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Department of Pharmacology & Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania
Received November 10, 2005; accepted November 17, 2005
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Abstract |
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 Top Abstract References |
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; Lester et al., 2004). All members of the LGIC family are believed to be pentameric in structure, with one or more different homologous subunits arranged around a central pore that forms the channel itself. Binding of the appropriate agonist to the receptor induces a conformational change in the protein, resulting in the opening of the ion channel, and initiating the sequelae of channel activation.
So far, a large number of subunits for each type of LGIC have been cloned (Table 1), and many studies have been carried out using heterologous expression systems such as Xenopus laevis oocytes or transfected mammalian cells to understand how subunit composition affects the functional properties of the receptor. For example, coexpression of the GABAAR 
1 and 
1 subunits in human embryonic kidney 293 cells results in receptors that display much of the pharmacology of bona fide GABAARs, including potentiation by barbiturates, but these receptors are insensitive to benzodiazepines. However, coexpression of the 
2 subunit with the 1 and 1 subunits produces receptors with benzodiazepine binding sites and functional responses to benzodiazepines (Pritchett et al., 1989), demonstrating the crucial role that subunits play in GABAAR function.
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An equally important goal of the expression studies is to duplicate various receptor subunit combinations found in vivo to use these refined systems as tools for developing receptor (sub)subtype-selective pharmacological agents. These tools could then be used not only for analysis of the properties of receptors themselves but also for the development of novel therapeutic agents targeted toward a specific receptor (sub)subtype (Gotti et al., 2000; Bunnelle et al., 2004). However, for this approach to be truly fruitful, one must first determine the subunit stoichiometry of a given target and then develop methods for the expression of receptors with this exact stoichiometry and subunit arrangement. Neither of these tasks is by any means easy or straightforward.
Consider the simplest nontrivial situation—a pentameric receptor that contains two different subunits, A and B. There are six possible subunit stoichiometries (A5,A4B, A3B2,A2B3, AB4, and B5). With identical handedness governed by l-amino acid chirality, there are eight possible unique subunit arrangements in the complex (Fig. 1). In the absence of a detailed understanding of the "rules" for receptor assembly, it is not possible a priori to predict the exact subunit stoichiometry and/or subunit arrangement in the complex when two different types of subunits could be incorporated into the complex. When there are more than two different subunits that could potentially be incorporated into the receptor (as is more often the case; see Table 1), the situation becomes even more complex. In either situation, experimental techniques must be developed to address the issue of subunit stoichiometry and subunit arrangement.
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; Li and De Blas, 1997; Turner and Kellar, 2005). Although this approach can certainly determine which subunits are associated with each other in a complex, it may not be able to identify all complexes that occur naturally because of the sensitivity of detection methods and may not be quantitative enough to unambiguously determine subunit stoichiometry. Nonetheless, studies of this type have identified a number of potential subunit combinations and stoichiometries of naturally occurring receptors, and provide the groundwork for studies in heterologous expression systems using more defined subunit composition.
Heterologous expression systems, such as X. laevis oocytes or transfected mammalian cells, have been used to determine subunit stoichiometry and to create receptors of defined subunit composition by introducing RNA or DNA for one or more subunits. Subunit stoichiometry can then be determined by analyzing the properties of the expressed receptors using a variety of techniques, including immunoprecipitation with subunit-selective antisera (Anand et al., 1991; Kellenberger et al., 1996; Knight et al., 2000), analysis of the effects of mutations in the pore-forming domain that alter receptor properties (Cooper et al., 1991; Backus et al., 1993; Chang et al., 1996; Boorman et al., 2000; Burzomato et al., 2003), and, in one case, atomic force microscopy (Barrera et al., 2005). Studies such as these provide most of what we know about subunit stoichiometry of receptors containing various subunits.
However, although one assumes/hopes that the receptors expressed in these systems will be faithful surrogates for naturally occurring receptors, there is some evidence that in heterologous expression systems, the ratio of subunits in a given receptor complex depends on the ratios of exogenous subunit RNA or DNA introduced into the expression system, resulting in variable subunit stoichiometry (Hedblom and Kirkness, 1997; Zwart and Vijverberg, 1998; Nelson et al., 2003). In addition, it is clear that these systems can produce receptors that probably do not exist in vivo—either homomeric receptors (Beato et al., 2002) or subunit-deficient complexes (Jackson et al., 1990; Charnet et al., 1992). Furthermore, the presence of endogenous subunits in the expression system may further complicate the analysis of the effects of altering subunit composition on receptor function (Buller and White, 1990). Therefore, these systems may not always produce the type of receptors expected, and conclusions obtained from such studies may not be as firm as one would like.
One method to obtain the expression of receptors of known/predefined subunit stoichiometry and even subunit orientation is the use of concatameric subunit constructs. This forces subunits to be in a particular stoichiometry and spatial arrangement by expressing a polyprotein containing more than one subunit sequences in the mature protein. This is essentially what is seen naturally in some voltage-gated channels. In the case of K+ channels, the channel is a true tetramer formed from four individual subunits (MacKinnon, 1991; Doyle et al., 1998), whereas in Na+ and Ca2+ channels, the "tetramer" is formed by four homologous repeat domains within a single large polypeptide chain encoding the main subunit (Catterall, 1995).
Originally applied to voltage-gated K+ channels (Liman et al., 1992), this approach has been used for several members of the LGIC family (Im et al., 1995; Baumann et al., 2001; Zhou et al., 2003; Grudzinska et al., 2005). In most cases, tandem dimeric subunits were created with a polyglutamine linker containing 10 to 25 glutamines between the two subunits. At this length, polyglutamine is water-soluble and assumes a random coil conformation (Altschuler et al., 1997), making it an ideal linker for this purpose. Expression of the dimeric construct with one or more individual subunits can then be used to determine not only the subunit stoichiometry but also the spatial arrangement of the subunits in the complex. For example, if expression of A
B tandem subunits (where the carboxyl terminus of the A subunit is joined to the amino terminus of the B subunit via the linker) alone does not produce functional receptors, then one can rule out dimers, tetramers, or hexamers as forming the entire receptor complex (which would not be surprising given the fact that numerous studies analyzing receptors from both tissues and heterologous systems have demonstrated that these receptors are pentamers). If coexpression of the AB dimer with the B subunit formed functional receptors but coexpression of A subunit with the dimer did not, then (assuming a pentameric complex) one would conclude that the stoichiometry of the receptor was A2B3 and the subunit arrangement in the complex was ABABB (and then back to the initial "A" in the sequence to complete the loop).
The above analysis assumes that both parts of the dimeric subunit are incorporated into the same receptor complex. However, two recent studies suggest that this may not always be the case, complicating the analysis of the results. Through the use of 42 and 24 nicotinic AChR receptor subunit dimer constructs, Zhou et al. (2003) showed that in some cases, the subunits in the dimer may be incorporated into two separate complexes, resulting in a dimer of pentameric complexes held together by the linker. Groot-Kormelink et al. (2004) used a wide variety of dimers containing various -subunits of neuronal AChRs combined with various -subunits in both orientations (i.e., and ) and analyzed the properties of the expressed receptors. Surprisingly, they found that some dimer constructs expressed alone in X. laevis oocytes gave rise to functional receptors, which is at odds with the known pentameric structure of the receptor. Further analysis using mutations in the subunit led the authors to conclude that in some cases, only one subunit in the dimer was incorporated into the complex, with the remaining subunit "floating in the wind" as an appendage outside of the complex.
These two studies demonstrate that the use of tandem dimeric subunits is not as clean as originally anticipated, and set the groundwork for the study by Groot-Kormelink et al. (2006) in the current issue. In this work, the authors expressed not tandem dimers, but concatameric pentamers containing 3 and 4 nicotinic AChR subunits in the order 44343 and compared the functional properties of these receptors with those expressed from the monomeric 3 and 4 subunits together. The functional properties of the receptors expressed from the pentameric construct were essentially identical to those expressed from the monomers. Through the coexpression of 3 or 4 subunits with a mutation in the pore domain that alters channel gating (L9'T; Revah et al., 1991; Filatov and White, 1995; Labarca et al., 1995) as a probe for incorporation of monomeric subunits, the authors were able to rule out proteolytic breakdown of the pentameric construct and subsequent reincorporation of monomeric subunits into the complex. Thus, the pentameric construct produces a fully functional receptor.
In a demonstration of the usefulness of this technique, the authors constructed a receptor in which only one of the two 3 subunits in the complex contained the L9'T mutation. Previous work suggested that when multiple subunits contained this change that the mutation in each subunit made more or less equal contributions to the overall change in receptor gating (Filatov and White, 1995; Labarca et al., 1995). However, because it was not possible in the original studies to ensure that only a single mutant subunit was incorporated into the complex, this notion could not be tested in an unambiguous fashion. In the present study, the authors were able to ensure incorporation of a single mutant 3 subunit into the receptor, leaving the other 3 subunit as a wild type, and they found that mutations in each subunit did not produce equivalent effects. This type of experiment could never be carried out without the use of the pentameric constructs.
Although this study provides interesting AChR-specific findings as well as a general approach that should be applicable to other LGICs, two types of additional experiments should be carried out before this method is elevated to panacea status. First, do other linear sequences that should produce the same arrangement of subunits in the complex (e.g., 34434) give the same results? One might expect that they would, but it would be nice to demonstrate this. If the alternate sequences do not result in the formation of functional receptors, this might provide an opening to a more detailed analysis of the contributions of particular regions of each subunit that contribute to the subunit-subunit interactions that hold the complex together. Second, do other subunit arrangements (e.g., 44433) not produce functional receptors? If the receptor has a unique stoichiometry and subunit arrangement, concatamers such as this should not work. If other constructs do work, it would demonstrate that there is not a unique arrangement of a given group of subunits and lays the groundwork for future studies of how subunit arrangement affects receptor function.
The construction of pentameric subunit concatamers now allows several types of studies that could not be carried out before. First, as demonstrated in this study, one can incorporate a mutation into just one subunit of the receptor to examine the role of individual subunits—even those that may be present in multiple copies in the complex. Although one might consider this a situation that would occur only in experiments designed to probe receptor structure-function relationships, this also might be observed in the case of polymorphisms in heterozygotes for a particular mutation. Application of this approach to the ligand-binding site regions [which are believed to be located at subunit-subunit interfaces (Karlin, 2004)] should allow the delineation of the role of residues on either half of a specific ligand-binding site in the receptor in the actions of agonists and antagonists. Second, and perhaps of much wider interest, one can now create receptors of predefined subunit stoichiometry and arrangement and use these not only to test which stoichiometries/arrangements produce functional receptors but also to create well defined targets for the development of novel experimental probes and therapeutic agents with receptor (sub)subtype selectivity. It is now time for the fun stuff to begin!
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Footnotes |
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Please see the related article on page 559
ABBREVIATIONS: LGIC, Cys-loop ligand gated ion channel; AChR, nicotinic acetylcholine receptor; GABAAR, GABAA receptor.
Address correspondence to: Dr. Michael M. White, Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA 19102. E-mail: mikewhite{at}drexel.edu
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