BACKGROUND
it has been well established that the amygdala is important in the acquisition and maintenance of fear/anxiety-related behaviors  <cit> .
strychnine-sensitive glycine receptors have recently been found in the adult rat basolateral amygdala  using whole cell and intracellular electrophysiology  <cit> .
reverse transcription polymerase chain reaction on whole bla tissue and single cells revealed a prominent expression of α <dig> mrna; and these receptors are likely to be α2β heteromers due to their low picrotoxin sensitivity  <cit> .
this finding is consistent with prominent bla 'general' immunoreactivity for α/β subunit protein but no apparent α1-specific protein expression  <cit> .
a similar enrichment of α2/β heteromers is also evident in striatal cholinergic interneurons  <cit> .
it is quite possible then that the α2β strychnine-sensitive glycine receptors present in the adult bla and other forebrain areas represents a receptor population that could be functionally distinguished from those found in the spinal cord.
because the bla regulates a number of anxiety- or fear-related behaviors  <cit> , it is possible that this population of strychnine-sensitive glycine receptors may represent a novel therapeutic target for anxiety disorders.
to insure that novel α2β compounds possess an appropriate therapeutic index, the pharmacology of these forebrain glycine receptors must be elucidated and extensively compared with the spinal isoform.
there have been conflicting reports regarding the details of glycine receptor pharmacology when expressed in heterologous systems.
for example, taurine acts as a partial agonists  for glyrα <dig> expressed in xenopus ooctyes  <cit>  whereas it shows nearly full agonist efficacy for glyrα <dig> expressed in hek  <dig> cells  <cit> .
compared to glyrα <dig>  taurine efficacy is even weaker for glyrα <dig>  when expressed in xenopus oocytes  <cit> .
however, native glyrα2β receptors expressed by bla neurons possess >50% efficacy for taurine and almost full efficacy for β-alanine  <cit> .
while these results might initially be dismissed as expression system-dependent phenomena, brain region-specific effects are also evident in the literature.
taurine has markedly different efficacies at glycine receptors expressed by isolated adult lateral/basolateral amygdala neurons  <cit> , adult hypothalamic magnocellular neurons  <cit> , and juvenile spinal cord neurons  <cit> .
it is therefore possible that the mechanisms regulating brain region-specific effects are related to those governing the divergence among heterologous expression systems.
however, such mechanisms have not been systematically investigated, despite their potential usefulness in understanding region-to-region pharmacologic heterogeneity evident for some native receptors.
this study utilizes whole-cell patch clamp electrophysiology to examine the influence of distinct heterologous expression systems on the β-amino acid pharmacology of glycine receptors composed of distinct subunit combinations.
we have focused on the α <dig> and α2β receptors since these appear to be the predominate isoforms found in the embryonic and adult forebrain, respectively.
our results provide potentially important insight into the types of mechanisms that may govern brain region-to-brain region variation in glycine receptor pharmacology.
several aspects of this work have appeared in abstract form  <cit> .
RESULTS
subunit- and system-dependent effects on glycine pharmacology
given the variation of glycine receptor partial agonist pharmacology in the literature, we specifically sought to identify any role that expression system may play in their pharmacological profiles.
first, glycine concentration-response relationships were established for glyrα <dig>  and glurα2/β in hek- <dig> cells and in l-cell fibroblasts.
glycine-gated responses for each receptor isoform were elicited in a dose-dependent manner in both cell types .
the apparent ec <dig> of glycine hek cells was  <dig> μm and  <dig> μm for α <dig>  and α2β , respectively.
glyr subunits expressed in l-cells displayed a similar pharmacological profile.
however, the apparent glycine ec <dig> of both glyrα <dig>  and glyrα2β  appeared lower than apparent affinities for the same subunits when expressed in l-cells.
two-way anova on the log  values  indicated a significant effect of system .
however, the presence of the β-subunit did not significantly affect glycine apparent affinity in either system nor was there a significant interaction between system and subunit composition.
these results indicate that glycine is less potent for receptors expressed in l cells compared to hek cells.
a – efficacy relative to maximal glycine response b – p <  <dig>  for system but not subunit using two-way anova c – estimate due to low efficacy
subunit- and system-dependent effects on β-alanine pharmacology
with the glycine pharmacological profile established, we next examined the pharmacology of the partial agonists, β-alanine and taurine.
the efficacy and potency of these β-amino acids were compared to glycine by normalizing the current response at each concentration to a maximal glycine response in that same cell.
in hek cells , the average β-alanine ec <dig> values calculated from individual cells were  <dig> μm  and  <dig> μm  for α <dig> and α2/β receptors, respectively.
in l-cells, β-alanine also elicited currents in a dose-dependent manner.
and, like glycine, β-alanine appeared to be less potent in these cells compared to hek cells.
ec <dig> values for α <dig> and α2/β receptors were  <dig>  mm  and  <dig>  mm , respectively.
two-way anova on logec <dig> values from these studies indicate a significant effect of the expression system on β-alanine potency .
there was a trend for the presence of the β-subunit to influence potency but this was not significant nor was there any significant interaction between expression system and subunit composition.
β-alanine efficacy was also examined in these same experiments by normalizing the maximal β-alanine response as a fraction of a maximal glycine response.
in hek cells, the α <dig> and α2β isoforms had efficacies of  <dig> ± 6% and  <dig> ± 7% of the maximal glycine response, respectively.
a similar trend was noted in l-cells with the α <dig> and α2β isoform with β-alanine efficacies being  <dig> ± 8% and  <dig> ± 5% of the maximal glycine response.
two-way anova analysis of these data indicate that both expression system and subunit composition had a significant influence on β-alanine efficacy .
there was no significant interaction between these variables.
these data demonstrate that the presence of the β subunit reduced β-alanine efficacy of α2-containing receptors and that this efficacy was substantially smaller l-cells compared to hek cells.
subunit- and system-dependent effects on taurine pharmacology
similar analysis of taurine pharmacology in hek and l-cells revealed more dramatic effects of system and subunit on this partial agonist .
in hek cells, the apparent ec <dig> for taurine was  <dig> μm for glyrα <dig>  and  <dig> mm for glyrα2β .
because of its remarkably low efficacy in l-cells , we can only provide estimates of taurine potency in this expression system.
regardless, apparent taurine affinity for both glyrα <dig> and glyrα2β expressed in l cells were ~ <dig> mm for both isoforms .
we did not compare l cell data with that obtained from hek cells due to the uncertainty surrounding the fits.
however, there was no significant difference in apparent taurine potency between the α <dig> and α2β receptors expressed in hek cells .
taurine efficacy was obviously quite different between the two expression systems.
in hek cells, taurine efficacy was  <dig> ± 12% of glycine for glyrα <dig> and  <dig> ± 4% of glycine for the glyrα2β isoform.
efficacy for these same receptors was reduced to approximately  <dig> ± 1% and  <dig> ±  <dig> % of glycine when they were expressed in l-cells in these particular studies.
the system difference was significant with two-way anova  with no substantial effects of subunit composition or interactions between these variables.
expression level and system-dependent pharmacology
the preceding results suggest that there may be a complex interaction between subunit composition and the expression system in which the receptor is produced.
specifically, the system-dependent agonist pharmacology could be related to differences in the relative expression levels between various systems.
expression level has clearly been demonstrated to influence agonist pharmacology for g protein-coupled receptors , where the levels of g-protein bound to receptor and thus the relative levels of high affinity receptor can vary from system to system.
however, the influence of expression level on ligand-gated channel function has not been extensively explored .
unfortunately, it is problematic to compare expression levels between hek and l-cells since the relative efficiency of transfection varied widely between these systems.
indeed, liposome-mediated transfection is remarkably efficient in hek  <dig> cells  but only marginally effective in l-cells .
to get around these differences in transfection efficiency, we examined the relative expression level of glyrα <dig> protein using western analysis of total lysate derived from the same number of gfp+ hek  <dig> or l cells from transfected cultures .
for this experiment, cells were harvested under native conditions, gfp+ cells were counted, and volumes of lysate corresponding to equivalent numbers of gfp+ cells was loaded onto the gel.
western blots from two separate experiments demonstrate that transfected hek  <dig> cells expressed 4- to 5-fold more glyrα <dig> protein than transfected l-cells.
the mean optical density from the two experiments was  <dig> ±  <dig> units for hek cells and  <dig> ±  <dig> units for l-cells.
maximal conductance is an independent measure of functional expression and was also larger for both α <dig> and α2β receptors expressed in hek cells compared to receptors expressed in l cells .
across all experiments where maximal glycine concentrations were assayed, the conductance of α <dig> receptors expressed in l-cells was  <dig> ±  <dig> ns and was  <dig> ±  <dig> ns in hek cells.
similarly, l-cells expressed α2β receptors at  <dig> ±  <dig> ns while hek cells expressed this isoform at  <dig> ±  <dig> ns.
two-way anova using subunit and system as variables revealed a significant effect of system  but not subunit, nor was there a significant interaction between variables.
results from both westerns and functional experiments therefore indicate that relative expression levels of glycine receptor were different between hek and l-cells.
to further explore the interaction between expression level and partial agonist efficacy, both current density and taurine efficacy were compared for α <dig> and α2β glycine receptors in a number of different heterologous systems, as well as for native receptors expressed in rat lateral/basolateral amygdala.
in addition to hek and l-cells, the heterologous systems included mouse 3t <dig> fibroblasts and mdck kidney cells.
α2β receptors expressed in mouse 3t <dig> fibroblasts had twice the current density  of the mouse l-cells  but had a similar taurine efficacy .
similarly, α2β receptors expressed in hek <dig> cells had a current density similar to glyrs expressed in 3t <dig> fibroblasts  but had a taurine efficacy compared to glycine of  <dig> ± 3%.
this efficacy was similar to glycine receptors expressed by acutely isolated adult rat basolateral amygdala neurons  although the current density in this native system was only  <dig> ±  <dig> pa/pf.
note that the channels expressed by these neurons are composed primarily of α2+β subunits  <cit> .
canine kidney mdck cells expressed the lowest α2β current density ; yet the channels expressed by this system had the highest taurine efficacy of any cell tested .
for α <dig> glyrs, the rank order of glycine receptor density was 3t <dig> > hek cell > l-cell ; while the rank order of taurine efficacy for these same receptors was hek > 3t <dig> > l-cells .
across all subunit combinations and systems, there was no significant correlation  between taurine efficacy and glycine current density .
indeed, no correlation between expression level and taurine efficacy was evident within any given population of cells whether the receptors were expressed in native or heterologous systems.
for example, the correlation coefficients for α2β receptors between taurine efficacy and glycine current density in individual systems were  <dig> ,  <dig> ,  <dig> ,  <dig> , and  <dig>  for hek, l-cells, 3t <dig> cells, mdck cells, and amygdala neurons, respectively .
thus, while there is clearly a difference in expression level between both the systems as well as between individual cells in a given system, this particular characteristic cannot account for the apparent taurine efficacy.
the agonist-binding site is not affected by expression system
there are a variety of possible mechanisms to account for the disparities in partial agonist pharmacology between two expression systems.
one way to address this is to examine competitive antagonist binding properties in hek and l-cells.
we therefore examined the potency of the glycine receptor competitive antagonist strychnine in both systems.
following a  <dig> second pretreatment with the antagonist  <cit> , we co-applied strychnine and an ec <dig> concentration of glycine.
the strychnine kb was estimated for hek and l-cells expressing either the glyrα <dig> or glyrα2+β subunits using the cheng-prusoff relationship .
this relationship takes into account the divergent hill-slope and potencies for glycine found in these two expression systems.
receptors composed of the glyrα <dig> subunit  had very similar kb values when expressed in either hek  or l-cells .
the same was true for cells expressing the glyrα2β subunits where strychnine apparent affinity was  <dig> ±  <dig> nm  in hek cells and  <dig> ±  <dig> nm  in l-cells.
two-way anova did not reveal any significant effect of either system or subunit composition.
since strychnine is a competitive antagonist and site-directed mutation studies suggests that strychnine and glycine interact with overlapping regions of the receptor  <cit> , our results strongly suggest that functional strychnine affinity, and hence the general structure of the agonist binding pocket, was not substantially influence by expression system.
cytoskeletal components influence glycine receptor pharmacology
a third possible mechanism for reduced efficacy in l-cells compared to hek cells or neurons could be related to intracellular factors that influence channel gating  <cit> .
this hypothesis was examined by disrupting the cytoskeletal protein tubulin, which has been shown to be important for glycine receptor localization  <cit> .
direct application of  <dig> μm colchicine did not elicit any membrane currents.
furthermore, acute application of  <dig> μm colchicine and an ec <dig> concentration of glycine  did not significantly affect glycine-gated currents themselves.
glycine currents were  <dig>  ±  <dig>  pa/pf while glycine+colchicine currents were  <dig>  ±  <dig>  pa/pf .
the relative efficacy of β-alanine and taurine was examined in hek cells expressing α2β subunits following  <dig> min incubation with  <dig> μm colchicine at 37°c, enough time to allow irreversible tubulin disruption  <cit> .
as an additional control, γ-lumicolchicine, an inactive analog of colchicine  <cit> , was also used to treat α2β-expressing hek cells .
these brief treatments had no obvious effect on the survival of untransfected cells.
there was a trend for colchicine treatment to reduce the overall current density at  <dig> μm glycine,  <dig>  ±  <dig>  pa/pf in control cells ,  <dig>  ±  <dig>  pa/pf in colchicine-treated cells , and  <dig>  ±  <dig>  pa/pf  in γ-lumicolchicine-treated cells; however, this was not significant  and was probably not related to any direct action of colchicine given that the glycine current density was also slightly reduced in α2β-expressing cells exposed to γ-lumicolchicine compared to controls.
however, the efficacy of both taurine  and β-alanine  were significantly decreased by colchicine but not γ-lumicolchicine treatment.
taurine efficacy was  <dig> ± 6% of glycine in controls,  <dig> ± 3% following colchicine, and  <dig> ± 3% following γ-lumicolchicine.
similarly, β-alanine efficacy was  <dig> ± 7% of glycine in controls,  <dig> ± 6% following colchicine, and  <dig> ± 7% following γ-lumicolchicine.
similar treatment of α2-expressing hek cells with colchicine  did not reveal any significant effect on glycine current density , on taurine efficacy , or on β-alanine efficacy .
cholchicine treatment also significantly reduced β-alanine efficacy in l-cells expressing glyrα2β  but not in glyrα2-expressing l-cells .
we did not attempt to examine taurine in l-cells treated with colchicine given the exceptionally low efficacy of receptors expressed in this cell line.
because the glycine receptor- and tubulin-binding protein gephyrin provides an obvious link between the receptor and the tubulin cytoskeleton, we used western analysis of hek and l-cell lysates with a gephyrin monoclonal antibody specific for the c-terminus.
these experiments revealed that gephyrin-like immunoreactivity was expressed in both expression systems .
notably, a ca.
<dig> kd band dominated the hek cell gephyrin immunoreactivity, while multiple bands of varying intensity could be seen in lysate from l-cells.
when taken with our colchicine data, differences in glycine receptor pharmacology between α2β receptors expressed in hek and l-cells may be partially due to distinct, system-dependent interactions with distinct isoforms of the cytoskeletal protein gephyrin.
discussion
we have expressed several the 'embryonic'  and 'forebrain'  isoforms in two distinct expression systems to understand the influence of endogenous and exogenous factors on receptor partial agonist pharmacology.
although the pharmacology of the 'embryonic' glyrα <dig> isoform and the 'adult spinal' isoform  have been explored more frequently in the literature, the pharmacology of glyrα2β receptors has remained largely unexplored.
despite this, there is strong evidence that the adult 'forebrain' isoforms, specifically in the rat basolateral amygdala, is indeed α2β  <cit> .
the current study indicates a general trend for decreased apparent affinity and reduced relative efficacy of agonists when receptors consist of the α2β subunits compared to their homomeric α <dig> counter parts.
we were particularly surprised to find that receptors expressed in different expression systems possessed markedly different partial agonist efficacies.
the remainder of our study focused on identifying extrinsic factors that influence difference in ligand-gated receptor pharmacology in distinct expression systems.
while differences in efficiency of cdna expression/transfection between systems could explain such differences, the expression levels of glyrα2β receptors measured by current density was not correlated with taurine efficacy across several different cell types or within any given system.
importantly, the efficacy of β-alanine and taurine in hek cells agree with previous findings where cells expressing glyrα <dig> show almost full efficacy for taurine and β-alanine  <cit> .
similarly, distinct ligand binding characteristics of receptors expressed in different expression systems seemed to be another possible mechanism governing agonist efficacy or potency.
for the glycine receptor, the binding site for the competitive antagonist strychnine is believed to be adjacent to the agonist-binding site, sterically hindering agonist binding.
a gross alteration in the agonist binding pocket, particularly one that hindered agonist binding, would most likely affect strychnine binding as well.
to examine this, strychnine kb was calculated for glyrα <dig> and glyrα2β isoforms expressed in both hek and l-cells.
in order to decrease the error in estimating kb, a derivation of the cheng-prusoff equation was used that takes in account variations in the slopes of the inhibition curves  <cit> .
differences in kb were negligible between expression systems, indicating the strychnine binding site, and presumably the agonist binding site, was altogether similar in these different systems.
differences in pharmacology between systems therefore cannot be explained by substantial alterations in the agonist/competitive antagonist binding pocket.
receptor gating is another mechanism by which receptor function may be altered.
cytoskeletal elements have been shown to play a crucial role in neurotransmitter receptor clustering  <cit>  and may have a role in receptor function as well.
for example, cytoskeletal stabilization has been shown to reduce ca++-dependent inactivation of ca++ channels in snail ganglia  <cit> ; and, actin has been shown to modulate several different types of membrane ion channel  <cit> .
cytoskeletal depolymerization has also been found to inhibit the function of gabaa receptors, which share significant sequence homology and functional characteristics with strychnine-sensitive glycine receptors  <cit> .
and the tubulin-gephyrin-glycine receptor interaction is critical for establishing functional glycinergic synapses  <cit> .
the current study suggests that cytoskeletal elements may play a functional role in α2β glycine receptor pharmacology as well.
β-containing glycine receptors are intimately associated with the tubulin-associated protein gephyrin  <cit>  via the gephyrin binding site that lies within the intracellular domain of this subunit  <cit> .
in our studies, the efficacy of both taurine and β-alanine were reduced in cells expressing glyrα2β subunits.
this despite the finding that gephyrin-like immunoreactivity in l-cells was apparently distinct from that in hek cells, suggesting that distinct cytoskeletal components in these systems may have profound influence over glyr α2β pharmacology.
this is further supported by suggestions that gephyrin may exist in multiple, tissue-specific isoforms with potentially distinct functional roles  <cit> .
it should be noted however that colchicine treatment of α2β-expressing hek cells did not suppress partial agonist efficacy to a level that approached that found in l-cells or 3t3-fibroblasts.
given that colchicine had no perceptible effect of α2-homomeric channels expressed in hek cells, our results suggest that additional system-dependent factors may have a more pronounced influence on the partial agonist pharmacology of strychnine-sensitive glycine receptors.
CONCLUSIONS
it is of particular interest that the β subunit appears to play a functional role in the pharmacology of α2-containing receptors regardless of expression system.
for example, the beta subunit decreased the apparent efficacy of the partial agonists.
since the β-subunit itself does not appreciably interact with the competitive antagonist strychnine  <cit> , these results are at least consistent with some allosteric interaction between the β-subunit and the agonist binding site present the α subunit.
this may indicate that α2β glycine receptors in the forebrain may be distinguishable from other receptor isoforms given the appropriate pharmacologic agent.
although cytoskeletal components potentially play some role for these 'forebrain' receptors, there appear to be other 'extrinsic' factors governing expression system-dependent effects on agonist pharmacology.
since it is conceivable that such factors may be differentially distributed between different forebrain regions, the large apparent differences between glycine receptor pharmacology reported by various studies may not necessarily depend upon differential expression of glycine receptor subunits per se.
at the very least, our findings suggest that great care should be taken when utilizing different expression systems to develop screens for novel pharmacophores acting on this receptor.

 it has been well established that the amygdala is important in the acquisition and maintenance of fear/anxiety-related behaviors. strychnine-sensitive glycine receptors have recently been found in the adult rat basolateral amygdala using whole cell and intracellular electrophysiology. reverse transcription polymerase chain reaction on whole bla tissue and single cells revealed a prominent expression of α mrna; and these receptors are likely to be α2β heteromers due to their low picrotoxin sensitivity. this finding is consistent with prominent bla'general' immunoreactivity for α/β subunit protein but no apparent α1-specific protein expression. a similar enrichment of α2/β heteromers is also evident in striatal cholinergic interneurons. it is quite possible then that the α2β strychnine-sensitive glycine receptors present in the adult bla and other forebrain areas represents a receptor population that could be functionally distinguished from those found in the spinal cord. because the bla regulates a number of anxiety- or fear-related behaviors, it is possible that this population of strychnine-sensitive glycine receptors may represent a novel therapeutic target for anxiety disorders. to insure that novel α2β compounds possess an appropriate therapeutic index, the pharmacology of these forebrain glycine receptors must be elucidated and extensively compared with the spinal isoform. there have been conflicting reports regarding the details of glycine receptor pharmacology when expressed in heterologous systems. for example, taurine acts as a partial agonists for glyrα expressed in xenopus ooctyes whereas it shows nearly full agonist efficacy for glyrα expressed in hek cells. compared to glyrα taurine efficacy is even weaker for glyrα when expressed in xenopus oocytes. however, native glyrα2β receptors expressed by bla neurons possess >50% efficacy for taurine and almost full efficacy for β-alanine. while these results might initially be dismissed as expression system-dependent phenomena, brain region-specific effects are also evident in the literature. taurine has markedly different efficacies at glycine receptors expressed by isolated adult lateral/basolateral amygdala neurons, adult hypothalamic magnocellular neurons, and juvenile spinal cord neurons. it is therefore possible that the mechanisms regulating brain region-specific effects are related to those governing the divergence among heterologous expression systems. however, such mechanisms have not been systematically investigated, despite their potential usefulness in understanding region-to-region pharmacologic heterogeneity evident for some native receptors. this study utilizes whole-cell patch clamp electrophysiology to examine the influence of distinct heterologous expression systems on the β-amino acid pharmacology of glycine receptors composed of distinct subunit combinations. we have focused on the α and α2β receptors since these appear to be the predominate isoforms found in the embryonic and adult forebrain, respectively. our results provide potentially important insight into the types of mechanisms that may govern brain region-to-brain region variation in glycine receptor pharmacology. several aspects of this work have appeared in abstract form.  subunit- and system-dependent effects on glycine pharmacology given the variation of glycine receptor partial agonist pharmacology in the literature, we specifically sought to identify any role that expression system may play in their pharmacological profiles. first, glycine concentration-response relationships were established for glyrα and glurα2/β in hek- cells and in l-cell fibroblasts. glycine-gated responses for each receptor isoform were elicited in a dose-dependent manner in both cell types. the apparent ec of glycine hek cells was μm and μm for α and α2β, respectively. glyr subunits expressed in l-cells displayed a similar pharmacological profile. however, the apparent glycine ec of both glyrα and glyrα2β appeared lower than apparent affinities for the same subunits when expressed in l-cells. two-way anova on the log values indicated a significant effect of system. however, the presence of the β-subunit did not significantly affect glycine apparent affinity in either system nor was there a significant interaction between system and subunit composition. these results indicate that glycine is less potent for receptors expressed in l cells compared to hek cells. a – efficacy relative to maximal glycine response b – p < for system but not subunit using two-way anova c – estimate due to low efficacy subunit- and system-dependent effects on β-alanine pharmacology with the glycine pharmacological profile established, we next examined the pharmacology of the partial agonists, β-alanine and taurine. the efficacy and potency of these β-amino acids were compared to glycine by normalizing the current response at each concentration to a maximal glycine response in that same cell. in hek cells, the average β-alanine ec values calculated from individual cells were μm and μm for α and α2/β receptors, respectively. in l-cells, β-alanine also elicited currents in a dose-dependent manner. and, like glycine, β-alanine appeared to be less potent in these cells compared to hek cells. ec values for α and α2/β receptors were mm and mm, respectively. two-way anova on logec values from these studies indicate a significant effect of the expression system on β-alanine potency. there was a trend for the presence of the β-subunit to influence potency but this was not significant nor was there any significant interaction between expression system and subunit composition. β-alanine efficacy was also examined in these same experiments by normalizing the maximal β-alanine response as a fraction of a maximal glycine response. in hek cells, the α and α2β isoforms had efficacies of ± 6% and ± 7% of the maximal glycine response, respectively. a similar trend was noted in l-cells with the α and α2β isoform with β-alanine efficacies being ± 8% and ± 5% of the maximal glycine response. two-way anova analysis of these data indicate that both expression system and subunit composition had a significant influence on β-alanine efficacy. there was no significant interaction between these variables. these data demonstrate that the presence of the β subunit reduced β-alanine efficacy of α2-containing receptors and that this efficacy was substantially smaller l-cells compared to hek cells. subunit- and system-dependent effects on taurine pharmacology similar analysis of taurine pharmacology in hek and l-cells revealed more dramatic effects of system and subunit on this partial agonist. in hek cells, the apparent ec for taurine was μm for glyrα and mm for glyrα2β. because of its remarkably low efficacy in l-cells, we can only provide estimates of taurine potency in this expression system. regardless, apparent taurine affinity for both glyrα and glyrα2β expressed in l cells were ~ mm for both isoforms. we did not compare l cell data with that obtained from hek cells due to the uncertainty surrounding the fits. however, there was no significant difference in apparent taurine potency between the α and α2β receptors expressed in hek cells. taurine efficacy was obviously quite different between the two expression systems. in hek cells, taurine efficacy was ± 12% of glycine for glyrα and ± 4% of glycine for the glyrα2β isoform. efficacy for these same receptors was reduced to approximately ± 1% and ± % of glycine when they were expressed in l-cells in these particular studies. the system difference was significant with two-way anova with no substantial effects of subunit composition or interactions between these variables. expression level and system-dependent pharmacology the preceding results suggest that there may be a complex interaction between subunit composition and the expression system in which the receptor is produced. specifically, the system-dependent agonist pharmacology could be related to differences in the relative expression levels between various systems. expression level has clearly been demonstrated to influence agonist pharmacology for g protein-coupled receptors, where the levels of g-protein bound to receptor and thus the relative levels of high affinity receptor can vary from system to system. however, the influence of expression level on ligand-gated channel function has not been extensively explored. unfortunately, it is problematic to compare expression levels between hek and l-cells since the relative efficiency of transfection varied widely between these systems. indeed, liposome-mediated transfection is remarkably efficient in hek cells but only marginally effective in l-cells. to get around these differences in transfection efficiency, we examined the relative expression level of glyrα protein using western analysis of total lysate derived from the same number of gfp+ hek or l cells from transfected cultures. for this experiment, cells were harvested under native conditions, gfp+ cells were counted, and volumes of lysate corresponding to equivalent numbers of gfp+ cells was loaded onto the gel. western blots from two separate experiments demonstrate that transfected hek cells expressed 4- to 5-fold more glyrα protein than transfected l-cells. the mean optical density from the two experiments was ± units for hek cells and ± units for l-cells. maximal conductance is an independent measure of functional expression and was also larger for both α and α2β receptors expressed in hek cells compared to receptors expressed in l cells. across all experiments where maximal glycine concentrations were assayed, the conductance of α receptors expressed in l-cells was ± ns and was ± ns in hek cells. similarly, l-cells expressed α2β receptors at ± ns while hek cells expressed this isoform at ± ns. two-way anova using subunit and system as variables revealed a significant effect of system but not subunit, nor was there a significant interaction between variables. results from both westerns and functional experiments therefore indicate that relative expression levels of glycine receptor were different between hek and l-cells. to further explore the interaction between expression level and partial agonist efficacy, both current density and taurine efficacy were compared for α and α2β glycine receptors in a number of different heterologous systems, as well as for native receptors expressed in rat lateral/basolateral amygdala. in addition to hek and l-cells, the heterologous systems included mouse 3t fibroblasts and mdck kidney cells. α2β receptors expressed in mouse 3t fibroblasts had twice the current density of the mouse l-cells but had a similar taurine efficacy. similarly, α2β receptors expressed in hek cells had a current density similar to glyrs expressed in 3t fibroblasts but had a taurine efficacy compared to glycine of ± 3%. this efficacy was similar to glycine receptors expressed by acutely isolated adult rat basolateral amygdala neurons although the current density in this native system was only ± pa/pf. note that the channels expressed by these neurons are composed primarily of α2+β subunits. canine kidney mdck cells expressed the lowest α2β current density; yet the channels expressed by this system had the highest taurine efficacy of any cell tested. for α glyrs, the rank order of glycine receptor density was 3t > hek cell > l-cell; while the rank order of taurine efficacy for these same receptors was hek > 3t > l-cells. across all subunit combinations and systems, there was no significant correlation between taurine efficacy and glycine current density. indeed, no correlation between expression level and taurine efficacy was evident within any given population of cells whether the receptors were expressed in native or heterologous systems. for example, the correlation coefficients for α2β receptors between taurine efficacy and glycine current density in individual systems were,,,, and for hek, l-cells, 3t cells, mdck cells, and amygdala neurons, respectively. thus, while there is clearly a difference in expression level between both the systems as well as between individual cells in a given system, this particular characteristic cannot account for the apparent taurine efficacy. the agonist-binding site is not affected by expression system there are a variety of possible mechanisms to account for the disparities in partial agonist pharmacology between two expression systems. one way to address this is to examine competitive antagonist binding properties in hek and l-cells. we therefore examined the potency of the glycine receptor competitive antagonist strychnine in both systems. following a second pretreatment with the antagonist, we co-applied strychnine and an ec concentration of glycine. the strychnine kb was estimated for hek and l-cells expressing either the glyrα or glyrα2+β subunits using the cheng-prusoff relationship. this relationship takes into account the divergent hill-slope and potencies for glycine found in these two expression systems. receptors composed of the glyrα subunit had very similar kb values when expressed in either hek or l-cells. the same was true for cells expressing the glyrα2β subunits where strychnine apparent affinity was ± nm in hek cells and ± nm in l-cells. two-way anova did not reveal any significant effect of either system or subunit composition. since strychnine is a competitive antagonist and site-directed mutation studies suggests that strychnine and glycine interact with overlapping regions of the receptor, our results strongly suggest that functional strychnine affinity, and hence the general structure of the agonist binding pocket, was not substantially influence by expression system. cytoskeletal components influence glycine receptor pharmacology a third possible mechanism for reduced efficacy in l-cells compared to hek cells or neurons could be related to intracellular factors that influence channel gating. this hypothesis was examined by disrupting the cytoskeletal protein tubulin, which has been shown to be important for glycine receptor localization. direct application of μm colchicine did not elicit any membrane currents. furthermore, acute application of μm colchicine and an ec concentration of glycine did not significantly affect glycine-gated currents themselves. glycine currents were ± pa/pf while glycine+colchicine currents were ± pa/pf. the relative efficacy of β-alanine and taurine was examined in hek cells expressing α2β subunits following min incubation with μm colchicine at 37°c, enough time to allow irreversible tubulin disruption. as an additional control, γ-lumicolchicine, an inactive analog of colchicine, was also used to treat α2β-expressing hek cells. these brief treatments had no obvious effect on the survival of untransfected cells. there was a trend for colchicine treatment to reduce the overall current density at μm glycine, ± pa/pf in control cells, ± pa/pf in colchicine-treated cells, and ± pa/pf in γ-lumicolchicine-treated cells; however, this was not significant and was probably not related to any direct action of colchicine given that the glycine current density was also slightly reduced in α2β-expressing cells exposed to γ-lumicolchicine compared to controls. however, the efficacy of both taurine and β-alanine were significantly decreased by colchicine but not γ-lumicolchicine treatment. taurine efficacy was ± 6% of glycine in controls, ± 3% following colchicine, and ± 3% following γ-lumicolchicine. similarly, β-alanine efficacy was ± 7% of glycine in controls, ± 6% following colchicine, and ± 7% following γ-lumicolchicine. similar treatment of α2-expressing hek cells with colchicine did not reveal any significant effect on glycine current density, on taurine efficacy, or on β-alanine efficacy. cholchicine treatment also significantly reduced β-alanine efficacy in l-cells expressing glyrα2β but not in glyrα2-expressing l-cells. we did not attempt to examine taurine in l-cells treated with colchicine given the exceptionally low efficacy of receptors expressed in this cell line. because the glycine receptor- and tubulin-binding protein gephyrin provides an obvious link between the receptor and the tubulin cytoskeleton, we used western analysis of hek and l-cell lysates with a gephyrin monoclonal antibody specific for the c-terminus. these experiments revealed that gephyrin-like immunoreactivity was expressed in both expression systems. notably, a ca. kd band dominated the hek cell gephyrin immunoreactivity, while multiple bands of varying intensity could be seen in lysate from l-cells. when taken with our colchicine data, differences in glycine receptor pharmacology between α2β receptors expressed in hek and l-cells may be partially due to distinct, system-dependent interactions with distinct isoforms of the cytoskeletal protein gephyrin. discussion we have expressed several the'embryonic' and'forebrain' isoforms in two distinct expression systems to understand the influence of endogenous and exogenous factors on receptor partial agonist pharmacology. although the pharmacology of the'embryonic' glyrα isoform and the'adult spinal' isoform have been explored more frequently in the literature, the pharmacology of glyrα2β receptors has remained largely unexplored. despite this, there is strong evidence that the adult'forebrain' isoforms, specifically in the rat basolateral amygdala, is indeed α2β. the current study indicates a general trend for decreased apparent affinity and reduced relative efficacy of agonists when receptors consist of the α2β subunits compared to their homomeric α counter parts. we were particularly surprised to find that receptors expressed in different expression systems possessed markedly different partial agonist efficacies. the remainder of our study focused on identifying extrinsic factors that influence difference in ligand-gated receptor pharmacology in distinct expression systems. while differences in efficiency of cdna expression/transfection between systems could explain such differences, the expression levels of glyrα2β receptors measured by current density was not correlated with taurine efficacy across several different cell types or within any given system. importantly, the efficacy of β-alanine and taurine in hek cells agree with previous findings where cells expressing glyrα show almost full efficacy for taurine and β-alanine. similarly, distinct ligand binding characteristics of receptors expressed in different expression systems seemed to be another possible mechanism governing agonist efficacy or potency. for the glycine receptor, the binding site for the competitive antagonist strychnine is believed to be adjacent to the agonist-binding site, sterically hindering agonist binding. a gross alteration in the agonist binding pocket, particularly one that hindered agonist binding, would most likely affect strychnine binding as well. to examine this, strychnine kb was calculated for glyrα and glyrα2β isoforms expressed in both hek and l-cells. in order to decrease the error in estimating kb, a derivation of the cheng-prusoff equation was used that takes in account variations in the slopes of the inhibition curves. differences in kb were negligible between expression systems, indicating the strychnine binding site, and presumably the agonist binding site, was altogether similar in these different systems. differences in pharmacology between systems therefore cannot be explained by substantial alterations in the agonist/competitive antagonist binding pocket. receptor gating is another mechanism by which receptor function may be altered. cytoskeletal elements have been shown to play a crucial role in neurotransmitter receptor clustering and may have a role in receptor function as well. for example, cytoskeletal stabilization has been shown to reduce ca++-dependent inactivation of ca++ channels in snail ganglia; and, actin has been shown to modulate several different types of membrane ion channel. cytoskeletal depolymerization has also been found to inhibit the function of gabaa receptors, which share significant sequence homology and functional characteristics with strychnine-sensitive glycine receptors. and the tubulin-gephyrin-glycine receptor interaction is critical for establishing functional glycinergic synapses. the current study suggests that cytoskeletal elements may play a functional role in α2β glycine receptor pharmacology as well. β-containing glycine receptors are intimately associated with the tubulin-associated protein gephyrin via the gephyrin binding site that lies within the intracellular domain of this subunit. in our studies, the efficacy of both taurine and β-alanine were reduced in cells expressing glyrα2β subunits. this despite the finding that gephyrin-like immunoreactivity in l-cells was apparently distinct from that in hek cells, suggesting that distinct cytoskeletal components in these systems may have profound influence over glyr α2β pharmacology. this is further supported by suggestions that gephyrin may exist in multiple, tissue-specific isoforms with potentially distinct functional roles. it should be noted however that colchicine treatment of α2β-expressing hek cells did not suppress partial agonist efficacy to a level that approached that found in l-cells or 3t3-fibroblasts. given that colchicine had no perceptible effect of α2-homomeric channels expressed in hek cells, our results suggest that additional system-dependent factors may have a more pronounced influence on the partial agonist pharmacology of strychnine-sensitive glycine receptors.  it is of particular interest that the β subunit appears to play a functional role in the pharmacology of α2-containing receptors regardless of expression system. for example, the beta subunit decreased the apparent efficacy of the partial agonists. since the β-subunit itself does not appreciably interact with the competitive antagonist strychnine, these results are at least consistent with some allosteric interaction between the β-subunit and the agonist binding site present the α subunit. this may indicate that α2β glycine receptors in the forebrain may be distinguishable from other receptor isoforms given the appropriate pharmacologic agent. although cytoskeletal components potentially play some role for these'forebrain' receptors, there appear to be other'extrinsic' factors governing expression system-dependent effects on agonist pharmacology. since it is conceivable that such factors may be differentially distributed between different forebrain regions, the large apparent differences between glycine receptor pharmacology reported by various studies may not necessarily depend upon differential expression of glycine receptor subunits per se. at the very least, our findings suggest that great care should be taken when utilizing different expression systems to develop screens for novel pharmacophores acting on this receptor. 
similar results were obtained for β-alanine. our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. however, taurine efficacy was dramatically reduced in l-cell fibroblasts. the efficacy of these partial agonists was also strongly reduced by the beta subunit. finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. while exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. we conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. the β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in hek cells. nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. there were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha + beta heteromeric channels. this subunit composition is distinct from the alpha + beta channels found throughout the adult spinal cord.
 strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha + beta heteromeric channels. this subunit composition is distinct from the alpha + beta channels found throughout the adult spinal cord. unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to'neonatal' alpha homomeric channels or'spinal' alpha1beta heteromers. in addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. to identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels.  while exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. the β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in hek cells. however, taurine efficacy was dramatically reduced in l-cell fibroblasts. similar results were obtained for β-alanine. the efficacy of these partial agonists was also strongly reduced by the beta subunit. there were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion.  our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. in the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. we conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. 