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NEUROSCIENCE |
1 Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
2 Department of Neurobiology, UAB School of Medicine, Birmingham, AL 35294, USA
3 Solae, St Louis, MO 63102, USA
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Abstract |
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, 
or 
subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two subunits, two subunits and one subunit, it is not clear how many and subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant or subunits with their wild-type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6' mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered.
(Received 1 September 2006;
accepted after revision 18 September 2006;
first published online 21 September 2006)
Corresponding author D. S. Weiss: Department of Physiology, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. Email: weissd{at}uthscsa.edu
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Introduction |
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In a previous study, we demonstrated that mutation of a highly conserved threonine residue at the 6' position in the second transmembrane domain of the 1, 2 or 2 subunits of 122 GABA receptors abolished antagonism by picrotoxin (Gurley et al. 1995). At the time of that study, the stoichiometry of the GABA receptor was unknown. Since then, the stoichiometry has been determined to be two subunits, two subunits and one subunit (Chang et al. 1996; Tretter et al. 1997). Although this implies that the 6' TM2 mutation in the single subunit was sufficient to abolish picrotoxin sensitivity, the same could not be said for the and mutations, for which there are two copies in each functional pentameric receptor. In the present study, by coexpressing combinations of wild-type and mutant subunits at defined ratios we directly address this fundamental issue. Our data unambiguously demonstrate that any one of the five subunits carrying this 6' TM2 mutation can impart picrotoxin resistance. Implications for the mechanism of action and the use of this system for probing native receptor function, stoichiometry and regulation are discussed .
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Methods |
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Rat 1, 2 and 2 cDNAs were subcloned into the pGEMHE vector (Liman et al. 1992). Mutations 1T260F and 2T256F were generated using the overlap extension method (Kammann et al. 1989). All constructs were verified by cDNA sequencing. The DNAs were linearized with Nhe I (New England BioLabs Inc., Ipswich, MA, USA), and run-off capped cRNA was transcribed from linearized cDNAs with standard in vitro transcription procedures using the T7 mMessage mMachine Kit (Ambion, Austin, TX, USA). Integrity and yield of the cRNA were verified on a 0.8% agarose gel.
Xenopus oocyte expression
Xenopus laevis (Xenopus I, Ann Arbor, MI, USA) were anaesthetized by placing them in a 0.2% solution of MS-222. The oocytes were surgically removed and placed in a solution that consisted of 85.5 mM NaCl, 2.5 mM KCl, 5 mM Hepes, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 50 i.u. ml–1 penicillin and 50 mg ml–1 streptomycin, pH 7.5, adjusted with NaOH. The frog was then allowed to recover fully before being returned to a recovery tank, after which time it was observed daily (for one week) to ensure that no complications had arisen as a result of the surgery. This procedure was in accordance with The Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and the NIH office of Laboratory Animal Welfare.
Oocytes were dispersed in this solution without Ca2+, but in the presence of 0.3% collagenase A (Roche Diagnostics, Indianapolis, IN, USA). After isolation, stage VI oocytes were thoroughly rinsed and maintained at 18°C in the above-mentioned solution plus 1 mM Ca2+. Micropipettes for injecting cRNA were pulled on a Sutter P87 horizontal puller. To match the cRNA concentrations, wild-type and mutant cRNAs were diluted 2- to 10-fold with diethyl pyrocarbonate (DEPC)-treated water and electrophoresed in a 0.8% agarose gel. Based on relative intensity of the bands, the cRNA concentrations were readjusted to be equal. The maximum GABA-activated currents recorded from oocytes injected with the same amount of the wild-type and mutant cRNAs were approximately the same. The experiments presented in this study were performed on several different batches of cRNA. For experiments involving coexpression of wild-type and mutant receptors, instead of matching concentrations of cRNA by comparing intensity of cRNA bands on the gel, we compared the maximum GABA-activated currents for control oocytes injected with the same amounts of wild-type or mutant cRNA. The ratios we provide in the figures reflect recalculation based on these maxima.
Voltage clamp of oocytes
Two-electrode voltage-clamp procedures were used for current recording 2 or 3 days after cRNA injection. Each oocytes was placed on a 300 µm nylon mesh suspended in a small-volume chamber (< 100 µl). The oocyte was perfused continuously with a solution containing 92.5 mM NaCl, 2.5 mM KCl, 5 mM Hepes, 1 mM CaCl2 and 1 mM MgCl2, pH 7.5, adjusted with NaOH. The solution was switched to the test solution, which was identical to the perfusion solution with the addition of drug (e.g. GABA). Typical interval between applications (GABA or GABA plus picrotoxin) was 5 min to allow sufficient recovery from desensitization as well as unbinding of picrotoxin. All experiments were performed at room temperature. Recording microelectrodes were pulled on a P87 Sutter horizontal puller and filled with 3 M KCl. The electrode resistances ranged from 1 to 3 M
. Standard two-electrode voltage-clamp techniques (GeneClamp 500; Molecular Devices, Sunnyvale, CA, USA) were used to record currents at a holding potential of –70 mV. On-line digitization of the signal at 20 Hz was carried out by using the ITC-16 data acquisition board (Instrutech, Long Island, NY, USA) and Igor software (Wavemetrics, Lake Oswego, OR, USA).
Data analysis
Agonist concentration–response relationships were fitted (least-squares) with the following sigmoidal equation:
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| (1) |
To assess the sensitivity of block by picrotoxin, the peak amplitudes were measured and normalized to the GABA-activated current in the absence of picrotoxin. The time-dependent decay was a combination of desensitization and block, so the measured block was slightly underestimated. While this approach may alter the accuracy of the determined antagonist sensitivity of the wild-type receptor, the effects would be minimal on the mutant receptors, where little antagonism was observed. The IC50 (concentration of PTX which decreased the agonist-mediated current by 50%) for each receptor was determined by a least-squares fit of the following relationship to the data:
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| (2) |
Statistical comparison of models
All statistical analyses were performed using R-2.3.1 (http://www.R-project.org). The following non-linear models were developed to predict the relative efficiency of inhibition from the proportion of wild-type and mutant isoforms for both and subunits:
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| (3) |
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| (4) |
or both subunits must be mutant to impart picrotoxin resistance, while model II assumes that only one or one subunit must be mutant to impart resistance. Models with a better fit were selected based on values of residual standard errors and results of goodness-of-fit tests (Bates & Watts, 1988; Neter et al. 1996). Structural modelling
ICM (Molsoft; La Jolla, CA, USA) was used to build a representation of the TM2 region of the GABAA receptor. The homology model was built by working from the 4Å resolution structure of the nicotinic acetylcholine receptor (nAChR) transmembrane domain (Miyazawa et al. 2003). Alignments were constructed by pairing GABA and nAChR subunits on the putative basis of their role in ligand binding. By this model, the two GABAA
1 subunits correspond to nAChR and 
, the two GABAA
2 subunits correspond to the two nAChR subunits and the GABAA
2 subunit corresponds to the nAChR subunit. The overall model was then energy minimized to eliminate intra-/intersubunit clashes. Introduction of mutations into the receptor was followed by further energy minimization to ensure that this perturbation did not impart any instability to the TM2 region.
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Results |
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1 subunit and position 256 in the subunit were switched to phenylalanine. Both of these residues are in the homologous 6' position of TM2. In all subsequent discussion, we will refer to the T260F mutation as m and the T256F mutation as m. Figure 1B plots the GABA dose–response relationship for wild-type receptors (
) as well as the m (
) and m receptors (). The continuous lines are fits of the Hill equation (eqn (1)) to the dose–response relationships and yielded EC50 values of 40.8 ± 21.0, 65.2 ± 17.0 and 38.6 ± 10.2 for , m and m, respectively (n
= 5 for all). This pore mutation did not significantly alter the sensitivity of the receptor to GABA (unpaired t test, n.s.).
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m and m receptors. These data are plotted in Fig. 2B, and the continuous line is the best fit of an inhibition function (eqn (2)) that yielded an IC50 for picrotoxin of 1.1 ± 0.3 µM (n
= 3) in the wild-type receptor. Note that antagonism by picrotoxin, at concentrations as high as 100 µM, was completely eliminated by the and subunit mutations. Similar picrotoxin resistance was seen in the homologous subunit mutation (Gurley et al. 1995).
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and subunits must carry the mutation to impart a picrotoxin resistance, we coexpressed various ratios of wild-type and mutant subunits. The top of Table 1 shows the possible combinations of receptors when either the wild-type or subunits (N6'T) are coexpressed with their corresponding mutants (N6'F). We can consider the possibilities for the subunit, but the identical argument applies to the subunit. Functionally, if we coexpress and m, there are three combinations to consider: those with two wild-type subunits, those with two mutant subunits and those with one wild-type and one mutant subunit. We can then use the binomial distribution to predict the ratio of these three combinations. In this analysis, we make two assumptions. First, we assume that the subunits assemble with roughly equivalent efficiency and indeed we do observe comparable levels of expression with comparable cRNA injections. In addition, we will describe an analysis later that takes into account any differences in expression between wild-type and mutant subunits. The second assumption is that we can ignore subunit order in terms of the actions of picrotoxin and the binomial calculations. Indeed, these receptors are asymmetric in terms of their subunit order and there is evidence indicating corresponding asymmetric properties (Tretter et al. 1997; Baumann et al. 2002, 2003; Boulineau et al. 2005; Baur et al. 2006). If there are differences in the sensitivity to picrotoxin depending on the position of the single mutant subunit within the pentamer, this should not influence the analysis of our experimental data here, since we are basing our results on the maximal block rather than the picrotoxin sensitivities. It is possible that receptors with, for example, one mutant and one wild-type subunit show partial block (partial resistance), but as will be demonstrated subsequently, this scenario adds an unnecessary layer of complexity.
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m cRNA compared with wild-type cRNA. In this case, 0.79 of the receptors will be double mutant, 0.01 would be double wild-type and 0.20 would have one wild-type and one mutant subunit. If two mutant subunits were required to eliminate picrotoxin sensitivity, then 0.79 of the current would be picrotoxin resistant (model II in Methods). Alternatively, if one mutant subunit imparts picrotoxin resistance, then 0.99 of the current would be picrotoxin resistant (model I in Methods). For each ratio of wild-type and mutant cRNAs injected (leftmost column in Table 1), one can predict in this same manner the fraction of current blocked for the model in which only one subunit needs to be mutant for picrotoxin resistance or the model in which both subunits must be mutant for picrotoxin resistance.
Figure 3A shows picrotoxin-mediated antagonism for GABA-activated currents (40 µM GABA) with varying ratios of injected wild-type and mutant cRNA, and Fig. 3B shows picrotoxin-mediated antagonism for GABA-activated currents (40 µM GABA) with varying ratios of injected wild-type and mutant cRNA. The fraction of wild-type receptors is indicated to the left of each set of traces. Note the decreasing inhibition as the fraction of wild-type subunits decreases (top to bottom). To obtain the fraction of current that is not blocked by picrotoxin, dose–response relationships for the various ratios were fitted with an inhibition function (eqn (2); Fig. 4A and B). Extrapolation of the fitted function provided the fraction of current resistant to picrotoxin. (The resulting IC50 values are provided in Table 1) Again, note that as the relative amount of mutant cRNA was reduced, the fraction of current blocked by picrotoxin decreased.
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subunit. The filled circles plot the fractional inhibition as a function of the cRNA ratio (each experiment plotted individually), while the open circles plot the best prediction of model I, and the open triangles plot the best prediction of model II. Figure 5B is a similar graph for the subunit. Model II clearly fits the data better than model I. This is supported by lower residual standard error values: 0.303 for model I versus 0.077 for model II for the subunit and 0.429 for model I versus 0.097 for model II for the subunit. Goodness-of-fit tests indicate that model II is significantly better than model I for both and data sets (P values < 2.2 x 10–16). These data, along with our previously published results on the subunit, unambiguously support the model in which any one subunit of the 122 GABA receptor carrying a mutation in this particular 6' TM2 threonine residue is resistant to picrotoxin.
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Discussion |
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It is worth mentioning that picrotoxin-mediated antagonism of the homologous glycine receptor, in stark contrast to the GABAA receptor, is competitive and not use dependent (Lynch et al. 1995). While this could be interpreted as a fundamental difference in the mechanism and/or site of action of picrotoxin between glycine and GABA receptors, the corresponding 6' mutation in the glycine receptor does eliminate antagonism by picrotoxin (Hawthorne & Lynch, 2005). This observation suggests a common site of action for GABA and glycine receptors. A possible explanation for the observed differences in the wild-type GABA and glycine receptors (competitive and use dependent) would be that the picrotoxin binding site is more accessible in the closed state for the glycine receptor and more accessible in the open state for the GABA receptor (Hawthorne & Lynch, 2005). In this scenario, the 15' TM2 mutation (Dibas et al. 2002) and the mutations in the TM2–TM3 extracellular linker (Hawthorne & Lynch, 2005) that convert picrotoxin block of the glycine receptor to use dependent could be explained by a structural rearrangement in TM2 that enhances open state accessibility by the blocker.
Modelling studies using Monte Carlo minimization give support to this TM2 site of action and postulate that picrotoxin interacts with the cytoplasmic half of the pore and is stabilized by all five TM2 domains (Zhorov & Bregestovski, 2000). In this model, picrotoxin is also stabilized by hydrogen bonds with the 6' threonine residue. This is the same threonine originally identified to be a major determinant of the action of picrotoxin on GABA and glycine receptors (Pribilla et al. 1992; Gurley et al. 1995) and the same mutation we use in the present study. If a single picrotoxin molecule were stabilized by multiple hydrogen bonds with the 6' hydroxyl, at least simplistically, one would not predict a complete elimination with the removal of one of the five hydroxyls. Alternatively, the substituted phenylalanine at any one of the five positions could provide a steric hindrance for the pictrotoxin molecule, thereby preventing access to its binding site at a more cytoplasmic position in TM2. In an effort to distinguish these two possibilities, we homology modelled the TM2 domains of the GABAA receptor, based on the homologous nACh receptor (Miyazawa et al. 2003). Figure 6A shows the ribbon structure of the TM2 domains and the 6' threonine side-chains after optimization (see Methods). The picrotoxin molecule (space filled) is superimposed on this structure to indicate its size relative to this position of the pore. In Fig. 6B, the 6' threonine of one subunit has been mutated to a phenylalanine. This preliminary structural analysis indicates that the large ringed side-chain of phenylalanine, if positioned as shown in Fig. 6B, could not only prevent access of picrotoxin beyond this position but could also prevent picrotoxin from sitting at this position were it the location of picrotoxin when it blocks the receptor. This could certainly account for the ability of a single phenylalanine mutation in any one of the five subunits to prevent picrotoxin block. A series of mutations of different side-chain sizes at this position are planned in an attempt to test this possibility further.
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subunit, present in one copy per functional pentamer, eliminated picrotoxin sensitivity (Gurley et al. 1995). However, there are certainly well-documented cases where subunit asymmetries exist in the GABA receptor (Baumann et al. 2003; Boulineau et al. 2005) and thus it is necessary to test for symmetry in the actions of picrotoxin. It is convenient that the mutation that eliminates picrotoxin sensitivity has a minimal effect on receptor sensitivity. Expression of a 6' mutant subunit in neurons or neuronal slices that endogenously express GABA receptors, along with electrophysiological recording in the presence of picrotoxin, can be used to help delineate the role of particular subunit combinations in shaping GABA-mediated inhibition in the brain. Another potential use of these findings is that they may provide a mechanism for delineating relative ratios of native subunits. For example, both GABA and glycine receptors have subunits that impart a native picrotoxin resistance (Pribilla et al. 1992; Zhang et al. 1995). Assessing the fraction of picrotoxin-sensitive current, in conjunction with the binomial analysis we have presented, could enable a determination of the relative expression of the resistance-imparting subunit.
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Acknowledgements |
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