BACKGROUND
nicotinic acetylcholine receptors  are excitatory neurotransmitter receptors that are found in both vertebrate and invertebrate species.
in insects, nachrs are expressed throughout the nervous system and are the site of action for economically important insecticides such as spinosyns and neonicotinoids  <cit> .
detailed information is available concerning the structure of nachrs, as a consequence of studies conducted with receptors purified from the electric organ of the marine ray torpedo <cit>  and from x-ray crystallographic studies conducted with nachr fragments  <cit>  and also with the closely related acetylcholine binding protein  <cit> .
nicotinic receptors are assembled from five subunits arranged around a central cation-selective pore  <cit> .
conventional agonists, such as acetylcholine, activate the receptor by binding at an extracellular site located at the interface between two subunits  <cit> , although recent evidence indicates that nachrs can also be activated by ligands binding to an allosteric transmembrane site  <cit> .
ten nachr subunits  have been identified in the model insect drosophila melanogaster and a similar number of subunits have been identified in other insect species  <cit> .
despite considerable efforts, there has been only limited success in expressing insect nachrs in artificial expressions systems  <cit>  and, where functional expression has been achieved, ion channel currents have tended to be small or have been generated in response to relatively high agonist concentrations  <cit> .
experimental approaches that have had some success in overcoming problems associated with expression of insect nachrs include the expression of subunit chimeras containing domains from other neurotransmitter receptors  <cit> , co-expression of insect nachrs with vertebrate subunits  <cit>  or a combination of these approaches  <cit> .
co-expression with vertebrate nachr subunits is an approach that has been used in the characterization of nachr subunits cloned from insect pest species such as the aphid myzus persicae <cit>  and the brown planthopper nilaparvata lugens <cit> .
however, for most insect species for which nachrs have been cloned, there have been no reports of successful heterologous expression.
this includes nachrs cloned from the honeybee apis mellifera <cit> , diamondback moth plutella xylostella <cit> , house fly musca domestica <cit> , locust locusta migratoria <cit> , mosquito anopheles gambiae <cit> , red flour beetle tribolium castaneum <cit> , silkworm bombyx mori <cit>  and tobacco hornworm manduca sexta <cit> .
ric- <dig> is a nachr-associated molecular chaperone that was originally characterised in the nematode caenorhabditis elegans <cit>  but has also been identified in several other species, including mammals and insects  <cit> .
it is a transmembrane protein that is able to enhance maturation  of several nachr subtypes  <cit> .
for example, co-expression of ric- <dig> with the vertebrate nachr α <dig> subunit enhances levels of functional expression in xenopus oocytes  <cit>  and is able to facilitate the functional expression of α <dig> nachrs in mammalian cell lines that are otherwise non-permissive for expression of α <dig>  <cit> .
in some cell types it has been found that the α <dig> subunit can be expressed  but, in the absence of ric- <dig>  is unable to fold into a conformation that can be detected by radioligand binding or form functional nachrs  <cit> .
in addition, some success has been achieved in overcoming difficulties associated with expression of insect nachrs by the co-expression with ric- <dig>  <cit> .
the dα <dig>  dα <dig> and dα <dig> subunits of drosophila show close sequence similarity to one another  and also have close similarity to the vertebrate nachr α <dig> subunit .
of the three drosophila subunits, dα <dig> and dα <dig> have the closest sequence similarity to one another and dα <dig> has the highest sequence similarity to the vertebrate α <dig>  <cit> .
in the present study, we report the molecular cloning of the dα <dig> subunit, the only drosophila nachr subunit for which a full-length cdna clone was not previously available in our laboratory.
heterologous expression studies with dα <dig>  dα <dig> and dα <dig> are described in three host cell types: drosophila s <dig> cells, human tsa <dig> cells and xenopus oocytes.
functional expression of several subunit combinations has been achieved in xenopus oocytes and has enabled the pharmacological properties of recombinant nachrs to be examined.
evidence is provided that demonstrates the ability of subunits to form both homomeric and heteromeric nachrs.
of particular note is evidence that dα <dig> can generate functional homomeric channels and that dα <dig> can form both homomeric and heteromeric channels.
we are not aware of any previous studies demonstrating the ability of dα <dig> and dα <dig> subunits to generate such recombinant nachrs, either with subunits cloned from drosophila or with analogous nachr subunits from other insect species.
RESULTS
molecular cloning of dα5
a full-length cdna encoding the drosophila nachr dα <dig> subunit was isolated from a preparation of drosophila embryo mrna.
the dα <dig> cdna encodes an open reading frame of  <dig> amino acids corresponding to the previously described dα <dig> isoform b  <cit> .
in agreement with previous studies  <cit> , the dα <dig> cdna isolated in this study contains an open reading frame encoding an unusually large n-terminal domain, extending some  <dig> amino acids upstream of the start methionine in most nachr subunits.
heterologous expression of dα <dig> in drosophila and human cell lines
the full-length coding sequence of the dα <dig> cdna was sub-cloned into the drosophila expression vector prmha <dig>  and into prk <dig> .
in cells transfected with prmha3-dα <dig> or prk5-dα <dig> alone, no evidence of specific high-affinity binding of nicotinic radioligands  could be detected.
the dα <dig> subunit was also co-expressed with an extensive series of drosophila nachr subunit combinations.
expression studies with more than  <dig> different subunit combinations  have been examined in our laboratory.
however, no specific binding was detected with any these combinations .
to illustrate the extent of these studies, details of all drosophila nachr subunit combinations containing the dα <dig> subunit are listed in table  <dig>  it is possible that the lack of radioligand binding is a consequence of the expressed subunit proteins failing to undergoing appropriate maturation  due to a requirement for specific chaperone proteins, as has been reported for other nachr subunits  <cit> , or due to a requirement for additional nachr subunits.
to illustrate the extent of radioligand binding studies undertaken, the table lists all subunit combinations containing dα <dig> that were examined in transfected drosophila s <dig> cells.
binding studies were performed with -α-bungarotoxin  and -epibatidine .
combinations of drosophila nachr subunit cdnas were transfected in the absence or presence of ric- <dig> cdna.
data indicating presence or absence of specific binding are derived from at least  <dig> independent experiments.
previous studies have shown that, when co-expressed with a vertebrate β <dig> subunit, some drosophila nachr α-subunits can generate functional recombinant nachrs and form high-affinity binding sites for nicotinic radioligands .
however, when dα <dig> was co-expressed with vertebrate β <dig> in drosophila s <dig> cells or in human tsa <dig> cells, no specific radioligand binding could be detected.
these findings with dα <dig> are similar to those conducted previously with the closely related drosophila dα <dig> and dα <dig> subunits  <cit> .
however, in control experiments conducted in parallel, high levels of specific radioligand binding were detected after co-expression of drosophila dα <dig> and dα <dig> subunits with the rat β <dig>  subunit.
this is in agreement with previous studies conducted with the dα2 + rβ <dig> and dα3 + rβ <dig> subunit combinations  <cit> .
dα5/5ht3a subunit chimera
as has been described previously for the dα <dig> and dα <dig> subunits  <cit> , a chimera was constructed containing the n-terminal ligand-binding domain of the dα <dig> subunit fused to the transmembrane and c-terminal regions of the mouse 5-ht3a subunit .
despite the inability of the intact dα <dig> subunit to be detected by 125i]-α-bungarotoxin binding when expressed in drosophila s <dig>  expression of the dα5/5ht3a chimera resulted in high levels of cell-surface 125i]-α-bungarotoxin binding .
these data from recombinant subunit chimeras is consistent with evidence derived from native drosophila nachrs that dα <dig> forms part of an α-bungarotoxin binding nachr  <cit> .
expression studies with the intact and chimeric dα <dig> subunit indicate that, in common with the dα <dig> and dα <dig> subunits, inefficient folding and assembly can be attributed to domains present in the c-terminal subunit domain.
similar conclusions have also been made concerning the closely related vertebrate α <dig> subunit  <cit> .
the influence of co-expressing combinations of subunit chimeras was also examined.
in comparisons to the level of 125i]-α-bungarotoxin binding detected with dα5/5ht3a alone, higher levels of specific cell-surface binding were detected when the dα <dig> chimera was co-expressed with other subunit chimeras .
however, the levels of specific binding detected were not significantly higher than would have been expected from a possible additive effect of co-expressing these chimeras.
consequently, this data cannot be used as evidence to support the possibility of heteromeric co-assembly, as was the case previously for studies conducted with the dα <dig> and dα <dig> subunit chimeras  <cit> .
heterologous expression with ric-3
previous studies have demonstrated that the molecular chaperone protein ric- <dig> can enhance maturation of several nachrs  <cit> .
this finding has prompted us to examine the influence of co-expressing dα <dig> with ric- <dig> in cultured cell lines, as we have done previously for other drosophila nachr subunits  <cit> .
various combinations  of dα <dig>  dα <dig> and dα <dig> were expressed with either ceric- <dig> or dmric- <dig> in both drosophila s <dig> cells and human tsa <dig> cells.
no specific cell-surface 125i]-α-bungarotoxin binding was detected when any of these subunits were expressed individually with ric- <dig>  however, specific binding was detected when dα <dig> was co-expressed with dα <dig> , albeit at a lower level than seen with the subunit chimeras .
interestingly, no specific binding was detected when these two subunits were also co-expressed with dα <dig> , suggesting that dα <dig> may co-assemble with dα <dig> or dα <dig> and, in doing so, impair receptor assembly or maturation.
similar results were obtained in both cell types examined , although specific binding was detected in mammalian cells only when they were cultured at a temperature lower than 37°c.
as has been reported previously  <cit> , lowering the temperature of transfected mammalian cells from 37°c to 25°c for  <dig> hours  facilitates receptor assembly and cell-surface expression.
as has been discussed previously with respect to insect nachr subunits  <cit> , the detection of specific radioligand binding only in mammalian cells cultured at 25°c is likely to be a consequence of more efficient subunit folding and assembly at lower temperatures.
expression in xenopus oocytes
xenopus oocytes were injected with crna encoding various combinations of the drosophila nachr subunits dα <dig>  dα <dig> and dα <dig>  no evidence of functional expression was detected for any subunit combination in the absence of co-expressed ric- <dig>  however, when co-expressed with ceric- <dig>  functional responses to acetylcholine were detected in oocytes injected with either the dα <dig> or the dα <dig> subunit, indicating the presence of functional homomeric dα <dig> and dα <dig> nachrs .
however, even when co-expressed with ric- <dig>  functional expression was somewhat inconsistent, being observed in some but not all batches of oocytes tested .
dose–response curves indicate that acetylcholine has a similar ec <dig> for these two homomeric receptor subtypes .
in contrast, no functional expression was detected when dα <dig> was co-expressed with ceric- <dig>
expression of pairwise subunit combinations  gave dose–response curves that were not significantly different  to that of homomeric dα <dig> or dα <dig> nachrs .
consequently, it was not possible to conclude whether pairwise heteromeric receptors were expressed.
one pairwise combination  failed to generate consistent responses, an indication that co-assembly of dα <dig> with dα <dig> blocks formation of functional nachrs in oocytes.
however, when all three subunits  were co-expressed with ceric- <dig>  dose–response data indicated a single population of receptors with a significantly higher  apparent affinity for acetylcholine  than that of either of the two homomeric nachrs  or any of the putative pairwise subunit combinations .
for all subunit combinations examined , functional responses to acetylcholine were completely blocked by a 10 min pre-incubation with 100nm α-bungarotoxin.
this block was completely reversible but occurred on a slow timescale, recovery taking, typically, about  <dig> minutes .
no significant differences were observed in pharmacological properties when nachrs were co-expressed with dmric- <dig>  <cit> , rather than ceric- <dig> .
note, in all cases, nachr subunits were co-expressed with ceric- <dig>
* note, ec <dig> value for dα5 + dα6 + dα <dig> is expressed as nm, rather than μm.
**ec <dig> and hill slopes are means ± sem of separate fits to dose–response curves derived from independent experiments .
† relatively small currents were detected with all subunit combinations, as indicated by the size of the maximum current that was detected with each subunit combination  and the mean maximum current  from between 6– <dig> different oocytes.
‡ subunit combinations that failed to generate functional responses are indicated by a dash.
this is based on studies conducted with at least  <dig> batches of oocytes that generated functional nachrs with other subunit combinations and at least  <dig> oocytes from each batch .
discussion
the dα <dig>  dα <dig> and dα <dig> subunits differ from other drosophila nachr subunits in their close sequence similarity to the vertebrate α <dig> nachr subunit  <cit> , a subunit that is notable for its ability to form both homomeric and heteromeric nachrs  <cit> .
in addition to being one of the best characterised homomeric nachrs, the vertebrate α <dig> subunit can co-assemble into heteromeric nachrs by co-assembly with the α <dig> subunit   <cit> .
although an α <dig> subunit is not present in mammals, recent evidence indicates that the mammalian α <dig> subunit can also form functional heteromeric nachrs by co-assembly with β <dig>  <cit> .
relatively limited information is available about the physiological roles of the dα <dig>  dα <dig> and dα <dig> subunits in drosophila, or about the role of analogous subunits in other insect species.
there is, however, evidence from studies of native nachrs in drosophila that dα <dig> forms part of a nachr that is sensitive to α-bungarotoxin  <cit> , dα <dig> forms part of the spinosad-sensitive nachr  <cit>  and that dα <dig> is required for the visually-mediated cholinergic escape response  <cit> .
as has been discussed elsewhere  <cit> , difficulties have been encountered in the efficient functional expression of insect nachrs.
here we report the cloning of a full-length cdna of the drosophila dα <dig> subunit corresponding to a previously described isoform b  <cit> .
other isoforms of dα <dig> described previously   <cit>  are a consequence of alternative splicing and have fewer exons than isoform b. isoform a lacks exon  <dig>  which codes for part of the second transmembrane domain, whilst isoform c lacks exon  <dig>  which codes for the region containing the extracellular cys-loop.
the cloning of the dα <dig> subunit was first reported in  <dig>  <cit>  but no expression studies were described at that time.
more recently, it has been reported that dα <dig> does not generate functional homomeric nachrs when expressed in xenopus oocytes, even when co-expressed with ric- <dig>  <cit> .
functional expression was, however, reported in the same study when dα <dig> was co-expressed with dα <dig> and ric- <dig>  <cit> .
in the present study, we have detected functional responses when dα <dig> is co-expressed with dα <dig> but, in contrast to the previous study  <cit> , we have also obtained evidence for the functional expression of homomeric dα <dig> nachrs.
similarly, we have demonstrated that dα <dig> can form both homomeric and heteromeric nachrs.
as far as we are aware, there have been no previous reports of the successful functional expression of dα <dig>  as either a homomeric or a heteromeric nachr.
given the difficulties encountered in obtaining reproducible functional expression of insect recombinant nachrs, it is not surprising that there may be some apparent differences in subunit combinations found to generate functional receptors in this and previous studies, particularly since the focus of the most detailed previous study was the identification of a spinosyn-sensitive nachrs  <cit> .
our studies conducted in cell lines provided evidence that the pairwise combination dα5 + dα <dig> generates a high affinity radioligand binding site, a finding that agrees with previous studies demonstrating functional expression of dα5 + dα <dig> nachrs in oocytes  <cit> .
interestingly, we have found no evidence of specific binding when dα <dig> was co-expressed with dα <dig> and dα <dig> in the same cell lines.
this lack of specific binding would seem to suggest that, in the two cell lines examined, co-assembly of dα <dig> with the dα <dig> and dα <dig> subunit interferes with the formation of correctly assembled complexes.
we observed a somewhat similar situation in oocytes, where expression of dα <dig> alone generates functional nachrs but it fails to do so when co-expressed with dα <dig>  this may reflect a tendency for dα <dig> and dα <dig> to assemble into non-functional complexes.
the one situation where this tendency is not dominant is when all three subunits  are co-expressed with ric- <dig> in oocytes, where they are able to form a functional ‘triplet’ nachr with high apparent affinity for acetylcholine.
the present findings suggest that the environment provided by the host cell exerts a substantial effect on the assembly of these nachr subtypes, a phenomena that has been reported previously for other nachrs  <cit> .
previous studies by another research group  <cit>  support the conclusion that co-assembly of dα5 + dα <dig> nachrs is somewhat inefficient.
not only was functional expression of the dα5 + dα <dig> subunit combination found to be inconsistent in the previous study, but it also appeared to be dependent on the ratio of crnas injected  <cit> .
perhaps this inconsistent functional expression reflects a tendency for some subunit combinations to assemble into non-functional complexes and that this may be more prevalent in certain subunit stoichiometries.
it is possible that, in the native cellular environment, factors determining efficiency of subunit assembly and maturation may differ, perhaps as a consequence of a different array of endogenous chaperone proteins.
this conclusion is supported by previous studies that have indicated that influence of ric- <dig> on maturation of nachrs is influenced by the host cell  <cit>  and may help to explain the differences that we have observed in the ability of some subunit combinations to assemble into nachrs in different expression systems.
the data obtained from expression studies in drosophila and human cell lines is broadly similar.
however, successful expression in human cells required incubation at a temperature lower than they would normally be maintained at  .
previous studies have demonstrated that the folding and assembly of the nachrs from insects  <cit>  and from some other non-insect species, such as the cold-water ray torpedo <cit> , can be influenced by temperature.
this temperature dependence appears to be a consequence of inefficient protein folding and/or subunit assembly at higher temperatures.
previously, due to difficulties in expression of dα <dig> and dα <dig> nachr subunits, we examined the ability of subunit chimeras to assemble into complexes capable of binding 125i]-α-bungarotoxin  <cit> .
from such studies, it was possible to conclude that the dα <dig> and dα <dig> subunits were capable of heterometic co-assembly.
in the present study the data from subunit chimeras is less clear cut.
although higher levels of 125i]-α-bungarotoxin were seen consistently when the dα <dig> chimera was co-expressed with either the dα <dig> and dα <dig> chimeras, it was not clear in all cases whether this was greater than an additive effect.
nevertheless these findings are consistent with the conclusion that dα <dig> is able to co-assemble into heteromeric complexes.
for all subunit combinations examined, responses to acetylcholine were completely blocked by α-bungarotoxin, a finding that is consistent with previous studies conducted with native nachrs purified from drosophila which demonstrated that dα <dig> is part of an α-bungarotoxin binding nachr  <cit> .
as mentioned above, a previous study has reported the functional expression of heteromeric dα5 + dα <dig> nachrs  in xenopus oocytes and also the inability of either dα <dig> or dα <dig> to form functional homomeric nachrs  <cit> .
significantly, the authors of this earlier study describe substantial difficulties in achieving reliable functional expression.
in the present study, despite demonstrating the functional expression of several combinations of the dα <dig>  dα <dig> and dα <dig> subunits, we have also encountered a much lower success rate than is typically achieved with other nachrs.
in both transfected cell lines and in xenopus oocytes, we occasionally failed to detect evidence of radioligand binding or functional expression, despite success with other nachrs that were expressed as positive controls .
the difficulties that we and others have encountered may be associated with a tendency for these subunits to co-assemble into non-functional complexes.
it is possible that this may reflect a requirement for additional chaperone proteins.
indeed, a study conducted with a c. elegans nachr has demonstrated a requirement for three different chaperone proteins for efficient functional heterologous expression  <cit> .
CONCLUSIONS
in summary, whereas it has been reported previously that dα <dig> and dα <dig> can form a functional heteromeric nachr  when expressed in xenopus oocytes  <cit> , this is the first evidence that either dα <dig> or dα <dig> can form functional homomeric nachrs.
it is also the first demonstration that dα <dig> can form a functional heteromeric nachr.
of particular interest is the evidence that the three subunits examined in this study can co-assemble to form a functional triplet  nachr with a high apparent affinity for acetylcholine.

 nicotinic acetylcholine receptors are excitatory neurotransmitter receptors that are found in both vertebrate and invertebrate species. in insects, nachrs are expressed throughout the nervous system and are the site of action for economically important insecticides such as spinosyns and neonicotinoids. detailed information is available concerning the structure of nachrs, as a consequence of studies conducted with receptors purified from the electric organ of the marine ray torpedo and from x-ray crystallographic studies conducted with nachr fragments and also with the closely related acetylcholine binding protein. nicotinic receptors are assembled from five subunits arranged around a central cation-selective pore. conventional agonists, such as acetylcholine, activate the receptor by binding at an extracellular site located at the interface between two subunits, although recent evidence indicates that nachrs can also be activated by ligands binding to an allosteric transmembrane site. ten nachr subunits have been identified in the model insect drosophila melanogaster and a similar number of subunits have been identified in other insect species. despite considerable efforts, there has been only limited success in expressing insect nachrs in artificial expressions systems and, where functional expression has been achieved, ion channel currents have tended to be small or have been generated in response to relatively high agonist concentrations. experimental approaches that have had some success in overcoming problems associated with expression of insect nachrs include the expression of subunit chimeras containing domains from other neurotransmitter receptors, co-expression of insect nachrs with vertebrate subunits or a combination of these approaches. co-expression with vertebrate nachr subunits is an approach that has been used in the characterization of nachr subunits cloned from insect pest species such as the aphid myzus persicae and the brown planthopper nilaparvata lugens. however, for most insect species for which nachrs have been cloned, there have been no reports of successful heterologous expression. this includes nachrs cloned from the honeybee apis mellifera, diamondback moth plutella xylostella, house fly musca domestica, locust locusta migratoria, mosquito anopheles gambiae, red flour beetle tribolium castaneum, silkworm bombyx mori and tobacco hornworm manduca sexta. ric- is a nachr-associated molecular chaperone that was originally characterised in the nematode caenorhabditis elegans but has also been identified in several other species, including mammals and insects. it is a transmembrane protein that is able to enhance maturation of several nachr subtypes. for example, co-expression of ric- with the vertebrate nachr α subunit enhances levels of functional expression in xenopus oocytes and is able to facilitate the functional expression of α nachrs in mammalian cell lines that are otherwise non-permissive for expression of α. in some cell types it has been found that the α subunit can be expressed but, in the absence of ric- is unable to fold into a conformation that can be detected by radioligand binding or form functional nachrs. in addition, some success has been achieved in overcoming difficulties associated with expression of insect nachrs by the co-expression with ric-. the dα dα and dα subunits of drosophila show close sequence similarity to one another and also have close similarity to the vertebrate nachr α subunit. of the three drosophila subunits, dα and dα have the closest sequence similarity to one another and dα has the highest sequence similarity to the vertebrate α. in the present study, we report the molecular cloning of the dα subunit, the only drosophila nachr subunit for which a full-length cdna clone was not previously available in our laboratory. heterologous expression studies with dα dα and dα are described in three host cell types: drosophila s cells, human tsa cells and xenopus oocytes. functional expression of several subunit combinations has been achieved in xenopus oocytes and has enabled the pharmacological properties of recombinant nachrs to be examined. evidence is provided that demonstrates the ability of subunits to form both homomeric and heteromeric nachrs. of particular note is evidence that dα can generate functional homomeric channels and that dα can form both homomeric and heteromeric channels. we are not aware of any previous studies demonstrating the ability of dα and dα subunits to generate such recombinant nachrs, either with subunits cloned from drosophila or with analogous nachr subunits from other insect species.  molecular cloning of dα5 a full-length cdna encoding the drosophila nachr dα subunit was isolated from a preparation of drosophila embryo mrna. the dα cdna encodes an open reading frame of amino acids corresponding to the previously described dα isoform b. in agreement with previous studies, the dα cdna isolated in this study contains an open reading frame encoding an unusually large n-terminal domain, extending some amino acids upstream of the start methionine in most nachr subunits. heterologous expression of dα in drosophila and human cell lines the full-length coding sequence of the dα cdna was sub-cloned into the drosophila expression vector prmha and into prk. in cells transfected with prmha3-dα or prk5-dα alone, no evidence of specific high-affinity binding of nicotinic radioligands could be detected. the dα subunit was also co-expressed with an extensive series of drosophila nachr subunit combinations. expression studies with more than different subunit combinations have been examined in our laboratory. however, no specific binding was detected with any these combinations. to illustrate the extent of these studies, details of all drosophila nachr subunit combinations containing the dα subunit are listed in table it is possible that the lack of radioligand binding is a consequence of the expressed subunit proteins failing to undergoing appropriate maturation due to a requirement for specific chaperone proteins, as has been reported for other nachr subunits, or due to a requirement for additional nachr subunits. to illustrate the extent of radioligand binding studies undertaken, the table lists all subunit combinations containing dα that were examined in transfected drosophila s cells. binding studies were performed with -α-bungarotoxin and -epibatidine. combinations of drosophila nachr subunit cdnas were transfected in the absence or presence of ric- cdna. data indicating presence or absence of specific binding are derived from at least independent experiments. previous studies have shown that, when co-expressed with a vertebrate β subunit, some drosophila nachr α-subunits can generate functional recombinant nachrs and form high-affinity binding sites for nicotinic radioligands. however, when dα was co-expressed with vertebrate β in drosophila s cells or in human tsa cells, no specific radioligand binding could be detected. these findings with dα are similar to those conducted previously with the closely related drosophila dα and dα subunits. however, in control experiments conducted in parallel, high levels of specific radioligand binding were detected after co-expression of drosophila dα and dα subunits with the rat β subunit. this is in agreement with previous studies conducted with the dα2 + rβ and dα3 + rβ subunit combinations. dα5/5ht3a subunit chimera as has been described previously for the dα and dα subunits, a chimera was constructed containing the n-terminal ligand-binding domain of the dα subunit fused to the transmembrane and c-terminal regions of the mouse 5-ht3a subunit. despite the inability of the intact dα subunit to be detected by 125i]-α-bungarotoxin binding when expressed in drosophila s expression of the dα5/5ht3a chimera resulted in high levels of cell-surface 125i]-α-bungarotoxin binding. these data from recombinant subunit chimeras is consistent with evidence derived from native drosophila nachrs that dα forms part of an α-bungarotoxin binding nachr. expression studies with the intact and chimeric dα subunit indicate that, in common with the dα and dα subunits, inefficient folding and assembly can be attributed to domains present in the c-terminal subunit domain. similar conclusions have also been made concerning the closely related vertebrate α subunit. the influence of co-expressing combinations of subunit chimeras was also examined. in comparisons to the level of 125i]-α-bungarotoxin binding detected with dα5/5ht3a alone, higher levels of specific cell-surface binding were detected when the dα chimera was co-expressed with other subunit chimeras. however, the levels of specific binding detected were not significantly higher than would have been expected from a possible additive effect of co-expressing these chimeras. consequently, this data cannot be used as evidence to support the possibility of heteromeric co-assembly, as was the case previously for studies conducted with the dα and dα subunit chimeras. heterologous expression with ric-3 previous studies have demonstrated that the molecular chaperone protein ric- can enhance maturation of several nachrs. this finding has prompted us to examine the influence of co-expressing dα with ric- in cultured cell lines, as we have done previously for other drosophila nachr subunits. various combinations of dα dα and dα were expressed with either ceric- or dmric- in both drosophila s cells and human tsa cells. no specific cell-surface 125i]-α-bungarotoxin binding was detected when any of these subunits were expressed individually with ric- however, specific binding was detected when dα was co-expressed with dα, albeit at a lower level than seen with the subunit chimeras. interestingly, no specific binding was detected when these two subunits were also co-expressed with dα, suggesting that dα may co-assemble with dα or dα and, in doing so, impair receptor assembly or maturation. similar results were obtained in both cell types examined, although specific binding was detected in mammalian cells only when they were cultured at a temperature lower than 37°c. as has been reported previously, lowering the temperature of transfected mammalian cells from 37°c to 25°c for hours facilitates receptor assembly and cell-surface expression. as has been discussed previously with respect to insect nachr subunits, the detection of specific radioligand binding only in mammalian cells cultured at 25°c is likely to be a consequence of more efficient subunit folding and assembly at lower temperatures. expression in xenopus oocytes xenopus oocytes were injected with crna encoding various combinations of the drosophila nachr subunits dα dα and dα no evidence of functional expression was detected for any subunit combination in the absence of co-expressed ric- however, when co-expressed with ceric- functional responses to acetylcholine were detected in oocytes injected with either the dα or the dα subunit, indicating the presence of functional homomeric dα and dα nachrs. however, even when co-expressed with ric- functional expression was somewhat inconsistent, being observed in some but not all batches of oocytes tested. dose–response curves indicate that acetylcholine has a similar ec for these two homomeric receptor subtypes. in contrast, no functional expression was detected when dα was co-expressed with ceric- expression of pairwise subunit combinations gave dose–response curves that were not significantly different to that of homomeric dα or dα nachrs. consequently, it was not possible to conclude whether pairwise heteromeric receptors were expressed. one pairwise combination failed to generate consistent responses, an indication that co-assembly of dα with dα blocks formation of functional nachrs in oocytes. however, when all three subunits were co-expressed with ceric- dose–response data indicated a single population of receptors with a significantly higher apparent affinity for acetylcholine than that of either of the two homomeric nachrs or any of the putative pairwise subunit combinations. for all subunit combinations examined, functional responses to acetylcholine were completely blocked by a 10 min pre-incubation with 100nm α-bungarotoxin. this block was completely reversible but occurred on a slow timescale, recovery taking, typically, about minutes. no significant differences were observed in pharmacological properties when nachrs were co-expressed with dmric-, rather than ceric-. note, in all cases, nachr subunits were co-expressed with ceric- * note, ec value for dα5 + dα6 + dα is expressed as nm, rather than μm. **ec and hill slopes are means ± sem of separate fits to dose–response curves derived from independent experiments. † relatively small currents were detected with all subunit combinations, as indicated by the size of the maximum current that was detected with each subunit combination and the mean maximum current from between 6– different oocytes. ‡ subunit combinations that failed to generate functional responses are indicated by a dash. this is based on studies conducted with at least batches of oocytes that generated functional nachrs with other subunit combinations and at least oocytes from each batch. discussion the dα dα and dα subunits differ from other drosophila nachr subunits in their close sequence similarity to the vertebrate α nachr subunit, a subunit that is notable for its ability to form both homomeric and heteromeric nachrs. in addition to being one of the best characterised homomeric nachrs, the vertebrate α subunit can co-assemble into heteromeric nachrs by co-assembly with the α subunit. although an α subunit is not present in mammals, recent evidence indicates that the mammalian α subunit can also form functional heteromeric nachrs by co-assembly with β. relatively limited information is available about the physiological roles of the dα dα and dα subunits in drosophila, or about the role of analogous subunits in other insect species. there is, however, evidence from studies of native nachrs in drosophila that dα forms part of a nachr that is sensitive to α-bungarotoxin, dα forms part of the spinosad-sensitive nachr and that dα is required for the visually-mediated cholinergic escape response. as has been discussed elsewhere, difficulties have been encountered in the efficient functional expression of insect nachrs. here we report the cloning of a full-length cdna of the drosophila dα subunit corresponding to a previously described isoform b. other isoforms of dα described previously are a consequence of alternative splicing and have fewer exons than isoform b. isoform a lacks exon which codes for part of the second transmembrane domain, whilst isoform c lacks exon which codes for the region containing the extracellular cys-loop. the cloning of the dα subunit was first reported in but no expression studies were described at that time. more recently, it has been reported that dα does not generate functional homomeric nachrs when expressed in xenopus oocytes, even when co-expressed with ric-. functional expression was, however, reported in the same study when dα was co-expressed with dα and ric-. in the present study, we have detected functional responses when dα is co-expressed with dα but, in contrast to the previous study, we have also obtained evidence for the functional expression of homomeric dα nachrs. similarly, we have demonstrated that dα can form both homomeric and heteromeric nachrs. as far as we are aware, there have been no previous reports of the successful functional expression of dα as either a homomeric or a heteromeric nachr. given the difficulties encountered in obtaining reproducible functional expression of insect recombinant nachrs, it is not surprising that there may be some apparent differences in subunit combinations found to generate functional receptors in this and previous studies, particularly since the focus of the most detailed previous study was the identification of a spinosyn-sensitive nachrs. our studies conducted in cell lines provided evidence that the pairwise combination dα5 + dα generates a high affinity radioligand binding site, a finding that agrees with previous studies demonstrating functional expression of dα5 + dα nachrs in oocytes. interestingly, we have found no evidence of specific binding when dα was co-expressed with dα and dα in the same cell lines. this lack of specific binding would seem to suggest that, in the two cell lines examined, co-assembly of dα with the dα and dα subunit interferes with the formation of correctly assembled complexes. we observed a somewhat similar situation in oocytes, where expression of dα alone generates functional nachrs but it fails to do so when co-expressed with dα this may reflect a tendency for dα and dα to assemble into non-functional complexes. the one situation where this tendency is not dominant is when all three subunits are co-expressed with ric- in oocytes, where they are able to form a functional ‘triplet’ nachr with high apparent affinity for acetylcholine. the present findings suggest that the environment provided by the host cell exerts a substantial effect on the assembly of these nachr subtypes, a phenomena that has been reported previously for other nachrs. previous studies by another research group support the conclusion that co-assembly of dα5 + dα nachrs is somewhat inefficient. not only was functional expression of the dα5 + dα subunit combination found to be inconsistent in the previous study, but it also appeared to be dependent on the ratio of crnas injected. perhaps this inconsistent functional expression reflects a tendency for some subunit combinations to assemble into non-functional complexes and that this may be more prevalent in certain subunit stoichiometries. it is possible that, in the native cellular environment, factors determining efficiency of subunit assembly and maturation may differ, perhaps as a consequence of a different array of endogenous chaperone proteins. this conclusion is supported by previous studies that have indicated that influence of ric- on maturation of nachrs is influenced by the host cell and may help to explain the differences that we have observed in the ability of some subunit combinations to assemble into nachrs in different expression systems. the data obtained from expression studies in drosophila and human cell lines is broadly similar. however, successful expression in human cells required incubation at a temperature lower than they would normally be maintained at. previous studies have demonstrated that the folding and assembly of the nachrs from insects and from some other non-insect species, such as the cold-water ray torpedo, can be influenced by temperature. this temperature dependence appears to be a consequence of inefficient protein folding and/or subunit assembly at higher temperatures. previously, due to difficulties in expression of dα and dα nachr subunits, we examined the ability of subunit chimeras to assemble into complexes capable of binding 125i]-α-bungarotoxin. from such studies, it was possible to conclude that the dα and dα subunits were capable of heterometic co-assembly. in the present study the data from subunit chimeras is less clear cut. although higher levels of 125i]-α-bungarotoxin were seen consistently when the dα chimera was co-expressed with either the dα and dα chimeras, it was not clear in all cases whether this was greater than an additive effect. nevertheless these findings are consistent with the conclusion that dα is able to co-assemble into heteromeric complexes. for all subunit combinations examined, responses to acetylcholine were completely blocked by α-bungarotoxin, a finding that is consistent with previous studies conducted with native nachrs purified from drosophila which demonstrated that dα is part of an α-bungarotoxin binding nachr. as mentioned above, a previous study has reported the functional expression of heteromeric dα5 + dα nachrs in xenopus oocytes and also the inability of either dα or dα to form functional homomeric nachrs. significantly, the authors of this earlier study describe substantial difficulties in achieving reliable functional expression. in the present study, despite demonstrating the functional expression of several combinations of the dα dα and dα subunits, we have also encountered a much lower success rate than is typically achieved with other nachrs. in both transfected cell lines and in xenopus oocytes, we occasionally failed to detect evidence of radioligand binding or functional expression, despite success with other nachrs that were expressed as positive controls. the difficulties that we and others have encountered may be associated with a tendency for these subunits to co-assemble into non-functional complexes. it is possible that this may reflect a requirement for additional chaperone proteins. indeed, a study conducted with a c. elegans nachr has demonstrated a requirement for three different chaperone proteins for efficient functional heterologous expression.  in summary, whereas it has been reported previously that dα and dα can form a functional heteromeric nachr when expressed in xenopus oocytes, this is the first evidence that either dα or dα can form functional homomeric nachrs. it is also the first demonstration that dα can form a functional heteromeric nachr. of particular interest is the evidence that the three subunits examined in this study can co-assemble to form a functional triplet nachr with a high apparent affinity for acetylcholine. 
we have demonstrated the functional expression, as homomeric nachrs, of the dα and dα subunits in xenopus oocytes by their co-expression with the molecular chaperone ric- also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nachr with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. a full-length cdna clone encoding the drosophila nachr dα subunit has been isolated and the properties of dα5-, dα6- and dα7-containing nachrs examined in a variety of cell expression systems. in addition, specific cell-surface binding of -α-bungarotoxin was detected in both drosophila and mammalian cell lines when dα was co-expressed with dα and ric- in contrast, co-expression of additional subunits with dα and dα prevented specific binding of -α-bungarotoxin in cell lines, suggesting that co-assembly with other nachr subunits can block maturation of correctly folded nachrs in some cellular environments. however, problems have been encountered in the successful functional expression of insect nachrs, although a number of strategies have been developed in an attempt to overcome such difficulties. ten nachr subunits have been identified in the model insect drosophila melanogaster and a similar number have been identified in other insect species. nicotinic acetylcholine receptors play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. in insects, nachrs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. the focus of the present study is the dα dα and dα subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α nachr subunit. data are presented demonstrating the ability of the drosophila dα and dα subunits to generate functional homomeric and also heteromeric nachrs.
 nicotinic acetylcholine receptors play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. in insects, nachrs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. however, problems have been encountered in the successful functional expression of insect nachrs, although a number of strategies have been developed in an attempt to overcome such difficulties. ten nachr subunits have been identified in the model insect drosophila melanogaster and a similar number have been identified in other insect species. the focus of the present study is the dα dα and dα subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α nachr subunit.  a full-length cdna clone encoding the drosophila nachr dα subunit has been isolated and the properties of dα5-, dα6- and dα7-containing nachrs examined in a variety of cell expression systems. we have demonstrated the functional expression, as homomeric nachrs, of the dα and dα subunits in xenopus oocytes by their co-expression with the molecular chaperone ric- also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nachr with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. in addition, specific cell-surface binding of -α-bungarotoxin was detected in both drosophila and mammalian cell lines when dα was co-expressed with dα and ric- in contrast, co-expression of additional subunits with dα and dα prevented specific binding of -α-bungarotoxin in cell lines, suggesting that co-assembly with other nachr subunits can block maturation of correctly folded nachrs in some cellular environments.  data are presented demonstrating the ability of the drosophila dα and dα subunits to generate functional homomeric and also heteromeric nachrs. 