inward currents  induced by consecutive 5 s pulses of 100 µm nmda + 10 µm glycine, with a 4 min interval.
the last image is an overlap of the first  and eighth  nmda-evoked current traces.
b inhibition of nmda-evoked inward current by the general nmdar antagonist -mk <dig> .
top shows traces of the nmda-evoked inward currents and below the normalized area of the nmda-evoked inward currents  for control ; and before, during two consecutive exposures to -mk <dig> and after removing -mk <dig> .
c inhibition of nmda-evoked inward current by the glun <dig> specific blocker ro 25- <dig> .
top shows the nmda-evoked inward currents in a cell before, during two consecutive exposures to ro 25- <dig>  and after removing ro 25- <dig>  bottom shows the normalized nmda-evoked inward currents for control ; and before and during four consecutive exposures to ro 25- <dig> .
significant different compared to the control group #p <  <dig> , +p <  <dig> , two-way anova with sidak’s multiple comparison post-test; n , control: n = 7; -mk801: n = 3; and ro 25-6981: n =  <dig> during the first three nmda stimulations and  <dig> for the last two nmda stimulations
statistics
when comparing two groups with various stimulations we used two-way anova  followed by sidak’s multiple comparison post-test.
when comparing more than two groups with various stimulations we used two-way anova followed by dunnett’s multiple comparison post-test, as recommended by graphpadprism  <dig> .
results are expressed as mean and standard error mean .
RESULTS
electrophysiological evaluation of ear <dig> and ear <dig> peptides on hippocampal primary cultures
primary hippocampal cultures were used between  <dig> and 20 days of culture, a period when the glun2b subunit has been shown to be highly expressed  <cit> .
when nmda stimulations were separated by a 4 min interval the magnitude of the nmda-evoked ca2+ currents was similar between consecutive stimulations , as reflected by the average behavior shown by the black squares in figs.
1b, c and 2a, b. the general nmda blockers mk- <dig> , blocked the nmda-evoked currents .
the magnitude of mk-801-mediated block increased with exposures to the blocker and it was irreversible , as previously reported in hippocampal neurons  <cit> .
in order to determine the contribution of the glun2b to the nmda-evoked response we exposed the hippocampal cells to a glun2b selective blocker ro 25- <dig> , at a concentration that is  <dig> fold higher that producing 50% inhibition of nmda/glun2b-mediated currents  <cit> .
at this concentration ro 25- <dig> has minimal or no effects on voltage-dependent na+ and ca+ <dig> channels  <cit> .
block with ro 25- <dig> showed no reversibility following 4 min of washout .
we observed that ro 25- <dig> blocked 70% of the nmda-evoked currents ; hence under our experimental conditions about 70% of the nmda-evoked currents in hippocampal neurons are mediated by activation of glun2b/nmdar.
moreover, the increase in the magnitude of the block observed following consecutive exposures to ro 25- <dig> is consistent with the blockers being use-dependent.fig.
<dig> ear <dig> and ear <dig> peptides inhibit nmda-evoked inward currents in hippocampal neurons.
a, b the inhibitory effect on nmda-evoked currents during consecutive exposures to either  <dig>   <dig> and 500 µm of ear <dig>  and ear <dig> .
the areas of the nmda-evoked inward currents were normalized to that measured prior to the addition of ear <dig>  .
significant different compared to the control group.
#p <  <dig> , +p< <dig> , @p <  <dig> , ^p <  <dig> , $p <  <dig> , two-way anova with dunnett’s multiple comparison post-test; n  for each pulse were for control ; for ear18: 10 µm , 100 µm ; 500 µm ; for ear16: 10 µm , 100 µm  and 500 µm .
c, d show nmda-evoked current traces before, in the presence of ear <dig>  and following washout.
the arrows indicate that upon the simultaneous removal of nmda + ear, there is a transient increase in the nmda-evoked inward current .
e dose–response, the data was fitted to y = bottom + /*hillslope)).
when the entire current was considered: bottom =  <dig> no current, top = − <dig> , the ic50 s were  <dig> and 241 µm for ear <dig> and ear <dig>  respectively
in hippocampal neurons, the effect of ear <dig> and ear <dig> on the nmda-evoked currents was accessed by applying each peptide for four consecutive nmda-evoked currents to determine whether these peptides blocked the nmda-evoked current and if so whether their block displayed use-dependence behavior.
ear <dig> inhibited nmda-evoked currents in a dose-dependent manner, and this inhibition was reversible .
ear <dig> had a similar inhibitory effect on nmda-evoked currents in hippocampal neurons .
for a given dose, the magnitude of the block mediated by ear <dig> or ear <dig> did not increase significantly with consecutive exposures ; hence these peptides show no clear use-dependence .
examination of the current traces, show that following the simultaneous removal of the nmda + ear <dig>  there was a relatively short-lived increase in the nmda-evoked current, suggesting that the off rate of ear <dig>  is fast enough that would allow binding of nmda early on during washout .
figure 2e shows the dose–response curves.
when considering the total current magnitude the estimated ic <dig> values were similar for both peptides being  <dig> and 242 µm for ear <dig> and ear <dig>  respectively .
no inhibitory action was observed with the control peptide at 500 µm .
ear <dig> displays higher selectivity than ear <dig> for the glun2b subunit over the glun2a subunit
to test the selectively of these peptides for the glun2b subunit, the blocking effect of these peptides was tested in hek- <dig> cells expressing recombinant nmdar comprised of either glun1a–glun2a or glun1a–glun2b subunits.
the rationale for doing so is that these are the predominant glun <dig> subunits in hippocampal neurons, moreover the amino acid sequence corresponding to the sequence used for the structural model of glun2b  is conserved  and of the lbd is highly conserved  between glun2a and glun2b  <cit> .
we selected 500 µm of each peptide to test their potency on hek <dig> cells expressing recombinant nmdar, since this peptide concentration produced a strong but not maximal block  on nmda-evoked currents in hippocampal neurons .
this should allow to determine whether or not these peptides displayed differences in potency between the glun2b and glun2a subunits.
figure  <dig> shows that as predicted ear <dig> at 500 µm blocked more strongly nmda-evoked currents in hek cells transfected with the glun1a–glun2b subunits  than those in cells transfected with the glun1a–glun2a subunits .
in contrast, ear <dig> at 500 µm blocked to a similar level the nmda-evoked currents in hek-cells transfected with either glun1a–glun2b  or glun1a–glun2a subunits .
the ic <dig> of each peptide for blocking glun1a–glun2b and glun1a–glun2a was estimated by using eq.
, where ‘x’ is the peptide concentration in log <dig> ; ‘y’ is the % inhibition at ‘x’ ; emax: is the maximal inhibitory effect which we are assuming to be 100%; ‘n’ is the slope of the fitting, which it is assumed to be “1” since there is only a single recombinant nmdar .
<dig> y=emax/1+ic50/xn the results are shown in fig.
3g, and indicate that ear <dig> has about  <dig> fold higher affinity for glun1a–glun2b than for glun1a–glun2a .
while ear <dig> displayed a comparable affinity for both glun1a–glun2b and glun1a–glun2a .fig.
<dig> ear <dig> but not ear <dig> shows selectivity for glun2b over glun2a.
a, b ear16-mediated inhibition of nmda-evoked inward currents on hek- <dig> cells expressing glun1a–glun2b subunits  or expressing glun1a–glun2a subunits .
d, e ear18-mediated inhibition of nmda-evoked inward currents on hek- <dig> cells expressing glun1a–glun2b  or in cells glun1a–glun2a subunits .
c, f the areas of the nmda-evoked inward currents were normalized to that measured prior to the addition of ear <dig>  or ear <dig>  .
no significant difference was found between “control-nmda” and “wash-nmda”.
significant different compared to “control-nmda”  or when compared to “wash-nmda”  #p <  <dig> , +p <  <dig> , @p <  <dig> , two-way anova with sidak’s multiple comparison post-test; n  ear <dig>  n =  <dig> for glun1a–glun2b, and n =  <dig> for glun1a–glun2a; ear <dig> n =  <dig> for glun1–glun2b  and n =  <dig> for glun1a–glun2a
as observed in hippocampal neurons, in hek- <dig> cells transfected with recombinant nmdar the simultaneous removal of the nmda + ear <dig>  also resulted in a short-lived increase in the nmda-evoked current, suggesting that the off rate of ear <dig>  was fast enough that would allow binding of nmda early on during washout.
docking of ear <dig> and ear <dig> with the lbd of the glun2b model
figure  <dig> shows the docking results of the interaction between the ear <dig> and the glun2b subunit.
ear <dig> binds to the glun2b through  <dig> hydrogen bridges,  <dig> of them are with residues located at the lbd in glun2b   <cit> .
the ear <dig> residues asp  <dig> and  <dig> contribute to the formation of these hydrogen bridges.
the residue met  <dig>  that has been reported to contribute to the molecular interaction between con-g and glun2b  <cit> , is predicted to form a hydrophobic interaction  with the leu  <dig> of ear <dig>  the majority of the interactions involve the amino terminal of ear <dig>  similar to what has been described for conantokin-g  <cit> , indicating that the substitution of the residue γ-carboxiglutamic acid  by asp appears to emulate the con-g interaction with the glun2b.fig.
<dig> schematic representation of docking of ear <dig> with the lbd of glun2b.
a, b docking of ear <dig> with the lbd  in the glun2b model.
c representation of the molecular surface of lbd in the glun2b, and the peptide conformation.
d amino acid residues and labels of glun2b  that form hydrogen bridges  with ear <dig>  the amino acid residues for ear <dig> are represented by element: carbon gray, nitrogen: dark blue, oxygen: red and, hydrogen: white; and the amino acid names in gray
in the case of ear <dig>  the interactions involve  <dig> hydrogen bridges ,  <dig> of them are with residues located at the lbd in glun2b .
the ear <dig> asp  <dig> residue plays an important role in establishing these  <dig> hydrogen bridges .
the tyr  <dig> of ear <dig> contributes to the formation of a hydrophobic interaction  with the ile  <dig> of the glun2b, and the ala  <dig> of ear <dig> is forming and hydrogen bridge with the lys  <dig> of the glun2b.
at the lbd, the ear <dig> conformation is less extended than that of the ear <dig>  allowing ear <dig> to interact with a larger number of residues in the glun2b.fig.
<dig> schematic representation of docking of ear <dig> with the lbd of glun2b.
a, b docking of ear <dig> with the lbd in the glun2b model.
c representation of the molecular surface of lbd in the glun2b, and the peptide conformation.
d amino acid residues and labels of glun2b  that form hydrogen bridges  with ear <dig>  the amino acid residues for ear <dig> are represented by element: carbon: gray, nitrogen: dark blue, oxygen: red, and hydrogen: white; and the amino acid names are in gray
discussion
in this study we demonstrated that the peptides ear <dig> and ear <dig> can inhibit nmda-evoked currents in hippocampal neurons in a dose-dependent and highly reversible manner.
we also demonstrated that these peptides inhibited nmda-evoked currents evoked by recombinant nmdar containing the glun1a–glun2b subunits.
however, only ear <dig> showed higher selectivity for glun1a–glun2b over glun1a–glun2a.
conantokin-g is a  <dig> amino acid peptide  blocks nmda-evoked currents in hippocampal  <cit>  and cortical  <cit>  neurons.
in expression systems, conantokin-g has been shown to be selective for nmdar containing the glun2b subunit  <cit> .
ear <dig> and ear <dig> were designed based on evaluations of in silico interactions by using point mutation on the conantokin-g sequence and by evaluating their binding capacity to an extracellular site of the glun2b receptor.
ear <dig> and ear <dig> also have  <dig> amino acids, between them their sequence differs in two amino acids.
both peptides have the same calculated isoelectric point of  <dig> , which is higher to that reported for conantokin-g of  <dig>   <cit> .
ear <dig> and ear <dig> peptides do not have γ-carboxiglutamic acid  which is present in conantokin-g. the latter was chosen to increase the flexibility of the peptide’s structure, with the rationale that this would facilitate their interactions with the conantokin-g binding pocket in the glun2b subunit.
the prediction for the three-dimensional structure for ear <dig> and ear <dig> was performed using a hydrophilic environment.
figure  <dig> shows the predicted structure for ear <dig> and ear <dig> and the one reported for conantokin-g  <cit> .
while conantokin-g adopts a helical conformation in more than 70% of its sequence  <cit> , ear <dig> and ear <dig> adopt a helical conformation in 50%  or less  of their sequence.
the high level of helical conformation in conantokin-g is due to the presence of gla, which coordinates binding of  <dig> calcium ions .
the decrease in the proportion of helical conformation will increase the molecular flexibility of ear <dig> and ear <dig> and this may contribute to the observed high reversibility of ear <dig> and ear <dig> .
conantokin-g displays a very slow and incomplete reversibility in cortical neurons, such that after 3 min of washout only about 10% of the nmda-evoked current was recuperated  <cit> .
in the same study, it was found that the reversibility of conantokin-g was faster and more complete in oocytes expressing glun1a–glun2b, such that after 4 min of washout the nmda-evoked currents displayed about 80% recovery  <cit> .
we observed that ear <dig> and ear <dig> both were highly reversible in both hippocampal and in hek cells expressing recombinant nmdar, but again it also appears that their reversibility was faster from the recombinant nmdar than from nmdar expressed in neuronal cells.
together, the results indicate that the conformation of the ligand binding domain  in the glun <dig> subunits may be affected by their neuronal environment.
regardless of such difference we found that ear <dig> and ear <dig> were highly reversible.
this high reversibility represents an advantage for the use of these peptides as potential pharmacological agents in comparison to other available nmdar blockers, including general nmdar blockers mk801) and those selective for glun2b .fig.
<dig> schematic representation  of peptides.
the predicted structures for ear <dig>  and ear <dig> , and the nmr reported structure for con-g   are shown.
the 3d predicted structure for ear <dig> and ear <dig> was performed using a hydrophilic environment, both peptides ear <dig> and ear <dig> shown a non-compact helical conformation compared with con-g. the n-terminal is represented in blue.
the images were rendered using ucsf chimera software
the met  <dig> residue in glun2b, is not present in the glun2a subunit and has been reported to be a molecular determinant for the selective antagonist of conantokin-g on glun2b over the glunn2a subunit  <cit> .
the docking results show the formation of a hydrophobic  interaction between the leu  <dig> of ear <dig> and the met 739-glun2b, such interaction is not observed for any of the residues of ear <dig>  this interaction may contribute to the apparent higher selectivity of ear <dig> over ear <dig> for the glun2b subunit over the glun2a subunit.
previously by testing various conantokin analogs, it was found that the presence and location of gla appeared to be important to confer to the conantokin analogs selectivity for the glun2b subunit over the glun2a subunit  <cit> .
we found that ear <dig>  in which gla was replaced by asp, did not display selectivity between glun2b and glun2a, which is consistent with the idea that gla is important to confer selectivity for glun2b.
however, we also found that ear <dig>  in which gla was also replaced by asp, showed higher selectivity for glun2b over glun2a, indicating that gla is not the only residue conferring selectivity for glun2b over glun2a.
there are only two amino acids difference between ear <dig> and ear <dig>  in ear <dig> these are a leucine  and an asparagine , which in ear <dig> correspond to tyrosine  and alanine .
our results are consistent with previous observation in which it has been reported that replacement of leu  <dig> with tyr in conantokin-g results in a reduction on the conantokin-g selectivity for glun2b  <cit> .
to determine whether the amino acid substitutions in ear <dig> results in selectivity for only the glun2b, additional studies are required measuring its selectivity for glun2b over the other two glun <dig> subunits, the glun2c and glun2d.
with respect to the predicted free energy of binding, one would expect that ear <dig>  would have a higher affinity and potentially a higher interaction with glun2b than ear <dig> .
however, their affinity is comparable, and ear <dig> shows an apparent higher selectivity for glun2b than glun2a, than ear <dig>  hence, neither the lower free energy of binding or the higher number of interactions of ear <dig> over ear  <dig> predict selectivity for glun2b with respect to glun2a.
the ic <dig> value for ear <dig> and ear <dig> is high as compared to that displayed by conantokin-g. the reported ic <dig> for conantokin-g is  <dig>  µm in hippocampal  <cit>  and cortical  <cit>  neurons, and  <dig>  µm in xenopus oocytes expressing the glun1a–glun2b subunits  <cit> .
hence our results with ear <dig> and ear <dig> suggest that the gla residue contributes to the high affinity of conantokin-g.
this study has several limitations.
first, we did not measure whether the peptide block was voltage-dependent; neither whether the peptide block was affected by the presence of physiological levels of mg2+.
second, we measured the selectivity of these peptides between glun2b and glun2a.
this was done in part because the amino acid sequence corresponding to that used for the glun2b model  is conserved  and of the lbd  is highly conserved  between glun2a and blun2b  <cit> .
however, although to a lower extent, the corresponding sequences in the glun2b subunit also show conservation with the glun2c  and with the glun2d  subunits  <cit> .
hence determining the potency of these peptides on recombinant nmdar containing other glun <dig> and glun <dig> subunits, would provide additional information about their level of selectivity which will help define their potential use as pharmacological agents.
CONCLUSIONS
we found that ear <dig> and ear <dig> block in a reversible manner nmda-evoked currents in hippocampal neurons.
moreover, ear <dig> showed selectivity for the glun2b over glun2a.
the high reversibility of both of these peptides and the selectivity of ear <dig> for glun2b, make them attractive as potential pharmacological agents.
abbreviations
nmda
n-methyl-d-aspartate
nmdar
n-methyl-d-aspartate receptor
ic50half maximal inhibitory concentration
emaxmaximal inhibitory effect
iglursinotropic glutamate receptors
atdamino terminal domain
abdagonist/ligand binding domain
lbdligand binding domain
tmdtrans-membrane domain
ctdcarboxyl terminal domain
m.w.molecular weight
ms hydrogen cation mass spectrometry analysis
ms sodium ion mass spectrometry analysis
ucsfuniversity of california san francisco
hplchigh-performance liquid chromatograph
pepstrpeptide tertiary structure prediction server
ampaα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
anovaanalysis of variance
glaγ-carboxiglutamic acid
authors’ contributions
earg prepared the hippocampal cultures, performed the electrophysiological measurements, data analysis, contributed to making the figures and in the preparation of the manuscript.
nvc contributed in training earg in cell culture and data discussions; earm contributed in data discussions, responsible for writing the manuscript.
erp trained and directly supervised the work of earg, performed the transfections with recombinant nmdar subunits, responsible for writing the manuscript, contributed to making the figures.
all authors read and approved the final manuscript.
publisher's note
springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

 n-methyl-d-aspartate receptors are inotropic glutamate receptors that contribute to multiple neuronal functions such as. however, excessive nmdar activity can lead to neuronal dysfunction and neuronal death. functional nmdar require two glun and two glun subunits. the glun subunit can be replaced by a glun subunit, but when present the glun subunit decreases the nmdar activity. there are four glun subunits. an increase in the expression of glun glun2a, and glun2b subunits in the hippocampus contribute to neurological problems that develop following traumatic brain injury. increase in the activity/expression of glun2b has been postulated to contribute to neuronal damage following stroke, as well as to the development of diseases such as parkinson, huntington, alzheimer and chronic neuropathic pain. nmdar are similar to other iglurs in that each subunit has four domains, which include the amino-terminal domain, an agonist/ligand binding domain, a trans-membrane domain and a carboxyl terminal domain. in glun subunits, the atd domain contains binding sites for allosteric inhibitors such as zinc and ifenprodil. the abd domain in glun and glun binds the coagonist glycine, whereas the abd domain in glun binds the agonist glutamate. some peptides isolated from marine cone snails have shown specificity for glun subunits of the nmdar. one member of the conantokin family, conantokin-g is a 17-residue peptide that shows selectivity for nmdar containing the glun2b subunit. although, conantokin-g shows high selectivity for glun2b, its reversibility appears to be very slow and incomplete on neuronal cells. we designed a number of peptides with potential glun2b–nmdar antagonistic activity, this was done by using point mutations on the conantokin-g sequence and evaluating their structure as well as their binding capacity to the ligand binding domain of the glun2b subunit by using various in silico evaluations. here we tested this prediction for two of these peptides, ear and ear and found that both of these peptides blocked in a highly reversible manner nmda-evoked currents in hippocampal neurons, and that ear was more selective for nmdars containing the glun2b than for those containing the glun2a subunit. the high reversibility of both of these peptides and the higher selectivity of ear for the nmdars containing the glun2b increase their potential as pharmacological agents. methods peptides these values were provided by the company apurity determine by high-performance liquid chromatograph bmw: molecular weight, theoretical cms: hydrogen cation mass spectrometry analysis dms: sodium ion mass spectrometry analysis peptide three-dimensional structure for each peptide a three-dimensional structure model was generated using ucsf chimera software, and a refined three-dimensional structure was obtained in an hydrophilic environment using pepstr as previously described. structural model of the glun2b details of this model are described in a separate publication. briefly, this model was obtained by using the amino acids 1- of the reported sequence of the glun2b subunit. this amino acid sequence includes the amino terminal and the ligand binding domains. the glun2b structural model was predicted using i-tasser, this software used several templates to determine the best model. the best model was obtained when the template used was the inotropic glutamate ampa receptor. this template has been previously used to elucidate other functions of glutamate receptors. the c-score usually ranges between − and the most positive the value the better the model topology, the glun2b model selected has a c-core of. the tm-score provides a value of the structural correspondence between two structures, values higher than indicate that the model topology is likely to be correct, the selected glun2b model has a tm-score of. the selected glun2b model was further evaluated using pdbsum, that provides various parameters including hydrophobicity profile, solvent accessibility, prediction of ligand binding sites. the secondary structure of the model was also evaluated using ramachandran plots, which provides the percentage of structural regions that are either “favorable”, “allowed” or “not-allowed”. a model with good stereo-chemistry should have close to 90% of the residues in structurally favorable regions. the selected model has % of the residues located in structurally “favorable” regions, and % of the residues in “allowed” regions. the model has structural topology comparable to that described for other inotropic glutamate receptors, including the atd, ldb and the transmembrane domains. the atd and lbd structural configurations are composed of mostly α helices and allows the binding of modulators an agonists. molecular docking molecular docking was performed with the obtained three-dimensional structures of the peptides and of the glun2b subunit by using “autodock vina”. a grid box was generated at the center of the lbd of the glun2b model to which the corresponding hydrogen atoms and chargers were assigned. the grid box was Å, in the software “completeness” was set to and the number of results per docking was set to for each peptide. the peptides corresponded to the ligands. the best conformations and orientations, which displayed the lowest values for binding energy, were visualized using autodock tools. the docking results were then analyzed with the rosetta flexpepdock web server, which refines the peptide-receptor interaction by optimizing the peptide orientation within the binding site using the monte-carlo method and focusing on energy minimization of the ligand with the receptor. with the starting structure flexpepdock realized independent simulations, simulations were done under high resolution. the other simulation included a step of pre-optimization at low resolution, followed by a method of refinement of high resolution. this resulted in models for each peptide, we selected the best one for each peptide based on the score provided by the punctuation function of rosetta. the docking images were generated using ucsf chimera.interactions between peptide-receptor were determine using discovery studio visualizer v software. hippocampal cultures pregnant rats were maintained and used following the guidelines approved by the new york university langone medical center institutional animal care and use committee. pregnant rats were killed using co followed by cervical dislocation. day embryos were removed by caesarian section. dissociated hippocampal cultures were prepared as previously described. cells were plated on 12 mm coverslips with neurobasal medium supplemented with b or with 2% bovine fetal serum and used between and 37 days in vitro; the data was collected from cells that had on average  days in vitro. coverslips were pre-treated by placing 50 µl of poly-d-lysine and laminin in the middle of the coverslip for 5 min at room temperature. transfection of hek- cells the plasmids pegfp-nr1a, pegfp-nr2b, and pegfp-nr2a were a gift from stefano vicini. hek- cells were grown on 12-mm coverslips in well plates. hek- cells were transfected using lipofectamine according to the product instructions, with a mixture of glun1a and glun2a subunits or a mixture of glun1a and glun2b subunits. the amount of cdna for each plasmid was  µg/well. cells were used 2–4 days following transfection. gfp signal was used to identify positively transfected cells. electrophysiology electrophysiological recordings were performed under voltage-clamp by using the whole cell conformation. intracellular solution: cs-gluconate, cscl, hepes, egta, 4 mg-atp, na-gtp, ph. extracellular solution: nacl, kcl, cacl hepes, d-glucose, ph. mg2+ was omitted to prevent the voltage-dependent block of the nmdar ion currents. the following blockers were added to extracellular solution:  μm tetrodotoxin to block na+ channels; 20 μm cnqx to block ampa and kainate receptors, 50 μm bicuculine and 100 μm picrotoxin to block gaba receptors, and 1 μm estricnin to block glycine channels. currents were measured using borosilicate pipettes with resistances of 1–3 mΩ. experiments were conducted at room temperature. currents were measured using a 2-electrode voltage clamp amplifier axopatch 200b. output from the voltage clamp amplifier was sent to a microcomputer using a data acquisition interface. currents were filtered at or 5 khz and sampled at 10–50 khz. for analysis the current traces were filtered at 1 khz. pclamp was used for data acquisition and data analyses. cells were held at −60 mv and the nmdar-evoked currents were obtained by applying a 5 s pulse of nmda followed by a 10 s pulse of bath solution through a puffer located directly on top of the cells. successive stimulations with nmda were done every 4 min, which under control conditions resulted in nmda-evoked ca2+ currents of similar magnitude over time. when the effects of mk- a general nmdar blocker, ro 25- a specific glun2b/nmdar blocker), and peptides were evaluated, the cells were first exposed to two or three consecutive nmda pulses to assure that the cell recording was stable, then the blocker was applied 10 s prior and during the following nmda pulse. in all cases the nmda pulses were followed by a 10 s pulse of bath solution through the puffer. the chamber was continuously perfused with the bath solution, except when the puffer was on. peptides were added at a final concentration of and 500 μm. the area of the nmda-evoked inward currents was measured over a period of 5 s, from the onset of the nmda-evoked inward current. the areas were normalized using the nmda-evoked inward current measured prior to the addition of the blocker or peptide. figures were done using graphpad prism v results are expressed as mean and standard error of mean.fig.  evoked nmda-currents in cultured hippocampal neurons are blocked by -mk and ro 25- a whole-cell nmda-evoked inward currents induced by consecutive 5 s pulses of 100 µm nmda + 10 µm glycine, with a 4 min interval. the last image is an overlap of the first and eighth nmda-evoked current traces. b inhibition of nmda-evoked inward current by the general nmdar antagonist -mk. top shows traces of the nmda-evoked inward currents and below the normalized area of the nmda-evoked inward currents for control; and before, during two consecutive exposures to -mk and after removing -mk. c inhibition of nmda-evoked inward current by the glun specific blocker ro 25-. top shows the nmda-evoked inward currents in a cell before, during two consecutive exposures to ro 25- and after removing ro 25- bottom shows the normalized nmda-evoked inward currents for control; and before and during four consecutive exposures to ro 25-. significant different compared to the control group #p < , +p < , two-way anova with sidak’s multiple comparison post-test; n, control: n = 7; -mk801: n = 3; and ro 25-6981: n =  during the first three nmda stimulations and for the last two nmda stimulations statistics when comparing two groups with various stimulations we used two-way anova followed by sidak’s multiple comparison post-test. when comparing more than two groups with various stimulations we used two-way anova followed by dunnett’s multiple comparison post-test, as recommended by graphpadprism. results are expressed as mean and standard error mean.  electrophysiological evaluation of ear and ear peptides on hippocampal primary cultures primary hippocampal cultures were used between and 20 days of culture, a period when the glun2b subunit has been shown to be highly expressed. when nmda stimulations were separated by a 4 min interval the magnitude of the nmda-evoked ca2+ currents was similar between consecutive stimulations, as reflected by the average behavior shown by the black squares in figs. 1b, c and 2a, b. the general nmda blockers mk-, blocked the nmda-evoked currents. the magnitude of mk-801-mediated block increased with exposures to the blocker and it was irreversible, as previously reported in hippocampal neurons. in order to determine the contribution of the glun2b to the nmda-evoked response we exposed the hippocampal cells to a glun2b selective blocker ro 25-, at a concentration that is fold higher that producing 50% inhibition of nmda/glun2b-mediated currents. at this concentration ro 25- has minimal or no effects on voltage-dependent na+ and ca+ channels. block with ro 25- showed no reversibility following 4 min of washout. we observed that ro 25- blocked 70% of the nmda-evoked currents; hence under our experimental conditions about 70% of the nmda-evoked currents in hippocampal neurons are mediated by activation of glun2b/nmdar. moreover, the increase in the magnitude of the block observed following consecutive exposures to ro 25- is consistent with the blockers being use-dependent.fig.  ear and ear peptides inhibit nmda-evoked inward currents in hippocampal neurons. a, b the inhibitory effect on nmda-evoked currents during consecutive exposures to either and 500 µm of ear and ear. the areas of the nmda-evoked inward currents were normalized to that measured prior to the addition of ear. significant different compared to the control group. #p < , +p<, @p < , ^p < , $p < , two-way anova with dunnett’s multiple comparison post-test; n for each pulse were for control; for ear18: 10 µm, 100 µm; 500 µm; for ear16: 10 µm, 100 µm and 500 µm. c, d show nmda-evoked current traces before, in the presence of ear and following washout. the arrows indicate that upon the simultaneous removal of nmda + ear, there is a transient increase in the nmda-evoked inward current. e dose–response, the data was fitted to y = bottom + /*hillslope)). when the entire current was considered: bottom =  no current, top = −, the ic50 s were and 241 µm for ear and ear respectively in hippocampal neurons, the effect of ear and ear on the nmda-evoked currents was accessed by applying each peptide for four consecutive nmda-evoked currents to determine whether these peptides blocked the nmda-evoked current and if so whether their block displayed use-dependence behavior. ear inhibited nmda-evoked currents in a dose-dependent manner, and this inhibition was reversible. ear had a similar inhibitory effect on nmda-evoked currents in hippocampal neurons. for a given dose, the magnitude of the block mediated by ear or ear did not increase significantly with consecutive exposures; hence these peptides show no clear use-dependence. examination of the current traces, show that following the simultaneous removal of the nmda + ear there was a relatively short-lived increase in the nmda-evoked current, suggesting that the off rate of ear is fast enough that would allow binding of nmda early on during washout. figure 2e shows the dose–response curves. when considering the total current magnitude the estimated ic values were similar for both peptides being and 242 µm for ear and ear respectively. no inhibitory action was observed with the control peptide at 500 µm. ear displays higher selectivity than ear for the glun2b subunit over the glun2a subunit to test the selectively of these peptides for the glun2b subunit, the blocking effect of these peptides was tested in hek- cells expressing recombinant nmdar comprised of either glun1a–glun2a or glun1a–glun2b subunits. the rationale for doing so is that these are the predominant glun subunits in hippocampal neurons, moreover the amino acid sequence corresponding to the sequence used for the structural model of glun2b is conserved and of the lbd is highly conserved between glun2a and glun2b. we selected 500 µm of each peptide to test their potency on hek cells expressing recombinant nmdar, since this peptide concentration produced a strong but not maximal block on nmda-evoked currents in hippocampal neurons. this should allow to determine whether or not these peptides displayed differences in potency between the glun2b and glun2a subunits. figure  shows that as predicted ear at 500 µm blocked more strongly nmda-evoked currents in hek cells transfected with the glun1a–glun2b subunits than those in cells transfected with the glun1a–glun2a subunits. in contrast, ear at 500 µm blocked to a similar level the nmda-evoked currents in hek-cells transfected with either glun1a–glun2b or glun1a–glun2a subunits. the ic of each peptide for blocking glun1a–glun2b and glun1a–glun2a was estimated by using eq., where ‘x’ is the peptide concentration in log; ‘y’ is the % inhibition at ‘x’; emax: is the maximal inhibitory effect which we are assuming to be 100%; ‘n’ is the slope of the fitting, which it is assumed to be “1” since there is only a single recombinant nmdar. y=emax/1+ic50/xn the results are shown in fig. 3g, and indicate that ear has about fold higher affinity for glun1a–glun2b than for glun1a–glun2a. while ear displayed a comparable affinity for both glun1a–glun2b and glun1a–glun2a.fig.  ear but not ear shows selectivity for glun2b over glun2a. a, b ear16-mediated inhibition of nmda-evoked inward currents on hek- cells expressing glun1a–glun2b subunits or expressing glun1a–glun2a subunits. d, e ear18-mediated inhibition of nmda-evoked inward currents on hek- cells expressing glun1a–glun2b or in cells glun1a–glun2a subunits. c, f the areas of the nmda-evoked inward currents were normalized to that measured prior to the addition of ear or ear. no significant difference was found between “control-nmda” and “wash-nmda”. significant different compared to “control-nmda” or when compared to “wash-nmda” #p < , +p < , @p < , two-way anova with sidak’s multiple comparison post-test; n ear n =  for glun1a–glun2b, and n =  for glun1a–glun2a; ear n =  for glun1–glun2b and n =  for glun1a–glun2a as observed in hippocampal neurons, in hek- cells transfected with recombinant nmdar the simultaneous removal of the nmda + ear also resulted in a short-lived increase in the nmda-evoked current, suggesting that the off rate of ear was fast enough that would allow binding of nmda early on during washout. docking of ear and ear with the lbd of the glun2b model figure  shows the docking results of the interaction between the ear and the glun2b subunit. ear binds to the glun2b through hydrogen bridges, of them are with residues located at the lbd in glun2b. the ear residues asp and contribute to the formation of these hydrogen bridges. the residue met that has been reported to contribute to the molecular interaction between con-g and glun2b, is predicted to form a hydrophobic interaction with the leu of ear the majority of the interactions involve the amino terminal of ear similar to what has been described for conantokin-g, indicating that the substitution of the residue γ-carboxiglutamic acid by asp appears to emulate the con-g interaction with the glun2b.fig.  schematic representation of docking of ear with the lbd of glun2b. a, b docking of ear with the lbd in the glun2b model. c representation of the molecular surface of lbd in the glun2b, and the peptide conformation. d amino acid residues and labels of glun2b that form hydrogen bridges with ear the amino acid residues for ear are represented by element: carbon gray, nitrogen: dark blue, oxygen: red and, hydrogen: white; and the amino acid names in gray in the case of ear the interactions involve hydrogen bridges, of them are with residues located at the lbd in glun2b. the ear asp residue plays an important role in establishing these hydrogen bridges. the tyr of ear contributes to the formation of a hydrophobic interaction with the ile of the glun2b, and the ala of ear is forming and hydrogen bridge with the lys of the glun2b. at the lbd, the ear conformation is less extended than that of the ear allowing ear to interact with a larger number of residues in the glun2b.fig.  schematic representation of docking of ear with the lbd of glun2b. a, b docking of ear with the lbd in the glun2b model. c representation of the molecular surface of lbd in the glun2b, and the peptide conformation. d amino acid residues and labels of glun2b that form hydrogen bridges with ear the amino acid residues for ear are represented by element: carbon: gray, nitrogen: dark blue, oxygen: red, and hydrogen: white; and the amino acid names are in gray discussion in this study we demonstrated that the peptides ear and ear can inhibit nmda-evoked currents in hippocampal neurons in a dose-dependent and highly reversible manner. we also demonstrated that these peptides inhibited nmda-evoked currents evoked by recombinant nmdar containing the glun1a–glun2b subunits. however, only ear showed higher selectivity for glun1a–glun2b over glun1a–glun2a. conantokin-g is a amino acid peptide blocks nmda-evoked currents in hippocampal and cortical neurons. in expression systems, conantokin-g has been shown to be selective for nmdar containing the glun2b subunit. ear and ear were designed based on evaluations of in silico interactions by using point mutation on the conantokin-g sequence and by evaluating their binding capacity to an extracellular site of the glun2b receptor. ear and ear also have amino acids, between them their sequence differs in two amino acids. both peptides have the same calculated isoelectric point of, which is higher to that reported for conantokin-g of. ear and ear peptides do not have γ-carboxiglutamic acid which is present in conantokin-g. the latter was chosen to increase the flexibility of the peptide’s structure, with the rationale that this would facilitate their interactions with the conantokin-g binding pocket in the glun2b subunit. the prediction for the three-dimensional structure for ear and ear was performed using a hydrophilic environment. figure  shows the predicted structure for ear and ear and the one reported for conantokin-g. while conantokin-g adopts a helical conformation in more than 70% of its sequence, ear and ear adopt a helical conformation in 50% or less of their sequence. the high level of helical conformation in conantokin-g is due to the presence of gla, which coordinates binding of calcium ions. the decrease in the proportion of helical conformation will increase the molecular flexibility of ear and ear and this may contribute to the observed high reversibility of ear and ear. conantokin-g displays a very slow and incomplete reversibility in cortical neurons, such that after 3 min of washout only about 10% of the nmda-evoked current was recuperated. in the same study, it was found that the reversibility of conantokin-g was faster and more complete in oocytes expressing glun1a–glun2b, such that after 4 min of washout the nmda-evoked currents displayed about 80% recovery. we observed that ear and ear both were highly reversible in both hippocampal and in hek cells expressing recombinant nmdar, but again it also appears that their reversibility was faster from the recombinant nmdar than from nmdar expressed in neuronal cells. together, the results indicate that the conformation of the ligand binding domain in the glun subunits may be affected by their neuronal environment. regardless of such difference we found that ear and ear were highly reversible. this high reversibility represents an advantage for the use of these peptides as potential pharmacological agents in comparison to other available nmdar blockers, including general nmdar blockers mk801) and those selective for glun2b.fig.  schematic representation of peptides. the predicted structures for ear and ear, and the nmr reported structure for con-g are shown. the 3d predicted structure for ear and ear was performed using a hydrophilic environment, both peptides ear and ear shown a non-compact helical conformation compared with con-g. the n-terminal is represented in blue. the images were rendered using ucsf chimera software the met residue in glun2b, is not present in the glun2a subunit and has been reported to be a molecular determinant for the selective antagonist of conantokin-g on glun2b over the glunn2a subunit. the docking results show the formation of a hydrophobic interaction between the leu of ear and the met 739-glun2b, such interaction is not observed for any of the residues of ear this interaction may contribute to the apparent higher selectivity of ear over ear for the glun2b subunit over the glun2a subunit. previously by testing various conantokin analogs, it was found that the presence and location of gla appeared to be important to confer to the conantokin analogs selectivity for the glun2b subunit over the glun2a subunit. we found that ear in which gla was replaced by asp, did not display selectivity between glun2b and glun2a, which is consistent with the idea that gla is important to confer selectivity for glun2b. however, we also found that ear in which gla was also replaced by asp, showed higher selectivity for glun2b over glun2a, indicating that gla is not the only residue conferring selectivity for glun2b over glun2a. there are only two amino acids difference between ear and ear in ear these are a leucine and an asparagine, which in ear correspond to tyrosine and alanine. our results are consistent with previous observation in which it has been reported that replacement of leu with tyr in conantokin-g results in a reduction on the conantokin-g selectivity for glun2b. to determine whether the amino acid substitutions in ear results in selectivity for only the glun2b, additional studies are required measuring its selectivity for glun2b over the other two glun subunits, the glun2c and glun2d. with respect to the predicted free energy of binding, one would expect that ear would have a higher affinity and potentially a higher interaction with glun2b than ear. however, their affinity is comparable, and ear shows an apparent higher selectivity for glun2b than glun2a, than ear hence, neither the lower free energy of binding or the higher number of interactions of ear over ear predict selectivity for glun2b with respect to glun2a. the ic value for ear and ear is high as compared to that displayed by conantokin-g. the reported ic for conantokin-g is  µm in hippocampal and cortical neurons, and  µm in xenopus oocytes expressing the glun1a–glun2b subunits. hence our results with ear and ear suggest that the gla residue contributes to the high affinity of conantokin-g. this study has several limitations. first, we did not measure whether the peptide block was voltage-dependent; neither whether the peptide block was affected by the presence of physiological levels of mg2+. second, we measured the selectivity of these peptides between glun2b and glun2a. this was done in part because the amino acid sequence corresponding to that used for the glun2b model is conserved and of the lbd is highly conserved between glun2a and blun2b. however, although to a lower extent, the corresponding sequences in the glun2b subunit also show conservation with the glun2c and with the glun2d subunits. hence determining the potency of these peptides on recombinant nmdar containing other glun and glun subunits, would provide additional information about their level of selectivity which will help define their potential use as pharmacological agents.  we found that ear and ear block in a reversible manner nmda-evoked currents in hippocampal neurons. moreover, ear showed selectivity for the glun2b over glun2a. the high reversibility of both of these peptides and the selectivity of ear for glun2b, make them attractive as potential pharmacological agents. abbreviations nmda n-methyl-d-aspartate nmdar n-methyl-d-aspartate receptor ic50half maximal inhibitory concentration emaxmaximal inhibitory effect iglursinotropic glutamate receptors atdamino terminal domain abdagonist/ligand binding domain lbdligand binding domain tmdtrans-membrane domain ctdcarboxyl terminal domain m.w.molecular weight ms hydrogen cation mass spectrometry analysis ms sodium ion mass spectrometry analysis ucsfuniversity of california san francisco hplchigh-performance liquid chromatograph pepstrpeptide tertiary structure prediction server ampaα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid anovaanalysis of variance glaγ-carboxiglutamic acid authors’ contributions earg prepared the hippocampal cultures, performed the electrophysiological measurements, data analysis, contributed to making the figures and in the preparation of the manuscript. nvc contributed in training earg in cell culture and data discussions; earm contributed in data discussions, responsible for writing the manuscript. erp trained and directly supervised the work of earg, performed the transfections with recombinant nmdar subunits, responsible for writing the manuscript, contributed to making the figures. all authors read and approved the final manuscript. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 
the effects of ear and ear in nmda-evoked currents were measured in cultured rat embryonic hippocampal neurons and in hek- cells expressing recombinant nmdar comprised of glun1a–glun2a or glun1a–glun2b subunits. the glun2b subunit of the n-methyl-d-aspartate receptor modulates many physiological processes including learning, memory, and pain. thus there is an interest in identifying nmdar antagonists that interact specifically with the glun2b subunit. recently based on structural analysis between the glun2b subunit and conantokin-g, a toxin that interacts selectively with the glun2b subunit, we designed various peptides that are predicted to act as nmdar antagonists by interacting with the glun2b subunit. in hippocampal neurons, ear and ear reduced the nmda-evoked calcium currents in a dose-dependent and reversible manner with comparable ic values of and 176 µm, respectively. nmdaconantokin-ghippocampal neuronsglun2bglun2acolciencias110156933188reyes-montaño edgar a. the anesthesia reserach fund nyuissue-copyright-statement© the author 2017 the newly designed ear and ear peptides were shown to block in reversible manner nmda-evoked currents, and ear showed a stronger selectivity for glun2b than for glun2a.
 the glun2b subunit of the n-methyl-d-aspartate receptor modulates many physiological processes including learning, memory, and pain. excessive increase in nmdar/glun2b activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. thus there is an interest in identifying nmdar antagonists that interact specifically with the glun2b subunit. recently based on structural analysis between the glun2b subunit and conantokin-g, a toxin that interacts selectively with the glun2b subunit, we designed various peptides that are predicted to act as nmdar antagonists by interacting with the glun2b subunit. in this study we tested this prediction for two of these peptides ear and ear  the effects of ear and ear in nmda-evoked currents were measured in cultured rat embryonic hippocampal neurons and in hek- cells expressing recombinant nmdar comprised of glun1a–glun2a or glun1a–glun2b subunits. in hippocampal neurons, ear and ear reduced the nmda-evoked calcium currents in a dose-dependent and reversible manner with comparable ic values of and 176 µm, respectively. at 500 µm, ear blocked more strongly the nmda-evoked currents mediated by the glun1a–glun2b than those mediated by the glun1a–glun2a subunits. at 500 µm, ear blocked to a similar extent the nmda-evoked currents mediated by the glun1a–glun2b and the glun1a–glun2a subunits.  the newly designed ear and ear peptides were shown to block in reversible manner nmda-evoked currents, and ear showed a stronger selectivity for glun2b than for glun2a. keywords nmdaconantokin-ghippocampal neuronsglun2bglun2acolciencias110156933188reyes-montaño edgar a. the anesthesia reserach fund nyuissue-copyright-statement© the author 2017 