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Education  |   December 2011
Use of Concatemers of Ligand-Gated Ion Channel Subunits to Study Mechanisms of Steroid Potentiation
Author Affiliations & Notes
  • Joe Henry Steinbach, Ph.D.
    *
  • Gustav Akk, Ph.D.
  • *Russell and Mary Shelden Professor of Anesthesiology, Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri. Assistant Professor of Anesthesiology, Department of Anesthesiology, Washington University School of Medicine.
Article Information
Education / Pharmacology
Education   |   December 2011
Use of Concatemers of Ligand-Gated Ion Channel Subunits to Study Mechanisms of Steroid Potentiation
Anesthesiology 12 2011, Vol.115, 1328-1337. doi:10.1097/ALN.0b013e318233046a
Anesthesiology 12 2011, Vol.115, 1328-1337. doi:10.1097/ALN.0b013e318233046a
MOST eukaryotic membrane channels are composed of several subunits assembled to make the functional channel, with the notable exceptions of the voltage-gated sodium and calcium channels that are translated as tandem repeats of four modules. The modular basis of the channels increases the diversity of possible functional properties, by allowing mixing and matching of various numbers and types of subunits. However, it also increases the difficulty in determining the molecular mechanisms for drug actions in two ways. First, it becomes necessary to control the number and position of particular subunits in the assembled channel. Otherwise, the concern is always present that a mixture of different subunit arrangements might result in a mixture of pharmacological effects. Second, if multiple copies of a subunit involved in a drug effect are present, it is difficult to distinguish the role or roles of the recognition or transduction regions in the positionally distinct copies. For many anesthetics, there is a third difficulty: they may act to potentiate responses to a transmitter. This mechanism requires that the transmitter and the anesthetic interact with each other through their separate interactions with the receptor. Does this mean that both must bind to the same subunit, or is the interaction mediated by a more global effect of the potentiator on the receptor?
How is it possible to create a receptor with defined subunits in defined places, given the problem that independent subunits assemble to create the final product? An experimental approach to controlling the number and position of subunits in a channel is to generate larger constructs that contain two or more subunits concatenated together. In a concatemer, the DNA coding for two or more separate subunits is covalently linked to result in the translation of a single, larger protein comprising the subunits in a defined linear order (see fig. 1for schematic views of single subunits, the pentameric receptor, and concatemers). This approach was first used to generate dimers of voltage-gated potassium channel subunits.1 Relatively soon after that it was extended to concatemers of γ-aminobutyric acid type A (GABAA) receptor subunits,2 and now has been used in studies of other members of the voltage-gated channel family,3  5 the nicotinic (or ligand-gated) ion channel family,6  9 and some other channel types.10,11 
Fig. 1. The structural features of ligand-gated ion channel family receptors. A  shows a schematic of the primary sequence for all subunits; the amino-terminus is at the left. The amino-terminal extracellular domain contains the loops contributing to the transmitter binding site, A through F. Three transmembrane domains, TM1, TM2, and TM3, then lead to the major cytoplasmic domain, also known as the TM3-TM4 loop. The subunit then ends with a fourth transmembrane domain and a short carboxyl-terminal domain. B  shows the folding of a single subunit with respect to the cell membrane. The orange oval  shows the approximate location of the transmitter-binding site. The second transmembrane domain (shown in red  ) forms the major portion of the ion channel lining. C  shows the arrangement of subunits in the assembled pentameric receptor, viewed from the extracellular side. The hatched circle  in the middle represents the ion channel. The subunits contributing the primary (positive or +) side of the transmitter-binding site are identified as 1o(gold circles  ), whereas the subunits contributing the complementary side are identified as 2oand are blue  . The locations of transmitter-binding sites are indicated by the red diamonds  . The fifth or structural subunit (indicated by X; unfilled circle  ) does not contribute to a transmitter-binding site but impacts the functional properties of the overall receptor and may contain sites for pharmacological agents. D  shows an image of a three-subunit concatemer. The blue-hatched segment  is the signal sequence of the first subunit, the black segments  are the “linkers” connecting subunits, and the subunits are schematized in red  with membrane-spanning regions cross-hatched. The signal sequence is cleaved from the mature subunit and is not present in the assembled receptor. E  shows a simplified version of a two-subunit concatemer in the membrane; other subunits are omitted for clarity. The cylinders outline the subunits, the red lines  indicate the general course of the peptide in the subunit, and the black line  indicates a hypothetical path for a linker to connect the subunits.
Fig. 1. The structural features of ligand-gated ion channel family receptors. A 
	shows a schematic of the primary sequence for all subunits; the amino-terminus is at the left. The amino-terminal extracellular domain contains the loops contributing to the transmitter binding site, A through F. Three transmembrane domains, TM1, TM2, and TM3, then lead to the major cytoplasmic domain, also known as the TM3-TM4 loop. The subunit then ends with a fourth transmembrane domain and a short carboxyl-terminal domain. B 
	shows the folding of a single subunit with respect to the cell membrane. The orange oval 
	shows the approximate location of the transmitter-binding site. The second transmembrane domain (shown in red 
	) forms the major portion of the ion channel lining. C 
	shows the arrangement of subunits in the assembled pentameric receptor, viewed from the extracellular side. The hatched circle 
	in the middle represents the ion channel. The subunits contributing the primary (positive or +) side of the transmitter-binding site are identified as 1o(gold circles 
	), whereas the subunits contributing the complementary side are identified as 2oand are blue 
	. The locations of transmitter-binding sites are indicated by the red diamonds 
	. The fifth or structural subunit (indicated by X; unfilled circle 
	) does not contribute to a transmitter-binding site but impacts the functional properties of the overall receptor and may contain sites for pharmacological agents. D 
	shows an image of a three-subunit concatemer. The blue-hatched segment 
	is the signal sequence of the first subunit, the black segments 
	are the “linkers” connecting subunits, and the subunits are schematized in red 
	with membrane-spanning regions cross-hatched. The signal sequence is cleaved from the mature subunit and is not present in the assembled receptor. E 
	shows a simplified version of a two-subunit concatemer in the membrane; other subunits are omitted for clarity. The cylinders outline the subunits, the red lines 
	indicate the general course of the peptide in the subunit, and the black line 
	indicates a hypothetical path for a linker to connect the subunits.
Fig. 1. The structural features of ligand-gated ion channel family receptors. A  shows a schematic of the primary sequence for all subunits; the amino-terminus is at the left. The amino-terminal extracellular domain contains the loops contributing to the transmitter binding site, A through F. Three transmembrane domains, TM1, TM2, and TM3, then lead to the major cytoplasmic domain, also known as the TM3-TM4 loop. The subunit then ends with a fourth transmembrane domain and a short carboxyl-terminal domain. B  shows the folding of a single subunit with respect to the cell membrane. The orange oval  shows the approximate location of the transmitter-binding site. The second transmembrane domain (shown in red  ) forms the major portion of the ion channel lining. C  shows the arrangement of subunits in the assembled pentameric receptor, viewed from the extracellular side. The hatched circle  in the middle represents the ion channel. The subunits contributing the primary (positive or +) side of the transmitter-binding site are identified as 1o(gold circles  ), whereas the subunits contributing the complementary side are identified as 2oand are blue  . The locations of transmitter-binding sites are indicated by the red diamonds  . The fifth or structural subunit (indicated by X; unfilled circle  ) does not contribute to a transmitter-binding site but impacts the functional properties of the overall receptor and may contain sites for pharmacological agents. D  shows an image of a three-subunit concatemer. The blue-hatched segment  is the signal sequence of the first subunit, the black segments  are the “linkers” connecting subunits, and the subunits are schematized in red  with membrane-spanning regions cross-hatched. The signal sequence is cleaved from the mature subunit and is not present in the assembled receptor. E  shows a simplified version of a two-subunit concatemer in the membrane; other subunits are omitted for clarity. The cylinders outline the subunits, the red lines  indicate the general course of the peptide in the subunit, and the black line  indicates a hypothetical path for a linker to connect the subunits.
×
We have been examining the ability of drugs to potentiate the agonist-elicited responses of receptors, with emphasis on the actions of neuroactive steroids. We will discuss the use of concatemers of subunits from two members of the ligand-gated ion channel family, a GABAAreceptor, and a neuronal nicotinic receptor. Concatemers have allowed us to selectively manipulate steroid- or transmitter-binding sites and thereby determine the role or roles of particular sites and possible interactions among sites in a receptor.
Research Overview
A ligand-gated ion channel gene family receptor is composed of five subunits, arranged in a pseudosymmetric rosette around the centrally located ion channel (fig. 1). There is an extensive N-terminal extracellular domain, which contains the binding sites for transmitters and several drugs. There are then three closely spaced transmembrane domains, followed by a variable length cytoplasmic loop. The subunit then finishes with a fourth transmembrane domain and a short extracellular C-terminal sequence (fig. 1). The major portion of the ion channel is formed by the second transmembrane domains from each of the five subunits. Channel gating is initiated by binding of transmitters to sites in the N-terminal extracellular domain and communicated to the gating region located in the transmembrane domains. Each of the subunits seems to be strongly coupled to the others in the receptor, in the sense that the process of channel opening appears to occur as a single, global step involving all subunits.
The agonist-binding site is located at an interface between two subunits, with each participating subunit contributing three loops. The assembled receptors we will discuss contain two agonist-binding sites per receptor. The conventional nomenclature is that the positive (primary or +) subunit contributes the A, B, and C loops, while the negative (complementary or −) subunit contributes the D, E, and F loops (fig. 1). For neuronal nicotinic receptors, the primary side is contributed by the α subunit and the complementary side by the β subunit. For the GABAAreceptor, the primary side is contributed by the β subunit and the complementary by the α. This discrepancy in nomenclature arose because subunits were initially named based on electrophoretic mobility.
We will discuss the actions of steroids to potentiate the responses of a prototypical GABAAreceptor composed of α1, β2, and γ2L subunits, and a neuronal nicotinic receptor composed of α4 and β2 subunits. These particular receptors are, respectively, the most common form of GABAAreceptor and the most common heteromultimeric neuronal nicotinic receptor in the brain. (We note that some steroids and analogues can inhibit GABAAand/or nicotinic receptors. We will not consider this action.)
Steroids and analogues are well known anesthetic drugs,12  14 and indeed an anesthetic comprising a mixture of alphaxalone and alphadolone was used clinically (Althesin®; Glaxo Laboratories Ltd., Greenford, England). A variety of studies have demonstrated that a number of neuroactive steroids potentiate the responses of GABAAreceptors to low concentrations of the transmitter, γ-aminobutyric acid (GABA).13,15,16 Further, there is a good correlation between the ability of steroids and analogues to anesthetize amphibian tadpoles and their ability to modulate GABAAreceptors,16 supporting the idea that the anesthesia produced by these agents reflects actions at the GABAAreceptor. The mechanism of action has been shown to result from an increase in the stability of the open-channel state of the receptor, rather than an increase in binding affinity or channel opening rate.17 Steroids are very hydrophobic molecules, and studies have indicated that the interaction between a steroid and a GABAAreceptor occurs in the cell membrane.18,19 A major advance was made when a binding site for steroids was identified in the transmembrane regions of the α1 subunit,20 and subsequently in other α subunits.21 It is possible to completely ablate steroid potentiation by mutations to a specific residue, α1 Q241, indicating the importance of this residue.20,22 There are two specific questions we have addressed using subunit concatemers. The first is concerned with the fact that the GABAAreceptor includes two copies of the α1 subunit, each containing a steroid-binding site: Does a steroid need to bind to both sites to potentiate, and are the sites equivalent? The second is concerned with the fact that potentiation reflects binding of both steroid and agonist: Does the same subunit need to participate in both binding interactions, or is potentiation a distributed response of the entire receptor?
The nicotinic α4β2 receptor is potentiated by the endogenous steroid, 17β-estradiol (although most steroids and analogues inhibit this receptor, by a distinct mechanism23,24). The mechanism for potentiation is not known, but it seems likely that it reflects stabilization of the open state.25 However, it is known that 17β-estradiol interacts with a specific sequence of four amino acids at the extreme C-terminus of the α4 subunit.24,25 We asked similar questions in studies of this receptor: Does the C-terminal domain need to be on a specific subunit, does the subunit need to participate in agonist binding, and does the copy number of domains affect potentiation?
Concatemers
The basic portions of a concatemer are the two or more subunits that are joined together and the linker or linkers, or the new protein sequence that is introduced to connect the subunits.
How to make a concatemer? Our approach has been to use constructs first made by others with only minor modifications, so these comments reflect the insights gained in their work. It is very helpful that both ends (the amino- and carboxy-termini) of these subunits are on the same side of the membrane (fig. 1), the extracellular side, which avoids problems of introducing (or removing) a membrane-spanning region. However, when initially translated each subunit has a “signal sequence” at the N-terminal end. The signal sequence assists in membrane insertion of the subunit during translation in the endoplasmic reticulum, and is subsequently cleaved so it is not present in the mature receptor. In the first concatemers, the signal sequence(s) were retained for all subunits in the concatemer, which resulted in a hybrid linker in which part was new protein and part was the existing signal sequence.2,7,9 Most subsequent work has removed all the internal signal sequences, from all subunits except the first.6,8,26 One comparison reported that removing internal signal sequences resulted in more normal pharmacological properties in pentameric concatemers.26 
The next question is what linker sequence should be used, and what secondary structure should it adopt? In general, the goal is to achieve a random coil, with the idea that this would place the least structural constraint on the concatemers as they assemble. The first linkers were simple repeats of glutamine residues.2 Subsequently, linkers incorporating repeats of other amino acids have been used (e.g.  , alanine-glycine-serine triplets).8 The rationale for avoiding long repeats of identical amino acids is that such tracts might result in local depletion of transfer RNA molecules and possible early termination of synthesis.
Finally, how long should the linking sequence be? A linker that is too short reduces expression of functional receptors, as might be expected if a short linker caused structural deformations in the subunit that prevent correct assembly.6,8,27 However, it appears that too long a linker can allow more variability in subunit arrangement in the pentamer.8,27 The best length seems to depend on the particular subunits being linked. Typically, the total length is estimated from the end of the fourth transmembrane region to the start of the following subunit mature sequence, and usually is in the range of 20 to 40 amino acid residues for well-behaved constructs.
A number of concatemers of differing length were produced and used in various laboratories, dimers,2,6  8 trimers,27,28 and full pentamers of subunits.26,27,29,30 The full pentamer allows the most complete definition of subunit composition, but shorter concatemers have value in terms of ease of generation and manipulation, and can be preferred for some experiments. In our laboratories we have used dimers and trimers of subunits, as we will describe.
Two general types of studies have used concatemers. The first type has the objective of defining the properties of receptors with a defined number and arrangement of subunits. In this case, concatemers are produced that incorporate the subunits in particular orders. This approach has been particularly valuable in providing models for possible endogenous receptors whose stoichiometry is unknown.27,31,32 For example, in the case of neuronal nicotinic receptors, it has provided the first evidence that receptors containing the α6 and β3 subunits can be studied in defined receptors.27 It has also produced some surprising results indicating that so-called “accessory” subunits, or subunits that are not thought to contribute to transmitter-binding interfaces, may assemble in unpredicted ways.31,32 In the case of the GABAAreceptor, the γ subunit is thought to assemble with 1 copy per receptor and to not contribute to a GABA-binding site. However, both the δ and ε subunits apparently can replace not only the γ subunit in the receptor, but also an α or a β subunit.31,32 
The second type of experiment has the objective of defining the physiologic or pharmacological properties of specific subunits in a receptor, such as the roles of the two primary subunits in transmitter binding.33 In this case, mutations are made in specific subunits in the concatemer to probe the similarities or differences in consequences for the overall receptor function. Our work so far has focused on this second area. One previous report has been made using concatemers to study receptor modulation, of the interaction between the benzodiazepine-binding site and the GABA-binding sites in GABAAreceptors.34 In this study, benzodiazepine binding could potentiate responses elicited by GABA binding to either GABA-binding site.
The end result is that several laboratories have succeeded in expressing functional receptors using subunit concatemers. The most successful expression has been in Xenopus oocytes. In general, concatemers express more poorly in other systems, such as HEK293 cells, although it has been possible to study some concatemers of GABAAsubunits in nonoocyte systems.9,29,35 The mechanisms determining the surface expression of membrane channels in different expression systems are not fully understood. Xenopus oocytes seem to be particularly adapted for the efficient expression of many proteins, as might be expected based on the extensive burst of initial development they undergo upon fertilization. In contrast, somatic cells are differentiated, to a greater or lesser extent. Indeed, it is known that coexpression of chaperone proteins can enhance expression of particular receptors; for example, the RIC3 protein in the case of the nicotinic α7 receptor36,37 or 14–3-3 protein for the nicotinic α4β2 receptor.26,38 In other cases, mutation of so-called “retention sequences,” which tend to reduce receptor trafficking from the initial site of synthesis to the surface membrane, can enhance surface expression.39 
Drawbacks to Using Concatemers
The first possible drawback to the use of concatemers is that the resulting receptors may not actually include the expected complement of subunits. This was recognized during studies of voltage-gated potassium channels.40 One obvious example of this problem is the finding that expression of a dimer or a trimer construct of a ligand-gated ion channel subunit can, by itself, result in the expression of functional channels.8 This is a problem, as the functional assembled receptor contains five subunits. Examination of the expressed protein, using immunoblotting, indicated that the concatemers are not degraded to separate monomeric subunits, so the question arises as to the location of excluded subunits. For nicotinic α4β2 receptors, it appeared that at least some of the receptors formed when a two-subunit concatemer was expressed were actually dimers of pentamers with a bridge contributed by one of the concatemers.8 In other cases, it has been proposed that some subunits may be in an undefined conformation that is “looped out” from the assembled receptor:7 A functional receptor is formed by five of the subunits assembling while the extra subunit is outside the pentamer. Other examples of unexpected assemblies have been identified by using “reporter” mutations, which result in a defined change in receptor function or pharmacology. The reporter mutation is made in a specific subunit in a concatemer to determine whether that subunit is functionally present in the receptor. In some cases, it appears that a subunit may not be expressed in the assembled receptor.7 The properties of defined receptors with these proposed abnormal subunit locations have not been extensively studied, so it is not clear how different they are from normal receptors. Instead, the focus of most research has been on defining conditions in which biochemical and functional tests indicate that the assembled receptors have the expected subunit stoichiometry.
The second possible drawback is that placing subunits in a concatemer may alter the physiologic or pharmacological properties of the resulting receptor. It has been reported that concatemers of GABAAreceptors often have a shift in the concentraton dependence for activation by GABA, requiring a higher concentration.28,41 For nicotinic α4β2 receptors, making a concatemer linked to the carboxy-terminus of the α4 subunit removed potentiation by 17β-estradiol.8,42 This is because the action of 17β-estradiol requires a free carboxy-terminal. However, in both of these cases other properties of the receptors were normal (see Neuronal Nicotinic receptors: potentiation by 17β-estradiol and GABAAReceptors: Potentiation by Neurosteroids).
What these observations require, then, is that any investigator who uses concatemers must perform the necessary controls to demonstrate that the concatemers are structurally intact, have reasonable functional and pharmacological properties, and behave in a consistent fashion.
The generation, properties, and uses of concatemers have been reviewed elsewhere, for those interested in pursuing the topic further.43  46 Although there are some clear artifacts that can arise in the use of concatemers, overall they provide the most direct means to answer some types of questions, as we will discuss in the rest of this article.
Neuronal Nicotinic Receptors: Potentiation by 17β-estradiol
The endogenous steroid 17β-estradiol potentiates activation of the nicotinic α4β2 receptor. Previous work has shown that the final four amino acid residues of the α4 subunit are required,24,25 and that potentiation is very sensitive to the location of these residues. For example, adding a single residue at the end abolishes potentiation, as does insertion or removal of a single residue present between the end of the fourth transmembrane region and the 17β-estradiol domain.24 This spatial sensitivity suggested that the domain had to be located in a precise location in the assembled receptor, and led to the hypotheses that the domain had to be on the α4 subunit and the α4 subunit that bound the steroid had to participate in binding of acetylcholine, contributing the primary side of the binding site.
We used concatemers containing two subunits, one α4 and one β2 subunit, to test these hypotheses.42 Two concatemers were produced, one with a β2 subunit at the amino-terminal end (β2-α4), and the other with α4 (α4-β2). The concatemers were based on the work of Zhou et al.  ,8 and we measured voltage-clamped responses from receptors expressed in Xenopus oocytes.42 Receptors were expressed by injecting a concatemer with a free subunit. We first confirmed that the receptors formed in this way had the properties expected from studies of free subunits; for example, by expressing a concatemer with free α4 subunit, so the resulting receptor had properties of a receptor containing three copies of the α4 subunit. Furthermore, we demonstrated that we could place a mutated subunit selectively in either an acetylcholine-binding or the structural position (fig. 2), using the two concatemers. The data clearly demonstrated that the functional, pentameric receptors assembled when a dimer was expressed with a free subunit behaved in the fashion expected for a receptor with two copies of the concatemer and one copy of the monomer.
Fig. 2. Studies of 17β-estradiol potentiation of nicotinic α4β2 receptors. A  and C  show an image of the pentameric receptors assembled when a concatemer assembles with a monomeric subunit, viewed from the extracellular side. The direction of the linker (amino- to carboxyl-terminus) is indicated by the arrowheads  , and the position of the free subunit is shown by the clear circle  . In the case of the α4-β2 concatemer, the free subunit (white  color, marked with “ACh”) assembles as a subunit contributing to a transmitter-binding site (shown by the gray diamonds  ); if the free subunit is β2, it contributes the complementary side, whereas if it is α4, it contributes the primary side. With the β2-α4 concatemer, both transmitter-binding sites are inside the concatemer copies, and the free subunit assembles in the structural position (“str”). B  shows the potentiation produced by 10 μM 17β-estradiol for sets of selected combinations of concatemers and free subunits. In each combination there were either zero (bar  labeled “none”) or only a single copy of the carboxyl-terminal binding domain (all other bars  ). For the middle pair of bars  , the single domain was on an α4 subunit or on a β2 subunit. The mean potentiation was slightly larger when the domain was placed on a β2 subunit. For the upper pair of bars  , potentiation was very similar when the domain was on an agonist-binding subunit or on a structural subunit. The data are the means of the average potentiation for each combination, +1 SEM. The legend to the right of each bar  gives the probability that the observed potentiation differs from 1 (where 1 indicates no potentiation; ns P  > 0.05, *P  < 0.05, ***P  < 0.001), followed by the number of combinations tested (e.g.  , a particular concatemer expressed with a particular free subunit) and the total number of oocytes studied for all combinations. D  shows the maximal potentiation ratio for 17β-estradiol as a function of the total number of binding domains in the pentamer. Combinations of constructs were chosen that would result in one, two, three, or five intact binding domains in the pentamer (two combinations for each number), and the concentration-effect relationship for potentiation by 17β-estradiol was determined. The fit maximum potentiation was estimated for each oocyte. The data are the mean maximal potentiation for each number of intact domains (five or six oocytes for each combination, two combinations per point) ±1 SE. The curves  show the best-fitting straight line (black dashed line  ) or geometric function (blue solid line  ), with ±1 SE (thin dashed lines  ). The geometric fit is significantly better than the linear fit (P  = 0.02, F test). For additional information, see the full publication.42 
Fig. 2. Studies of 17β-estradiol potentiation of nicotinic α4β2 receptors. A 
	and C 
	show an image of the pentameric receptors assembled when a concatemer assembles with a monomeric subunit, viewed from the extracellular side. The direction of the linker (amino- to carboxyl-terminus) is indicated by the arrowheads 
	, and the position of the free subunit is shown by the clear circle 
	. In the case of the α4-β2 concatemer, the free subunit (white 
	color, marked with “ACh”) assembles as a subunit contributing to a transmitter-binding site (shown by the gray diamonds 
	); if the free subunit is β2, it contributes the complementary side, whereas if it is α4, it contributes the primary side. With the β2-α4 concatemer, both transmitter-binding sites are inside the concatemer copies, and the free subunit assembles in the structural position (“str”). B 
	shows the potentiation produced by 10 μM 17β-estradiol for sets of selected combinations of concatemers and free subunits. In each combination there were either zero (bar 
	labeled “none”) or only a single copy of the carboxyl-terminal binding domain (all other bars 
	). For the middle pair of bars 
	, the single domain was on an α4 subunit or on a β2 subunit. The mean potentiation was slightly larger when the domain was placed on a β2 subunit. For the upper pair of bars 
	, potentiation was very similar when the domain was on an agonist-binding subunit or on a structural subunit. The data are the means of the average potentiation for each combination, +1 SEM. The legend to the right of each bar 
	gives the probability that the observed potentiation differs from 1 (where 1 indicates no potentiation; ns P 
	> 0.05, *P 
	< 0.05, ***P 
	< 0.001), followed by the number of combinations tested (e.g. 
	, a particular concatemer expressed with a particular free subunit) and the total number of oocytes studied for all combinations. D 
	shows the maximal potentiation ratio for 17β-estradiol as a function of the total number of binding domains in the pentamer. Combinations of constructs were chosen that would result in one, two, three, or five intact binding domains in the pentamer (two combinations for each number), and the concentration-effect relationship for potentiation by 17β-estradiol was determined. The fit maximum potentiation was estimated for each oocyte. The data are the mean maximal potentiation for each number of intact domains (five or six oocytes for each combination, two combinations per point) ±1 SE. The curves 
	show the best-fitting straight line (black dashed line 
	) or geometric function (blue solid line 
	), with ±1 SE (thin dashed lines 
	). The geometric fit is significantly better than the linear fit (P 
	= 0.02, F test). For additional information, see the full publication.42
Fig. 2. Studies of 17β-estradiol potentiation of nicotinic α4β2 receptors. A  and C  show an image of the pentameric receptors assembled when a concatemer assembles with a monomeric subunit, viewed from the extracellular side. The direction of the linker (amino- to carboxyl-terminus) is indicated by the arrowheads  , and the position of the free subunit is shown by the clear circle  . In the case of the α4-β2 concatemer, the free subunit (white  color, marked with “ACh”) assembles as a subunit contributing to a transmitter-binding site (shown by the gray diamonds  ); if the free subunit is β2, it contributes the complementary side, whereas if it is α4, it contributes the primary side. With the β2-α4 concatemer, both transmitter-binding sites are inside the concatemer copies, and the free subunit assembles in the structural position (“str”). B  shows the potentiation produced by 10 μM 17β-estradiol for sets of selected combinations of concatemers and free subunits. In each combination there were either zero (bar  labeled “none”) or only a single copy of the carboxyl-terminal binding domain (all other bars  ). For the middle pair of bars  , the single domain was on an α4 subunit or on a β2 subunit. The mean potentiation was slightly larger when the domain was placed on a β2 subunit. For the upper pair of bars  , potentiation was very similar when the domain was on an agonist-binding subunit or on a structural subunit. The data are the means of the average potentiation for each combination, +1 SEM. The legend to the right of each bar  gives the probability that the observed potentiation differs from 1 (where 1 indicates no potentiation; ns P  > 0.05, *P  < 0.05, ***P  < 0.001), followed by the number of combinations tested (e.g.  , a particular concatemer expressed with a particular free subunit) and the total number of oocytes studied for all combinations. D  shows the maximal potentiation ratio for 17β-estradiol as a function of the total number of binding domains in the pentamer. Combinations of constructs were chosen that would result in one, two, three, or five intact binding domains in the pentamer (two combinations for each number), and the concentration-effect relationship for potentiation by 17β-estradiol was determined. The fit maximum potentiation was estimated for each oocyte. The data are the mean maximal potentiation for each number of intact domains (five or six oocytes for each combination, two combinations per point) ±1 SE. The curves  show the best-fitting straight line (black dashed line  ) or geometric function (blue solid line  ), with ±1 SE (thin dashed lines  ). The geometric fit is significantly better than the linear fit (P  = 0.02, F test). For additional information, see the full publication.42 
×
The α4 and β2 subunits have one or two proline residues at the end of the fourth transmembrane region, which allowed a reference point for the transfer of the carboxy-terminal β-estradiol domain.
The results show that neither of our hypotheses is correct. We found that we could move the β-estradiol domain to a single β2 subunit, eliminate it on all α4 subunits, and have strong potentiation by 17β-estradiol (fig. 2B), so our first hypothesis is disproven. We also found that the domain could be on an acetylcholine-binding subunit or the structural subunit (either a β2 or an α4 subunit) with essentially equivalent potentiation (fig. 2B), so our second hypothesis is disproven. We could then construct receptors with various numbers of domains included in the pentamer: zero, one, two, three, or five. The estimated maximal potentiation increased more than linearly with the numbers of domains (fig. 2C).
What interpretations do we make of these data? The first is that 17β-estradiol does not have to interact with a subunit that also binds acetylcholine. This observation indicates that potentiation involves a process that affects the receptor as a whole. The second is that, although the domain is spatially very constrained, potentiation does not seem to require a particular subunit on either side of the subunit containing the domain. This suggests that the mechanism of potentiation only involves the single subunit that contains the estradiol domain. Finally, the maximal potentiation increases about 1.6-fold with each added copy of the domain. A geometric increase is consistent with a simple idea for the mechanism of potentiation. This idea is that when 17β-estradiol binds to the C-terminal domain on a specific subunit, it results in an alteration of the structure of that subunit, which stabilizes the open-channel state of the entire receptor by adding a stabilizing energy. If 17β-estradiol binds to another subunit in the same receptor, then there is the addition of an identical energy to stabilize the open state. Each additive energy contribution results in a multiplicative change in potentiation, because the total energy change is exponentiated to result in the change in equilibrium constant. The multiplicative increase in potentiation results from independent energetic contributions from each subunit. Because of the concerted nature of gating for the ligand-gated ion channels, these independent contributions from subunits have a global effect on receptor activation.
GABAAReceptors: Potentiation by Neurosteroids
A number of steroids potentiate activation of GABAAreceptors.15,16 The work by Hosie et al.  20 has shown that the amino acid residue at position 241 in the α1 subunit (α1 Q241) forms an essential part of the steroid-binding site. We initially explored the consequences of the fact that there are two copies of the α1 subunit in the assembled receptor.41 Does a steroid need to bind to more than one subunit to potentiate the receptor, and do the two steroid-binding sites have different pharmacological properties?
We constructed two concatemers, following the work of Baumann et al.  28 One contained β2-α1 subunits, and the other γ2L-β2-α1 subunits (fig. 3A). The two concatemers were expressed together, and assembled to form functional receptors. The results indicate that the presence of a single normal site conferred steroid potentiation (fig. 3B). When both sites were mutated, potentiation was lost. We tested the pharmacological properties of the sites by comparing the abilities of a 5α-reduced steroid and a 5β-reduced steroid to potentiate, and found that there was no selective effect on potentiation (fig. 3B). We then extended these observations by expressing receptors containing concatemers in HEK cells and examining single-channel currents elicited by GABA and modulated by steroids.35 The single-channel properties of receptors containing concatemers of wild-type subunits were qualitatively identical to those seen with receptors formed from free subunits. The only quantitative difference was a reduction in the affinity of the resting receptor for GABA, which is also reflected in the finding that the concentration of GABA needed to produce a half-maximal whole cell response is also increased for receptors containing these concatemers.28,35,41 For the wild-type concatemers, potentiation by the steroid allopregnanolone showed the same kinetic effects that we had seen in receptors composed of free subunits. We also obtained results for the receptors with the α1Q241L mutation in the γ-β-α concatemer; again, potentiation by allopregnanolone showed the same kinetic effects as for free subunits with intact steroid-binding sites. (Several of the concatemers expressed at such low levels that it was not possible to obtain adequate numbers of single-channel events.) Overall, the studies of single-channel currents indicate that the receptors composed of concatemers have normal functional properties and modulation by neurosteroids. The studies of whole cell responses demonstrate that the sites in the two α1 subunits have indistinguishable pharmacological properties, and that either can support potentiation by steroids. The results indicate that there is only a small decrease in the maximal potentiation produced by steroids when one site is removed.
Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A  shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B  shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars  ) and 5βTHDOC (blue bars  ), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars  show data for receptors with both steroid-binding sites intact; the top pair  shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs  show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar  gives the probability that the potentiation differs from 1 (ns P  > 0.05, *P  < 0.05, **P  < 0.01, ***P  < 0.001) and the number of oocytes studied. For additional information, see the full publication.41 C  shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D  summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars  summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair  is for combinations with all sites intact or with both steroid sites mutated. The middle group of three  shows combinations for which the steroid site in the “a” pair is intact, while the upper group  shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B  . For additional information, see the full publication.47 GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.
Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A 
	shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B 
	shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars 
	) and 5βTHDOC (blue bars 
	), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars 
	show data for receptors with both steroid-binding sites intact; the top pair 
	shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs 
	show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar 
	gives the probability that the potentiation differs from 1 (ns P 
	> 0.05, *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001) and the number of oocytes studied. For additional information, see the full publication.41C 
	shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D 
	summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars 
	summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair 
	is for combinations with all sites intact or with both steroid sites mutated. The middle group of three 
	shows combinations for which the steroid site in the “a” pair is intact, while the upper group 
	shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B 
	. For additional information, see the full publication.47GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.
Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A  shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B  shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars  ) and 5βTHDOC (blue bars  ), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars  show data for receptors with both steroid-binding sites intact; the top pair  shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs  show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar  gives the probability that the potentiation differs from 1 (ns P  > 0.05, *P  < 0.05, **P  < 0.01, ***P  < 0.001) and the number of oocytes studied. For additional information, see the full publication.41 C  shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D  summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars  summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair  is for combinations with all sites intact or with both steroid sites mutated. The middle group of three  shows combinations for which the steroid site in the “a” pair is intact, while the upper group  shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B  . For additional information, see the full publication.47 GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.
×
We then set out to answer a second question.47 Potentiation requires binding of both transmitter and potentiator. Is there a “privileged” relationship for particular subunits? For example, is potentiation enhanced when steroid binds to a subunit that also binds with a transmitter, or does potentiation apply equally to activation elicited by binding to either transmitter-binding site? For these studies we used a different pair of concatemers, one containing β2-α1 and the other β2-α1-γ2L (fig. 3C). We selectively ablated the GABA-binding site by the mutation β2Y205S48 and the steroid site by α1Q241L. The mutated constructs were expressed in all possible combinations (fig. 3D) to examine interactions between steroid- and transmitter-binding sites, using the neurosteroid allopregnanolone to modulate GABA-elicited responses. The results did not show any significant coupling between the GABA- and steroid-binding sites on a particular subunit (fig. 3D). Potentiation was seen whether the GABA- and steroid-binding occurred in the same α subunit or not. These results indicate that neurosteroid potentiation of GABAAreceptors is mediated by a generalized effect on the entire receptor.
It is interesting to note that it was possible to activate receptors in which both GABA-binding sites were ablated by using pentobarbital.48 The data47 indicated that either steroid site was able to potentiate responses elicited by pentobarbital, as in the case of GABA. Since the binding site for pentobarbital is presently unknown, it was not possible to examine interactions between pentobarbital and allopregnanolone.
Conclusions
Steroids, either neurosteroids acting on the GABAAreceptor or 17β-estradiol acting on the nicotinic α4β2 receptor, appear to act by affecting the gating properties of the receptor as a whole. Potentiation does not require that the subunit that binds a steroid also binds an agonist. For the nicotinic receptor, the steroid-binding domain is a remarkably discrete element that can be moved from one subunit to another. This may be analogous to our finding that the two copies of the α1 subunit in the GABAAreceptor are essentially equivalent to each other. One difference between the nicotinic and the GABAAreceptors is that potentiation increases steadily as we increase the number of copies of the binding domain in the nicotinic receptor, but does not change consistently when we reduce the number of intact domains in the GABAAreceptor from two to one.
Steroid potentiation is mediated by interactions in or very near to the membrane-spanning regions of these receptors. It is already known that there are mutations in the second transmembrane region that enhance the stability of the open channel state.49  51 It is interesting that these mutations appear to act irrespective of the subunit in which they are placed, either transmitter-binding or structural, and that the energetic contributions to stabilizing the open state appear to add. It is possible that the mechanism of steroid potentiation is to directly stabilize the channel in the open channel conformation by an effect on transmembrane domain interactions, either with other channel domains or with the surrounding lipid.
The results confirm that concatemers of subunits can be used to define the number and position of subunits in assembled and functional ligand-gated ion channel family receptors. By using concatemers, we have been able to ask and answer questions about the nature of steroid-interacting sites on these receptors and how interactions between potentiating drugs and transmitters may occur.
Potentiation of GABAAreceptors by steroids underlies their anesthetic actions. Neurosteroid potentiation is potent and efficacious, particularly for responses resulting from relatively low activation levels, by low concentrations of GABA or by receptors that have a low maximal activation. Potentiation reflects a global, receptor-wide effect, and the presence of multiple homologous binding sites in a single receptor provides an efficient and redundant mechanism for mediating steroid effects.
Future Directions
We are quite excited by the possibilities for future work that are opened up by the use of concatemers. In studies of anesthetic actions, there are several general areas we think are ripe for study. All of these will focus on the GABAAreceptor, as the more relevant target for studies of anesthetics.
The first is the study of additional classes of anesthetics. Other anesthetics have identified binding regions in the GABAAreceptor,52  56 which are present on subunits that have two copies in the assembled receptor. Analogous studies to the ones we have performed with anesthetic steroids will help to clarify the properties of individual anesthetic sites and interactions with GABA-binding sites.
Another is to extend studies using voltage-clamp fluorometry.57 By introducing the reporter fluorophore into specific locations in particular subunits, we hope to be able to examine the propagation of conformational changes induced by anesthetics.
Finally, concatemers provide the opportunity to study receptors of defined subunit stoichiometry and arrangement. Recent experiments31,32,58 have demonstrated that some subunits, notably the δ and ε subunits, can assemble in different positions in the pentamer and may be able to incorporate with differing numbers of copies per receptor. Receptors containing these subunits often contribute to nonsynaptic, “tonic” GABAergic responses. We plan to extend our studies of anesthetic actions to nonsynaptic receptors, but to do so we need to have a defined population of receptors. The use of concatemers provides a path to this goal.
References
Isacoff EY, Jan YN, Jan LY: Evidence for the formation of heteromultimeric potassium channels in Xenopus  oocytes. Nature 1990; 345:530–4
Im WB, Pregenzer JF, Binder JA, Dillon GH, Alberts GL: Chloride channel expression with the tandem construct of α6-β2 GABAAreceptor subunit requires a monomeric subunit of α6 or γ2. J Biol Chem 1995; 270:26063–6
Varnum MD, Zagotta WN: Subunit interactions in the activation of cyclic nucleotide-gated ion channels. Biophys J 1996; 70:2667–79
Wimmers S, Bauer CK, Schwarz JR: Biophysical properties of heteromultimeric erg K+channels. Pflugers Arch 2002; 445:423–30
Wang R, Zhang X, Cui N, Wu J, Piao H, Wang X, Su J, Jiang C: Subunit-stoichiometric evidence for kir6.2 channel gating, ATP binding, and binding-gating coupling. Mol Pharmacol 2007; 71:1646–56
Baumann SW, Baur R, Sigel E: Subunit arrangement of γ-aminobutyric acid type A receptors. J Biol Chem 2001; 276:36275–80
Groot-Kormelink PJ, Broadbent SD, Boorman JP, Sivilotti LG: Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors. J Gen Physiol 2004; 123:697–708
Zhou Y, Nelson ME, Kuryatov A, Choi C, Cooper J, Lindstrom J: Human α4β2 acetylcholine receptors formed from linked subunits. J Neurosci 2003; 23:9004–15
Boileau AJ, Pearce RA, Czajkowski C: Tandem subunits effectively constrain GABAAreceptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAAreceptor-associated protein. J Neurosci 2005; 25:11219–30
Zerhusen B, Zhao J, Xie J, Davis PB, Ma J: A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules. J Biol Chem 1999; 274:7627–30
Schorge S, Elenes S, Colquhoun D: Maximum likelihood fitting of single channel NMDA activity with a mechanism composed of independent dimers of subunits. J Physiol 2005; 569:395–418
Selye H: The anesthetic effects of steroid hormones. Proc Soc Exp Biol Med 1941; 46:116–21
Harrison NL, Vicini S, Barker JL: A steroid anesthetic prolongs inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurosci 1987; 7:604–9
Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM: Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 1986; 232:1004–7
Lambert JJ, Cooper MA, Simmons RD, Weir CJ, Belelli D: Neurosteroids: Endogenous allosteric modulators of GABAAreceptors. Psychoneuroendocrinology 2009; 34(Suppl 1):S48–58
Akk G, Covey DF, Evers AS, Steinbach JH, Zorumski CF, Mennerick S: Mechanisms of neurosteroid interactions with GABAAreceptors. Pharmacol Ther 2007; 116:35–57
Akk G, Bracamontes JR, Covey DF, Evers A, Dao T, Steinbach JH: Neuroactive steroids have multiple actions to potentiate GABAAreceptors. J Physiol 2004; 558:59–74
Akk G, Shu HJ, Wang C, Steinbach JH, Zorumski CF, Covey DF, Mennerick S: Neurosteroid access to the GABAAreceptor. J Neurosci 2005; 25:11605–13
Li P, Shu HJ, Wang C, Mennerick S, Zorumski CF, Covey DF, Steinbach JH, Akk G: Neurosteroid migration to intracellular compartments reduces steroid concentration in the membrane and diminishes GABAAreceptor potentiation. J Physiol 2007; 584:789–800
Hosie AM, Wilkins ME, da Silva HM, Smart TG: Endogenous neurosteroids regulate GABAAreceptors through two discrete transmembrane sites. Nature 2006; 444:486–9
Hosie AM, Clarke L, da Silva H, Smart TG: Conserved site for neurosteroid modulation of GABAAreceptors. Neuropharmacology 2009; 56:149–54
Li P, Covey DF, Steinbach JH, Akk G: Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant α1β2γ2 GABAAreceptors. Mol Pharmacol 2006; 69:2015–26
Paradiso K, Sabey K, Evers AS, Zorumski CF, Covey DF, Steinbach JH: Steroid inhibition of rat neuronal nicotinic α4β2 receptors expressed in HEK 293 cells. Mol Pharmacol 2000; 58:341–51
Paradiso K, Zhang J, Steinbach JH: The C terminus of the human nicotinic α4β2 receptor forms a binding site required for potentiation by an estrogenic steroid. J Neurosci 2001; 21:6561–8
Curtis L, Buisson B, Bertrand S, Bertrand D: Potentiation of human α4β2 neuronal nicotinic acetylcholine receptor by estradiol. Mol Pharmacol 2002; 61:127–35
Carbone AL, Moroni M, Groot-Kormelink PJ, Bermudez I: Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)(β2)(2) nicotinic acetylcholine receptors: Subunit arrangement determines functional expression. Br J Pharmacol 2009; 156:970–81
Kuryatov A, Lindstrom J: Expression of functional human α6β2β3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers. Mol Pharmacol 2011; 79:126–40
Baumann SW, Baur R, Sigel E: Forced subunit assembly in α1β2γ2 GABAAreceptors. Insight into the absolute arrangement J Biol Chem 2002; 277:46020–5
Baur R, Minier F, Sigel E: A GABAAreceptor of defined subunit composition and positioning: Concatenation of five subunits. FEBS Lett 2006; 580:1616–20
Groot-Kormelink PJ, Broadbent S, Beato M, Sivilotti LG: Constraining the expression of nicotinic acetylcholine receptors by using pentameric constructs. Mol Pharmacol 2006; 69:558–63
Bollan KA, Baur R, Hales TG, Sigel E, Connolly CN: The promiscuous role of the ε subunit in GABAAreceptor biogenesis. Mol Cell Neurosci 2008; 37:610–21
Baur R, Kaur KH, Sigel E: Diversity of structure and function of α1α6β3δ GABAAreceptors: Comparison with α1β3δ and α6β3δ receptors. J Biol Chem 2010; 285:17398–405
Baumann SW, Baur R, Sigel E: Individual properties of the two functional agonist sites in GABAAreceptors. J Neurosci 2003; 23:11158–66
Baur R, Sigel E: Benzodiazepines affect channel opening of GABAAreceptors induced by either agonist binding site. Mol Pharmacol 2005; 67:1005–8
Akk G, Li P, Bracamontes J, Steinbach JH: Activation and modulation of concatemeric GABAAreceptors expressed in human embryonic kidney cells. Mol Pharmacol 2009; 75:1400–11
Halevi S, McKay J, Palfreyman M, Yassin L, Eshel M, Jorgensen E, Treinin M: The C. elegans  ric-3 gene is required for maturation of nicotinic acetylcholine receptors. Embo J 2002; 21:1012–20
Lansdell SJ, Gee VJ, Harkness PC, Doward AI, Baker ER, Gibb AJ, Millar NS: RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol Pharmacol 2005; 68:1431–8
Jeanclos EM, Lin L, Treuil MW, Rao J, DeCoster MA, Anand R: The chaperone protein 14–3-3eta interacts with the nicotinic acetylcholine receptor α4 subunit. Evidence for a dynamic role in subunit stabilization J Biol Chem 2001; 276:28281–90
Srinivasan R, Pantoja R, Moss FJ, Mackey ED, Son CD, Miwa J, Lester HA: Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via  stoichiometry-dependent chaperoning. J Gen Physiol 2011; 137:59–79
McCormack K, Lin L, Iverson LE, Tanouye MA, Sigworth FJ: Tandem linkage of Shaker K+channel subunits does not ensure the stoichiometry of expressed channels. Biophys J 1992; 63:1406–11
Bracamontes JR, Steinbach JH: Steroid interaction with a single potentiating site is sufficient to modulate GABAAreceptor function. Mol Pharmacol 2009; 75:973–81
Jin X, Steinbach JH: A portable site: A binding element for 17β-estradiol can be placed on any subunit of a nicotinic α4β2 receptor. J Neurosci 2011; 31:5045–54
Sack JT, Shamotienko O, Dolly JO: How to validate a heteromeric ion channel drug target: Assessing proper expression of concatenated subunits. J Gen Physiol 2008; 131:415–20
Barrera NP, Edwardson JM: The subunit arrangement and assembly of ionotropic receptors. Trends Neurosci 2008; 31:569–76
White MM: Pretty subunits all in a row: Using concatenated subunit constructs to force the expression of receptors with defined subunit stoichiometry and spatial arrangement. Mol Pharmacol 2006; 69:407–10
Minier F, Sigel E: Techniques: Use of concatenated subunits for the study of ligand-gated ion channels. Trends Pharmacol Sci 2004; 25:499–503
Bracamontes J, McCollum M, Esch C, Li P, Ann J, Steinbach JH, Akk G: Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAAreceptor induced from either transmitter binding site. Mol Pharmacol 2011; 80:79–86
Amin J, Weiss DS: GABAAreceptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital. Nature 1993; 366:565–9
Revah F, Bertrand D, Galzi JL, Devillers-Thiéry A, Mulle C, Hussy N, Bertrand S, Ballivet M, Changeux JP: Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 1991; 353:846–9
Chang Y, Weiss DS: Allosteric activation mechanism of the α1β2γ2 γ-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine. Biophys J 1999; 77:2542–51
Moroni M, Zwart R, Sher E, Cassels BK, Bermudez I: α4β2 nicotinic receptors with high and low acetylcholine sensitivity: Pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine. Mol Pharmacol 2006; 70:755–68
Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MA, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL: Sites of alcohol and volatile anaesthetic action on GABAAand glycine receptors. Nature 1997; 389:385–9
Krasowski MD, Nishikawa K, Nikolaeva N, Lin A, Harrison NL: Methionine 286 in transmembrane domain 3 of the GABAAreceptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. Neuropharmacology 2001; 41:952–64
Bali M, Akabas MH: Defining the propofol binding site location on the GABAAreceptor. Mol Pharmacol 2004; 65:68–76
Li GD, Chiara DC, Sawyer GW, Husain SS, Olsen RW, Cohen JB: Identification of a GABAAreceptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J Neurosci 2006; 26:11599–605
Belelli D, Lambert JJ, Peters JA, Wafford K, Whiting PJ: The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci U S A 1997; 94:11031–6
Akk G, Steinbach LH: Structural studies of the actions of anesthetic drugs on the γ-aminobutyric acid type A receptor. ANESTHESIOLOGY. 2011; in press
Boileau AJ, Li T, Benkwitz C, Czajkowski C, Pearce RA: Effects of γ2S subunit incorporation on GABAAreceptor macroscopic kinetics. Neuropharmacology 2003; 44:1003–12
Fig. 1. The structural features of ligand-gated ion channel family receptors. A  shows a schematic of the primary sequence for all subunits; the amino-terminus is at the left. The amino-terminal extracellular domain contains the loops contributing to the transmitter binding site, A through F. Three transmembrane domains, TM1, TM2, and TM3, then lead to the major cytoplasmic domain, also known as the TM3-TM4 loop. The subunit then ends with a fourth transmembrane domain and a short carboxyl-terminal domain. B  shows the folding of a single subunit with respect to the cell membrane. The orange oval  shows the approximate location of the transmitter-binding site. The second transmembrane domain (shown in red  ) forms the major portion of the ion channel lining. C  shows the arrangement of subunits in the assembled pentameric receptor, viewed from the extracellular side. The hatched circle  in the middle represents the ion channel. The subunits contributing the primary (positive or +) side of the transmitter-binding site are identified as 1o(gold circles  ), whereas the subunits contributing the complementary side are identified as 2oand are blue  . The locations of transmitter-binding sites are indicated by the red diamonds  . The fifth or structural subunit (indicated by X; unfilled circle  ) does not contribute to a transmitter-binding site but impacts the functional properties of the overall receptor and may contain sites for pharmacological agents. D  shows an image of a three-subunit concatemer. The blue-hatched segment  is the signal sequence of the first subunit, the black segments  are the “linkers” connecting subunits, and the subunits are schematized in red  with membrane-spanning regions cross-hatched. The signal sequence is cleaved from the mature subunit and is not present in the assembled receptor. E  shows a simplified version of a two-subunit concatemer in the membrane; other subunits are omitted for clarity. The cylinders outline the subunits, the red lines  indicate the general course of the peptide in the subunit, and the black line  indicates a hypothetical path for a linker to connect the subunits.
Fig. 1. The structural features of ligand-gated ion channel family receptors. A 
	shows a schematic of the primary sequence for all subunits; the amino-terminus is at the left. The amino-terminal extracellular domain contains the loops contributing to the transmitter binding site, A through F. Three transmembrane domains, TM1, TM2, and TM3, then lead to the major cytoplasmic domain, also known as the TM3-TM4 loop. The subunit then ends with a fourth transmembrane domain and a short carboxyl-terminal domain. B 
	shows the folding of a single subunit with respect to the cell membrane. The orange oval 
	shows the approximate location of the transmitter-binding site. The second transmembrane domain (shown in red 
	) forms the major portion of the ion channel lining. C 
	shows the arrangement of subunits in the assembled pentameric receptor, viewed from the extracellular side. The hatched circle 
	in the middle represents the ion channel. The subunits contributing the primary (positive or +) side of the transmitter-binding site are identified as 1o(gold circles 
	), whereas the subunits contributing the complementary side are identified as 2oand are blue 
	. The locations of transmitter-binding sites are indicated by the red diamonds 
	. The fifth or structural subunit (indicated by X; unfilled circle 
	) does not contribute to a transmitter-binding site but impacts the functional properties of the overall receptor and may contain sites for pharmacological agents. D 
	shows an image of a three-subunit concatemer. The blue-hatched segment 
	is the signal sequence of the first subunit, the black segments 
	are the “linkers” connecting subunits, and the subunits are schematized in red 
	with membrane-spanning regions cross-hatched. The signal sequence is cleaved from the mature subunit and is not present in the assembled receptor. E 
	shows a simplified version of a two-subunit concatemer in the membrane; other subunits are omitted for clarity. The cylinders outline the subunits, the red lines 
	indicate the general course of the peptide in the subunit, and the black line 
	indicates a hypothetical path for a linker to connect the subunits.
Fig. 1. The structural features of ligand-gated ion channel family receptors. A  shows a schematic of the primary sequence for all subunits; the amino-terminus is at the left. The amino-terminal extracellular domain contains the loops contributing to the transmitter binding site, A through F. Three transmembrane domains, TM1, TM2, and TM3, then lead to the major cytoplasmic domain, also known as the TM3-TM4 loop. The subunit then ends with a fourth transmembrane domain and a short carboxyl-terminal domain. B  shows the folding of a single subunit with respect to the cell membrane. The orange oval  shows the approximate location of the transmitter-binding site. The second transmembrane domain (shown in red  ) forms the major portion of the ion channel lining. C  shows the arrangement of subunits in the assembled pentameric receptor, viewed from the extracellular side. The hatched circle  in the middle represents the ion channel. The subunits contributing the primary (positive or +) side of the transmitter-binding site are identified as 1o(gold circles  ), whereas the subunits contributing the complementary side are identified as 2oand are blue  . The locations of transmitter-binding sites are indicated by the red diamonds  . The fifth or structural subunit (indicated by X; unfilled circle  ) does not contribute to a transmitter-binding site but impacts the functional properties of the overall receptor and may contain sites for pharmacological agents. D  shows an image of a three-subunit concatemer. The blue-hatched segment  is the signal sequence of the first subunit, the black segments  are the “linkers” connecting subunits, and the subunits are schematized in red  with membrane-spanning regions cross-hatched. The signal sequence is cleaved from the mature subunit and is not present in the assembled receptor. E  shows a simplified version of a two-subunit concatemer in the membrane; other subunits are omitted for clarity. The cylinders outline the subunits, the red lines  indicate the general course of the peptide in the subunit, and the black line  indicates a hypothetical path for a linker to connect the subunits.
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Fig. 2. Studies of 17β-estradiol potentiation of nicotinic α4β2 receptors. A  and C  show an image of the pentameric receptors assembled when a concatemer assembles with a monomeric subunit, viewed from the extracellular side. The direction of the linker (amino- to carboxyl-terminus) is indicated by the arrowheads  , and the position of the free subunit is shown by the clear circle  . In the case of the α4-β2 concatemer, the free subunit (white  color, marked with “ACh”) assembles as a subunit contributing to a transmitter-binding site (shown by the gray diamonds  ); if the free subunit is β2, it contributes the complementary side, whereas if it is α4, it contributes the primary side. With the β2-α4 concatemer, both transmitter-binding sites are inside the concatemer copies, and the free subunit assembles in the structural position (“str”). B  shows the potentiation produced by 10 μM 17β-estradiol for sets of selected combinations of concatemers and free subunits. In each combination there were either zero (bar  labeled “none”) or only a single copy of the carboxyl-terminal binding domain (all other bars  ). For the middle pair of bars  , the single domain was on an α4 subunit or on a β2 subunit. The mean potentiation was slightly larger when the domain was placed on a β2 subunit. For the upper pair of bars  , potentiation was very similar when the domain was on an agonist-binding subunit or on a structural subunit. The data are the means of the average potentiation for each combination, +1 SEM. The legend to the right of each bar  gives the probability that the observed potentiation differs from 1 (where 1 indicates no potentiation; ns P  > 0.05, *P  < 0.05, ***P  < 0.001), followed by the number of combinations tested (e.g.  , a particular concatemer expressed with a particular free subunit) and the total number of oocytes studied for all combinations. D  shows the maximal potentiation ratio for 17β-estradiol as a function of the total number of binding domains in the pentamer. Combinations of constructs were chosen that would result in one, two, three, or five intact binding domains in the pentamer (two combinations for each number), and the concentration-effect relationship for potentiation by 17β-estradiol was determined. The fit maximum potentiation was estimated for each oocyte. The data are the mean maximal potentiation for each number of intact domains (five or six oocytes for each combination, two combinations per point) ±1 SE. The curves  show the best-fitting straight line (black dashed line  ) or geometric function (blue solid line  ), with ±1 SE (thin dashed lines  ). The geometric fit is significantly better than the linear fit (P  = 0.02, F test). For additional information, see the full publication.42 
Fig. 2. Studies of 17β-estradiol potentiation of nicotinic α4β2 receptors. A 
	and C 
	show an image of the pentameric receptors assembled when a concatemer assembles with a monomeric subunit, viewed from the extracellular side. The direction of the linker (amino- to carboxyl-terminus) is indicated by the arrowheads 
	, and the position of the free subunit is shown by the clear circle 
	. In the case of the α4-β2 concatemer, the free subunit (white 
	color, marked with “ACh”) assembles as a subunit contributing to a transmitter-binding site (shown by the gray diamonds 
	); if the free subunit is β2, it contributes the complementary side, whereas if it is α4, it contributes the primary side. With the β2-α4 concatemer, both transmitter-binding sites are inside the concatemer copies, and the free subunit assembles in the structural position (“str”). B 
	shows the potentiation produced by 10 μM 17β-estradiol for sets of selected combinations of concatemers and free subunits. In each combination there were either zero (bar 
	labeled “none”) or only a single copy of the carboxyl-terminal binding domain (all other bars 
	). For the middle pair of bars 
	, the single domain was on an α4 subunit or on a β2 subunit. The mean potentiation was slightly larger when the domain was placed on a β2 subunit. For the upper pair of bars 
	, potentiation was very similar when the domain was on an agonist-binding subunit or on a structural subunit. The data are the means of the average potentiation for each combination, +1 SEM. The legend to the right of each bar 
	gives the probability that the observed potentiation differs from 1 (where 1 indicates no potentiation; ns P 
	> 0.05, *P 
	< 0.05, ***P 
	< 0.001), followed by the number of combinations tested (e.g. 
	, a particular concatemer expressed with a particular free subunit) and the total number of oocytes studied for all combinations. D 
	shows the maximal potentiation ratio for 17β-estradiol as a function of the total number of binding domains in the pentamer. Combinations of constructs were chosen that would result in one, two, three, or five intact binding domains in the pentamer (two combinations for each number), and the concentration-effect relationship for potentiation by 17β-estradiol was determined. The fit maximum potentiation was estimated for each oocyte. The data are the mean maximal potentiation for each number of intact domains (five or six oocytes for each combination, two combinations per point) ±1 SE. The curves 
	show the best-fitting straight line (black dashed line 
	) or geometric function (blue solid line 
	), with ±1 SE (thin dashed lines 
	). The geometric fit is significantly better than the linear fit (P 
	= 0.02, F test). For additional information, see the full publication.42
Fig. 2. Studies of 17β-estradiol potentiation of nicotinic α4β2 receptors. A  and C  show an image of the pentameric receptors assembled when a concatemer assembles with a monomeric subunit, viewed from the extracellular side. The direction of the linker (amino- to carboxyl-terminus) is indicated by the arrowheads  , and the position of the free subunit is shown by the clear circle  . In the case of the α4-β2 concatemer, the free subunit (white  color, marked with “ACh”) assembles as a subunit contributing to a transmitter-binding site (shown by the gray diamonds  ); if the free subunit is β2, it contributes the complementary side, whereas if it is α4, it contributes the primary side. With the β2-α4 concatemer, both transmitter-binding sites are inside the concatemer copies, and the free subunit assembles in the structural position (“str”). B  shows the potentiation produced by 10 μM 17β-estradiol for sets of selected combinations of concatemers and free subunits. In each combination there were either zero (bar  labeled “none”) or only a single copy of the carboxyl-terminal binding domain (all other bars  ). For the middle pair of bars  , the single domain was on an α4 subunit or on a β2 subunit. The mean potentiation was slightly larger when the domain was placed on a β2 subunit. For the upper pair of bars  , potentiation was very similar when the domain was on an agonist-binding subunit or on a structural subunit. The data are the means of the average potentiation for each combination, +1 SEM. The legend to the right of each bar  gives the probability that the observed potentiation differs from 1 (where 1 indicates no potentiation; ns P  > 0.05, *P  < 0.05, ***P  < 0.001), followed by the number of combinations tested (e.g.  , a particular concatemer expressed with a particular free subunit) and the total number of oocytes studied for all combinations. D  shows the maximal potentiation ratio for 17β-estradiol as a function of the total number of binding domains in the pentamer. Combinations of constructs were chosen that would result in one, two, three, or five intact binding domains in the pentamer (two combinations for each number), and the concentration-effect relationship for potentiation by 17β-estradiol was determined. The fit maximum potentiation was estimated for each oocyte. The data are the mean maximal potentiation for each number of intact domains (five or six oocytes for each combination, two combinations per point) ±1 SE. The curves  show the best-fitting straight line (black dashed line  ) or geometric function (blue solid line  ), with ±1 SE (thin dashed lines  ). The geometric fit is significantly better than the linear fit (P  = 0.02, F test). For additional information, see the full publication.42 
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Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A  shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B  shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars  ) and 5βTHDOC (blue bars  ), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars  show data for receptors with both steroid-binding sites intact; the top pair  shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs  show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar  gives the probability that the potentiation differs from 1 (ns P  > 0.05, *P  < 0.05, **P  < 0.01, ***P  < 0.001) and the number of oocytes studied. For additional information, see the full publication.41 C  shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D  summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars  summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair  is for combinations with all sites intact or with both steroid sites mutated. The middle group of three  shows combinations for which the steroid site in the “a” pair is intact, while the upper group  shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B  . For additional information, see the full publication.47 GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.
Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A 
	shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B 
	shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars 
	) and 5βTHDOC (blue bars 
	), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars 
	show data for receptors with both steroid-binding sites intact; the top pair 
	shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs 
	show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar 
	gives the probability that the potentiation differs from 1 (ns P 
	> 0.05, *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001) and the number of oocytes studied. For additional information, see the full publication.41C 
	shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D 
	summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars 
	summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair 
	is for combinations with all sites intact or with both steroid sites mutated. The middle group of three 
	shows combinations for which the steroid site in the “a” pair is intact, while the upper group 
	shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B 
	. For additional information, see the full publication.47GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.
Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A  shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B  shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars  ) and 5βTHDOC (blue bars  ), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars  show data for receptors with both steroid-binding sites intact; the top pair  shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs  show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar  gives the probability that the potentiation differs from 1 (ns P  > 0.05, *P  < 0.05, **P  < 0.01, ***P  < 0.001) and the number of oocytes studied. For additional information, see the full publication.41 C  shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D  summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars  summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair  is for combinations with all sites intact or with both steroid sites mutated. The middle group of three  shows combinations for which the steroid site in the “a” pair is intact, while the upper group  shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B  . For additional information, see the full publication.47 GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.
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