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1 Subunit M1 Domain Reveal Unexpected Complexity for Modulation by Neuroactive SteroidsDepartment of Anesthesiology (G.A., P.L., J.B., J.H.S.), Mallinckrodt Institute of Radiology (D.E.R.), and Department of Developmental Biology (D.F.C.), Washington University School of Medicine, St. Louis, Missouri
Received for publication May 1, 2008.
Accepted for publication June 10, 2008.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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1Asn407/Tyr410 in the M4 transmembrane domain and residue 1Gln241 in the M1 domain. We examined the role of residues in the 1 subunit M1 domain in the modulation of the rat 1β2
2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the 1Q241W or 1Q241L mutations were insensitive to (3,5)-3-hydroxypregnan-20-one (35P), albeit with different underlying mechanisms. The 1Q241S mutant was potentiated by 35P, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the 1Q241L mutation had no effect on channel potentiation by (3,5)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, 1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5-pregnan-20-one) potentiated the wild-type receptor but not the 1Q241L mutant. The findings are consistent with a model in which the 1Ser240 and 1Gln241 residues shape the surface to which steroid molecules bind.
,5)-3-hydroxypregnan-20-one (35P) and (3,5β)-3-hydroxypregnan-20-one (35βP)] act on the GABA-A receptor by modifying the channel open and closed times, leading to an increase in the open probability of the channel, enhanced macroscopic peak current, and a slower current decay when exposure to agonist is terminated. The putative steroid binding site is located in the membrane-spanning regions of the subunit of the receptor, extending from the 1Gln241 residue in the M1 membrane-spanning region to the residues 1Asn407 and 1Tyr410 in the M4 domain (Hosie et al., 2006
). Mutations that reduce the H-bonding ability of these residues reduce receptor potentiation by both 5- and 5β-reduced steroids. It was proposed that a common interaction site mediates the effects of the two classes of steroids, the 1Gln241 residue acting as an H-bond acceptor to the 3-hydroxyl group of the steroid molecule and the 1Asn407/Tyr410 residues interacting with the ketone group in the side chain on the D ring of steroids (Hosie et al., 2006). Subsequent studies showed that mutations that disrupt channel potentiation by steroids also affect modulation by a tricyclic benz[e]indene neurosteroid analog (Li et al., 2006), enantiomers of natural steroids (Li et al., 2007a), and the marine cembranoid eupalmerin acetate (Li et al., 2008), suggesting that the site may function as a common interaction site for a number of GABA-A receptor modulators.
To fully understand the role of the 1Gln241, 1Asn407, and 1Tyr410 residues in steroid actions, it is essential to establish, first, the role of these residues in normal receptor activity. Many channel modulators act in a state-specific manner. Therefore, a lack of responsiveness to a drug application may result from changes in channel baseline kinetic properties rather than reflect the inability of the drug to interact with the receptor. In addition, previous single-channel work has demonstrated that steroids modify several kinetic parameters, effects that may be mediated by steroid interactions with two or more distinct binding sites (Akk et al., 2004; Li et al., 2007a). Consequently, it is of interest to assess the effect of mutations to the putative steroid site residues on the full spectrum of kinetic variables that are modified in the presence of neurosteroids.
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1 subunit M1 membrane-spanning domain on channel modulation by neuroactive steroids. A structural model showing the locations of the residues studied is shown in Fig. 1. We show that mutations to the 1Gln241 site strongly affect channel modulation by 35P, albeit through different kinetic mechanisms. Channel potentiation by (3,5)-3-hydroxymethylpregnan-20-one (3CH2OH5βP) was disrupted in the 1S240L but not 1Q241L mutant receptor. We also show that the steroid 5-pregnan-20-one (3deoxy5P) potentiates the wild-type receptor but not a receptor containing the 1Q241L mutation. The findings are most compatible with a model in which the steroids bind to a hydrophobic surface on the receptor 1 subunit, the 1Ser240 and 1Gln241 residues acting to shape the surface to accommodate a variety of structurally distinct steroids. |
Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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1β22L GABA-A receptors. The details of receptor expression and electrophysiology have been described in detail previously (Akk et al., 2001, 2004; Li et al., 2006). The mutations were generated using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA), and the mutated subunits were fully sequenced to confirm that only the desired mutation had been produced. The 1 subunit is epitope (FLAG)-tagged (Ueno et al., 1996) in the amino-terminal end of the subunit. Cells expressing high levels of receptors were determined using a bead-binding technique in which the presence of the FLAG peptide was detected with a mouse monoclonal antibody to the FLAG epitope (M2; Sigma-Aldrich, St. Louis, MO), which had been adsorbed to beads with a covalently attached goat anti-mouse IgG antibody (Invitrogen, Carlsbad, CA). The electrophysiological experiments were carried out using standard single-channel patch clamp and whole-cell voltage clamp methods. The bath solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4. In single-channel recordings, the pipet solution contained 120 mM NaCl, 5 mM KCl, 10 mM MgCl2, 0.1 mM CaCl2, 20 mM tetraethylammonium chloride, 5 mM 4-aminopyridine, 10 mM glucose, and 10 mM HEPES, pH 7.4. In whole-cell recordings, the pipet solution contained 140 mM CsCl, 4 mM NaCl, 4 mM MgCl2, 0.5 mM CaCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.4.
The agonist (GABA or piperidine-4-sulfonic acid) and steroid modulators were added to the pipet solution in single-channel recordings, or applied through the bath using a fast perfusion stepper system (SF-77B; Warner Instruments, Hamden, CT) in whole-cell experiments. The steroids were initially dissolved in DMSO at 5 to 10 mM concentration and diluted immediately before the experiment. The maximal DMSO concentration in diluted steroid solutions was 0.1%. We have found previously that channel activation by GABA is not affected by the presence of up to 0.3% DMSO (Li et al., 2007a). All experiments were carried out at room temperature (19-22°C).
The recording and analysis of single-channel currents have been described in detail previously (Akk et al., 2001, 2004). The pipet potential was held at +60 to +80 mV, which translates to an approximately -120 to -100 mV potential difference across the patch membrane. The channel activity was recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), low-pass-filtered at 10 kHz, and acquired with a Digidata 1320 series interface at 50 kHz using pClamp software (Molecular Devices). The key features of the analysis of single-channel currents are as follows. When possible, the analysis was limited to clusters (i.e., episodes of intense activity originating from the activation of a single ion channel or fragments of clusters containing no overlapping currents). The currents were low-pass-filtered at 2 to 3 kHz, and the data were idealized using the segmented-k-means algorithm (Qin et al., 1996). The open and closed times were estimated from the idealized currents using a maximum likelihood method that incorporates a correction for missed events (QuB Suite; http://www.qub.buffalo.edu). Under certain conditions (e.g., in the presence of P4S or low concentrations of GABA), no clear-cut clusters were observed. In these cases, episodes of activity containing no overlapping currents were used for analysis, and the analysis was limited to estimation of channel open time durations.
Throughout the manuscript, the open- and closed-time components, as determined from the respective histograms, are referrred to as OT1-3 and CT1-3, respectively. The numerical designation applies to the lifetime of the component (i.e., OT1 and CT1 are the briefest components in the respective histograms) but does not necessarily connote a specific state in the activation scheme. For example, the CT3 component at one GABA concentration does not have to match up with the CT3 component at another GABA concentration, and the components may involve dwells in different activation states. Thus, no mechanism is implied. In some cases, when we believe that a particular closed-time component can be associated with sojourns in a particular activation state, we have used additional nomenclature to illustrate the mechanistic implications. For example, a closed-time component whose duration inversely correlates with agonist concentration probably results from sojourns in the un-, mono- and diliganded closed states. We have designated the corresponding closed-time component CTβ.
The recording and analysis of whole-cell currents was carried out as described previously (Li et al., 2006). The cells were clamped at -60 mV. The cells were exposed to GABA and steroids for 4 s with 30-s washouts separating successive applications. The current traces were low-pass-filtered at 2 kHz and digitized at 10 kHz. The analysis of whole-cell currents was carried out using the pClamp 9.0 software package and was aimed at determining the peak amplitude. Each cell was, before testing the effects of steroids, examined using two GABA concentrations to determine the approximate GABA EC50 for the cell to verify the expression of subunit in the receptor complexes (Boileau et al., 2003).
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1 subunits were developed using the program Modeler (Sali and Blundell, 1993). The alignments used for the models were produced using the program MUSCLE (Edgar, 2004) by aligning the rat 1 sequence with the acetylcholine binding protein of Lymanea stagnalis (PDB code 1I9B
[PDB]
) (Brejc et al., 2001) and the nicotinic acetylcholine receptor of Torpedo marmorata (PDB code 2BG9
[PDB]
) (Unwin, 2005). To improve the quality of the models, the 72-residue cytoplasmic loop connecting the M3 and M4 domains was replaced with seven glycine residues. There are no conserved residues in the cytoplasmic loop, and only 30 amino acid residues could be aligned to positions on the templates. By replacing this region with seven glycines, optimal for stabilizing two helices (Liu et al., 2003), a large region of conformational uncertainty was removed. For each protein, a total of 10 models was produced using molecular dynamics-based refinement for each model. The model quality was then assessed using the default penalty function, and further scored with "DOPE" (Discrete Optimized Protein Energy), and the best model was then used for further analysis (Shen and Sali, 2006). Although DOPE was developed from a nonredundant set of 1472 structures from the PDB, perhaps biasing toward soluble proteins rather than membrane-bound proteins, the idealized reference state is independent of the composition of the protein and the scoring function represents the current state of the art. The molecular graphics images shown in this work were produced with the UCSF Chimera package (Pettersen et al., 2004) from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health grant P41-RR01081).
The synthesis of 3CH2OH5βP will be described in detail in future publications. The compound had spectroscopic properties consistent with the assigned structure. It was chromatographically pure and gave the correct elemental analysis. Steroid 3deoxy5P was obtained from Steraloids, Inc (Newport, RI). Other chemicals, including 35P, 35βP, and pregnenolone sulfate, were purchased from Sigma-Aldrich (St. Louis, MO).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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1Q241W Mutation on Channel Activation by GABA. We first examined the activation of the 1Q241W mutant receptor by GABA. The goal of the experiments was to establish the baseline kinetic properties of the mutant receptor for subsequent studies of steroid modulation.
In whole-cell recordings, the GABA dose-response relationship of the 1Q241W receptor was shifted to lower agonist concentrations compared with the wild-type receptor. The midpoint of the dose-response curve was at 2.5 ± 0.2 µM, and the Hill slope was 1.0 ± 0.1 (Fig. 2A).
The principal characteristics of single-channel activity were unchanged by the mutation. Similar to what we have observed previously for wild-type receptors (e.g., Steinbach and Akk, 2001; Li et al., 2007b), exposure to high concentrations of GABA led to channel activity in easily identifiable single-channel clusters. Figure 3 shows sample currents from the mutant receptor exposed to 2, 10, or 50 µM GABA. At 2 µM GABA, channel activity was characterized by isolated openings interspersed with brief bursts of activity, but no single-channel clusters were evident. When channel activity was elicited by 10 or 50 µM GABA, the openings were condensed into 1- to 10-s clusters of activity.
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The 1Q241W mutation resulted in an increase in the relative frequency of the longest-lived open-time component, OT3. At GABA concentrations evoking equivalent responses, the prevalence of OT3 is 13 ± 4% in the wild-type receptor (Li et al., 2007b) and 52 ± 12% in the 1Q241W mutant receptor (Table 1). In addition, there was a trend toward an increase in the duration of OT3 in the mutant receptor. As a result, the mean open duration was enhanced from 2.1 ± 0.3 to 5.6 ± 0.6 ms in the presence of the 1Q241W mutation.
In the presence of 10 to 100 µM GABA, the intracluster closed-time distributions contained three components. At 10 µM GABA, the mean durations of the closed-time components (see also Table 2) were 0.13 ± 0.01 ms (58%; CT1), 1.9 ± 0.4 ms (27%; CT2), and 20.3 ± 2.0 ms (15%; CT3). In the presence of 100 µM GABA, the mean durations and fractions of the closed times (n = 4 patches) were 0.18 ± 0.04 ms (63%; CT1), 1.7 ± 0.2 ms (29%; CT2), and 8.8 ± 2.7 ms (8%; CT3).
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We have previously postulated that the intracluster closed-time histograms from the wild-type receptor contain a component whose duration (but not prevalence) depends on agonist concentration, as well as several components whose durations are not modulated by changes in agonist concentration (Steinbach and Akk, 2001). The agonist concentration-dependent component (CTβ) results from dwells in unliganded, monoliganded, and diliganded closed states. The lifetime of the CTβ component inversely correlates with agonist concentration because the movement from states with lower ligation status to the diliganded open state involves the binding of agonist. The agonist concentration-independent closed states probably result from dwells in various blocked or short-lived desensitized states. The relative frequencies of the closed states are not affected by GABA concentration (Steinbach and Akk, 2001).
It is typically not feasible to resolve all states at a given GABA concentration; i.e., the CTβ state may overlap in duration with dwells in the blocked or short-lived desensitized states, but, by altering the agonist concentration, it is possible to manipulate the duration of the CTβ state and separate it from other closed-time components. For example, we previously showed that the single-channel clusters from the wild-type receptor contain a long-lived (10-20 ms) but infrequent (2% of all intracluster closed events) closed-time component (Steinbach and Akk, 2001). We associated this closed event with a short-lived desensitized state (CTSD), because it seemed to be related to the long-lived closed times observed among channel openings after a brief pulse of agonist (Jones and Westbrook, 1995). In the presence of 50 µM GABA, where the activation-related closed-time component is prolonged, the CTβ and CTSD states have indistinguishable life-times, and both states contribute to the longest-lived intracluster closed-time component (CT3). In contrast, at saturating GABA concentrations, where the activation-related closed times are brief, the infrequent CTSD state is the sole contributor to the long-lived CT3 closed-time component.
Single-channel currents from the 1Q241W mutant receptor indicated the presence of a similar, long-lived closed state. At 100 µM GABA (a saturating concentration), the longest-lived closed component (CT3) had a relative frequency of 8%, but when the receptors were activated by 10 µM GABA, the prevalence of CT3 was 15%. We suggest that the CT3 component at saturating GABA concentrations consists of dwells in the short-lived desensitized state (CTSD), whereas at 10 µM GABA, the CT3 component contains dwells in the CTSD state as well as sojourns in the mono- and unliganded closed states (i.e., CTβ). We can therefore estimate the prevalence of CTβ by subtracting the relative frequency of CTSD, determined at 100 µM GABA (8 ± 3%), from the relative frequency of CT3 measured at 10 µM GABA (15 ± 4%). Assuming that the relative frequencies of the two states are unaffected by changes in the GABA concentration, we get a prevalence of approximately 7% for the activation-related closed-time component. This value is lower than that in the wild-type receptor (27 ± 6%; Li et al., 2007b).
In sum, kinetic analysis of the single-channel data from the 1Q241W mutant receptor shows both similarities to and differences from the wild-type data. In the presence of elevated GABA concentrations, the mutant receptor single-channel activity consists of high-open-probability clusters that contain three open-time and three closed-time components. However, the properties of the open and closed states are dissimilar to those in the wild-type receptor. The prevalence and duration of the longest-lived open-time component (OT3) are increased, and the prevalence of the activation-related closed-time component (CTβ) is decreased compared with the wild-type receptor. These are the same kinetic parameters that are affected when the wild-type receptor is activated by GABA in the presence of neurosteroid 35P (Akk et al., 2005).
The 1Q241W Mutant Receptor Is Not Potentiated by 35P. Previous work (Hosie et al., 2006), confirmed by us (Fig. 2B), showed that 35P is ineffective at potentiating macroscopic currents from the 1Q241W mutant receptor. To determine the effect of the steroid on single-channel currents, we compared single-channel currents elicited by 10 µM GABA in the absence and presence of 1 µM 35P. This steroid concentration elicits maximal potentiation in the wild-type receptor. As predicted by macroscopic recordings, the presence of steroid did not affect the kinetic properties of single-channel activity. Sample currents are shown in Figs. 4, A and B, and the data are summarized in Tables 1 and 2.
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1β22L GABA-A receptor, 35P potentiates the currents by increasing the duration and prevalence of OT3 and by decreasing the prevalence of CT3 (Akk et al., 2005). As a result, the mean open time is enhanced and the mean intracluster closed time is reduced in the presence of steroid. Our findings indicate that none of these changes occur when the glutamine residue in the 241 position is replaced with tryptophan. But the baseline values for the duration and prevalence of OT3 and the prevalence of CT3 in the 1Q241W receptor are similar to those observed with the wild-type receptor in the presence of steroid, suggesting that the amino acid substitution mimics the presence of steroid.
The 1Q241W Mutation Modifies Channel Activation by Piperidine-4-sulfonic Acid. Piperidine-4-sulfonic acid (P4S) is a high-affinity, low-efficacy agonist of the GABA-A receptor. In macroscopic recordings, the concentration producing half-maximal response from wild-type 1β22L receptors is 16.5 ± 0.3 µM (i.e., similar to that for GABA), but the peak current elicited by saturating concentrations of P4S is only 63 ± 13% of that for GABA (data not shown). Unlike GABA, P4S, even at saturating concentrations, elicits single-channel currents best-characterized as monotonous, low open probability episodes of activity (Fig. 5A) (also Steinbach and Akk, 2001). The channel open-time histograms contain two components, with mean durations resembling those of OT1 and OT2 for receptors activated by GABA. The studies of closed times are typically inconclusive because the number of active receptors in the patch producing the single-channel activity is unknown.
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Coapplication of 35P with P4S cardinally changes the mode of activity. Instead of isolated openings, channel activity in the presence of the steroid takes place in easily identifiable clusters. Sample recordings from the wild-type receptor activated by 1 mM P4S + 1 µM 35P are shown in Fig. 5B. The analysis of intracluster open time histograms revealed the emergence of the third, long-lived open state. Averaged from 5 patches, the mean open times were 0.21 ± 0.04 ms (33%; OT1), 1.4 ± 0.5 ms (28%; OT2), and 8.4 ± 3.1 ms (40%; OT3).
The data described above suggested that in the presence of GABA, the 1Q241W mutation acts by mimicking the effects of steroid. We were interested in testing whether channel activation by P4S is similarly modified by the mutation. Accordingly, we next examined the activation of the 1Q241W mutant receptor by P4S. The major finding was that mutant receptors activated by P4S exhibited clear-cut clusters that contained three classes of open events. Sample single-channel activity is shown in Fig. 5C. At 1 mM P4S, the intracluster open- and closed-time histograms contained three components. The mean open times (averaged from four patches) were 0.41 ± 0.21 ms (23%; OT1), 3.1 ± 1.0 ms (66%; OT2), and 8.3 ± 3.5 ms (11%; OT3).
The presence of the long-lived (
8 ms) OT3 component in the 1Q241W receptor activated by P4S is qualitatively similar to the emergence of OT3 when the wild-type receptor is activated by P4S + 35P. We conclude that the 1Q241W mutation and the presence of steroid 35P have qualitatively similar effects on GABA-A receptor activation by P4S.
The 1Q241W Mutation Does Not Affect GABA-A Receptor Inhibition by Pregnenolone Sulfate. Pregnenolone sulfate is an endogenous neurosteroid that inhibits GABA-A receptor activation (Majewska et al., 1988). In macroscopic recordings, the effect manifests as an increase in the apparent rate of desensitization (Shen et al., 2000). Previous studies have suggested that distinct, nonoverlapping sites mediate the inhibitory effect of pregnenolone sulfate and the effects of potentiating neuroactive steroids (Park-Chung et al., 1999; Akk et al., 2001). Here, we sought to confirm this by probing the effect of the 1Q241W mutation on channel modulation by pregnenolone sulfate. We hypothesized that if the site mediating the effect of pregnenolone sulfate were distinct from the 35P binding site, then the 1Q241W mutation would be likely to have no effect on channel modulation by the inhibitory steroid.
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1Q241W mutant receptors. The receptors were activated by a saturating concentration of GABA (1 mM for wild type, 250 µM for the mutant) in the absence and presence of 2 to 50 µM pregnenolone sulfate. In the wild-type receptor, the major effect of pregnenolone sulfate was a dose-dependent enhancement in the apparent rate of desensitization. In control recordings from cells exposed to 1 mM GABA, the desensitization time constant was 6.3 ± 2.8 s (n = 5 cells). When 50 µM pregnenolone sulfate was coapplied with GABA, the decay time constant was 656 ± 172 ms (n = 5 cells). In cells expressing 1Q241W mutant receptors, the mode of action of pregnenolone sulfate was analogous. The decay time constant was 4.1 ± 1.8 s (n = 4 cells) in the presence of GABA and 413 ± 78 ms (n = 4 cells) in the presence of GABA + 50 µM pregnenolone sulfate. In addition, there was a slight (<10%) decrease in peak response in some cells exposed to pregnenolone sulfate. Sample current traces are shown in Fig. 6, A and B.
The rate of development of block in the presence of 50 µM pregnenolone sulfate can be estimated from the relationship k+PS = {1/
decay(PS) - 1/des}/50 µM, where des is the macroscopic current desensitization time constant in the presence of GABA, and decay(PS) is the decay time constant in the presence of GABA + pregnenolone sulfate. We estimate that the k+PS was 0.03 ± 0.01 µM-1s-1 in the wild-type receptor and 0.04 ± 0.01 µM-1s-1 in the 1Q241W mutant receptor.
We examined the concentration-dependence of the effect of pregnenolone sulfate by comparing the area of the response (total charge transfer) during a 4-s application of GABA and 2 to 50 µM steroid (Fig. 6C). The data show that pregnenolone sulfate had a half-maximal effect at 7.4 ± 0.4 and 7.7 ± 2.2 µM on wild-type and mutant receptors, respectively. In sum, we infer that the 1Q241W mutation is without effect on channel modulation by the inhibitory steroid pregnenolone sulfate, indicating that distinct sites underlie channel modulation by potentiating and inhibitory steroids.
Properties of Activation, and Lack of Modulation by 35P of the 1Q241L Mutant Receptor. We next examined the activation properties of the 1Q241L mutant receptor. This mutation also blocks potentiation of macroscopic currents by 35P (Hosie et al., 2006; Fig. 2B), and we were curious to see whether the leucine substitution, similar to the more bulky tryptophan substitution, mimicked the presence of the steroid.
At GABA concentrations of 50 to 1000 µM, clear-cut clusters were observed (Fig. 7, A and B). The intracluster open-time distributions contained two components. In the presence of 1 mM GABA (a saturating concentration), the open times were 0.79 ± 0.03 ms (47%) and 1.9 ± 0.3 ms (53%) (averaged from three patches). The open-time distributions were unchanged when the GABA concentration was reduced to 50 µM (Table 1). The intracluster closed-time histograms were best fit to three exponentials. When the receptors were exposed to 1 mM GABA, the closed-time components had mean durations and prevalence of 0.23 ± 0.07 (57%), 1.0 ± 0.2 ms (35%), and 13.3 ± 5.1 ms (8%). In the presence of 50 µM GABA, the prevalence of the CT3 component was 29% (Table 2). By applying the reasoning used above in the analysis of single-channel activity from the 1Q241W mutant receptor, we estimate that the prevalence of CTβ is approximately 21% in the 1Q241L receptor. This value is comparable with the prevalence of CTβ in the wild-type receptor (27 ± 6%; Li et al., 2007b), and we conclude that the leucine substitution, in contrast to the tryptophan substitution, does not decrease the fraction of CTβ. The intracluster open-time histograms from the 1Q241L mutant lacked the long-lived OT3 component, and it is thus not possible to directly compare the open-time data from the wild-type and 1Q241L receptors. Nonetheless, the 1Q241L mutation, unlike the 1Q241W, did not result in a frequent, long-lived open-time component.
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5P on channel activation elicited by 50 µM GABA. Sample currents are shown in Fig. 7C, and the data are summarized in Tables 1 and 2. The data indicate that the presence of steroid is without effect on the intracluster kinetic parameters. These findings are consistent with a model where the leucine substitution disrupts the interaction of 35P with the receptor.
The 1Q241S Mutation Modifies Channel Potentiation by 35P. In macroscopic recordings, the 1Q241S mutation shifts the steroid potentiation curve to higher steroid concentrations but has no effect on maximal potentiation (Hosie et al., 2006; Fig. 2B). The previous results (see above) had indicated that mutations to 1Gln241 produced changes in potentiation by neurosteroids and in the kinetic properties of single-channel currents elicited by GABA alone. Accordingly, it was of interest to examine the consequences of a mutation of this residue, which had relatively small effects on potentiation.
Single-channel clusters were recorded at 50 and 1000 µM GABA (Fig. 8, A and B). At 1 mM GABA, the open-time distributions of currents from the 1Q241S receptor contained two components with mean durations and fractions of 0.76 ± 0.23 ms (75%) and 2.0 ± 1.0 ms (25%) (n = 3 patches). Lowering the GABA concentration to 50 µM did not alter the open time distributions (Table 1).
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5P on channel activation elicited by 50 µM GABA. Sample currents are shown in Fig. 8C, and the summary of the effects of the presence of steroid is given in Tables 1 and 2. The data indicate that 35P has a complex effect on the intracluster open- and closed-time distributions. The mean duration of the longer-lived open-time component was increased in the presence of 35P. This effect is in qualitative agreement with the findings obtained from the wild-type receptor. However, we note that, in contrast to its effect on wild-type receptors, the application of 35P did not lead to a statistically significant increase in the prevalence of long-lived openings. The presence of 35P also did not affect the prevalence of CT3 in the 1Q241S mutant receptor. These findings indicate that the mutation modifies the mode of action of neurosteroid 35P on the GABA-A receptor.
The 1S240L but Not the 1Q241L Mutation Disrupts Channel Potentiation by a 3-Hydroxymethyl Steroid. Lack of potentiation by 35P in the 1Q241L mutant has been proposed to stem from the inability of the leucine residue to act as an H-bond acceptor to the C3-OH group of the steroid (Hosie et al., 2006). Previous studies have shown that steroid analogs with substitutions other than the hydroxyl group in the C3 position can be efficacious potentiators of the GABA-A receptor. For example, pregnane steroids with a carboxylic acid, or its amide derivative, at C3 are positive modulators of GABA-A receptor activity (Mennerick et al., 2001). The greater distance between the backbone of the steroid's A ring and the H-bonding group on C3 in such steroids as well as the rotational freedom of the C3 substituents suggests that the steroid may be interacting with a locus other than the one used by 35P, and so we hypothesized that such steroids may remain capable of potentiating receptors containing the 1Q241L mutation.
To test this hypothesis, we compared wild-type and 1Q241L mutant channel potentiation by the steroid 3CH2OH5βP (Fig. 9A) that has a hydroxymethyl group at C3. Sample currents and steroid dose-response curves are shown in Fig. 9, B to D. The data demonstrate that the 1Q241L mutation has minimal effect on channel potentiation by 3CH2OH5βP. The steroid, at 10 µM, potentiated macroscopic responses elicited by an EC20-25 concentration of GABA from the wild-type and mutant receptors by 2.7 ± 0.4-fold (n = 5 cells) and 2.2 ± 0.2-fold (n = 4 cells), respectively.
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1Gln241 on channel potentiation by 35P and 3CH2OH5βP. First, we tested the effect of substituting leucine for serine in position 240.
The GABA dose-response curve from the 1S240L mutant receptor was shifted to higher agonist concentrations, having an EC50 of 38 ± 4 µM (Fig. 10A). The steroid effects were examined in the presence of GABA concentrations producing peak responses equal to 10 to 20% of maximal current. We found that coapplication of 3CH2OH5βP with GABA did not lead to potentiation of peak response in the 1S240L receptor (Fig. 10, B-D). The peak response was 109 ± 39% of control (n = 8 cells) when 10 µM 3CH2OH5βP was coapplied with 10 or 25 µM GABA.
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In contrast, the mutation was essentially without effect on potentiation by 35P. Application of 3 µM 35P potentiated currents from the 1S240L mutant to 284 ± 103% of control (n = 7 cells) indicating that the 1Ser240 residue is critical in channel modulation by 3CH2OH5βP but not 35P.
Effects of Mutations to 1Trp245 and 1Ser243 Residues on Channel Potentiation by 3CH2OH5βP and 35P. We next examined the effects of leucine mutations to residues 1Trp245 and 1Ser240 on channel activation by GABA, and modulation by 3CH2OH5βP and 35P. In the 1W245L mutant receptor, the EC50 for GABA-elicited currents was 24 ± 2 µM (Fig. 10A). The effects of steroids were studied in the presence of 5 or 10 µM GABA. The data show that the steroids were ineffective at producing channel modulation (Fig. 10D). Coapplication of 10 µM 3CH2OH5βP with GABA resulted in peak current of 101 ± 15% of control (n = 9 cells). When 3 µM 35P was coapplied with GABA, the peak response was 108 ± 32% of control (n = 11 cells).
To test the possibility that the 1W245L mutation has a more global effect on receptor function preventing channel potentiation per se, we recorded whole-cell responses in the presence of pentobarbital. This GABA-A receptor modulator potentiates current responses at micromolar concentrations. The kinetic mechanism of action of pentobarbital resembles that of neuroactive steroids (e.g., Steinbach and Akk, 2001; Akk et al., 2004), which might imply commonality of the transduction elements involved in channel modulation by the two drugs, but the effects are considered to be mediated by drug interactions with distinct sites (Akk et al., 2004; Hosie et al., 2006). We reasoned that if the 1W245L mutation allows potentiation by pentobarbital, then this serves as indication of lack of global changes in receptor function. In five cells expressing the 1W245L mutant receptor, coapplication of 100 µM pentobarbital with 5 µM GABA (EC10) increased the peak response to 906 ± 254% of control. We interpret the findings to indicate the selective involvement of the 1Trp245 residue in steroid actions.
As a negative control, we examined the effect of the 1S243L mutation on channel modulation by potentiating steroids. The -helical configuration of the M1 membrane-spanning domain places the sidechain of this residue essentially in the opposite (compared with the 1Gln241 residue) surface of the domain, and we hypothesized that mutations to this residue would have minimal effect on channel modulation by either 35P or 3CH2OH5βP. The 1S243L mutation shifted the GABA dose-response curve by almost 10-fold to higher concentrations (EC50 = 80 ± 3 µM). Coapplication of 3 µM 35P or 10 µM 3CH2OH5βP with 30 µM GABA enhanced the peak response to 374 ± 41% (n = 6 cells) or 183 ± 11% (n = 8 cells) of control, respectively. We conclude that the 1S243L mutation has minimal effect on channel potentiation by 35P and 3CH2OH5βP.
Mutations to the M1 Domain Residues Affect Channel Modulation by 35βP. We next examined the effects of the mutations to the M1 membrane-spanning domain on channel modulation by 35βP. Single-channel experiments have shown that 35P and 35βP are kinetically similar as modulators of the GABA-A receptor currents (Akk et al., 2005; Li et al., 2007a). Previous work employing mutations to the 1Gln241 site has indicated that 35P and 35βP may interact with the same site to potentiate the GABA-A receptor (Hosie et al., 2006). In contrast, Mennerick et al. (2004) showed that a steroid analog (3,5)-17-phenylandrost-16-en-3-ol (17-PA) selectively antagonized channel potentiation and direct activation by 35P but not 35βP, suggesting that different sites mediate the effects of these steroids. Here, we have evaluated the effects of mutations 1S240L, 1Q241L, 1S243L, and 1W245L on channel potentiation by 35βP to compare the differential effect that these mutations have on channel potentiation by 5- and 5β-reduced steroids.
The summary of results is given in Fig. 11. Coapplication of 3 µM 35βP with GABA resulted in potentiation of peak current in 1β22L wild-type receptors and 1S243L mutant receptors but not when the receptor contained the 1S240L, 1Q241L, or 1W245L mutation. Potentiation of receptors containing the 1S243L mutation is in agreement with our data suggesting that the orientation of the 1Ser243 residue is such that substitutions here have little effect on modulation by steroids. The lack of potentiation in the 1W245L mutant is similar to the effect of this mutation on modulation by 35P, confirming our earlier conclusion that this residue is critical to channel potentiation by neuroactive steroids. The lack of potentiation by 35βP in the 1Q241L mutant is also similar to the effect of the mutation on modulation by 35P. But the 1S240L mutation had different effects on potentiation by 35P and 35βP. The mutation fully abolishes potentiation by 35βP (107 ± 19% of control, n = 5 cells) but is without effect on potentiation by 35P (Fig. 10B). Thus, mutations to the 1 subunit M1 domain differentially affect channel modulation by 35P and 35βP, suggesting that the two steroids are oriented differently in the binding pocket.
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-Pregnan-20-one. The data presented above on the effects of the mutations to the 1Ser240 and 1Gln241 residues on channel potentiation by 35P and 3CH2OH5βP could be interpreted as indicating that the hydroxyl group of 35P interacts, possibly via H-bonding, with the 1Gln241 residue, and the hydroxymethyl group of 3CH2OH5βP interacts with the 1Ser240 residue. On the other hand, the finding that potentiation by 35βP is sensitive to the nature of the residue in both the 240 and 241 positions and the single-channel data on the 1Q241S mutation that retains but modifies the mechanism of potentiation by 35P are not fully compatible with this simple model.
A possible explanation is that the 1Gln241 residue is not directly interacting with the C3-OH group of the steroid molecule but is rather a necessary component to appropriately shape the binding surface to accommodate the steroids 35P and 35βP. To explore this hypothesis, we examined receptor modulation by 3deoxy5P. This steroid (Fig. 12A) is devoid of H-bonding groups on C3 and is therefore predicted to not interact, at least via H-bonding, with the residues in the 1 subunit M1 domain. The interaction of 3deoxy5P with the wild-type receptor could be considered functionally analogous to 35P interaction with the 1Q241L mutant receptor.
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We examined macroscopic currents from the wild-type receptor exposed to 5 µM GABA in the absence and presence of 0.2 to 5 µM 3deoxy5P. Channel potentiation was observed at steroid concentrations 0.5 µM and higher, reaching maximal potentiation at 1 µM 3deoxy5P. Exposure to 1 µM 3deoxy5P increased the peak response to 202 ± 16% of control (n = 5 cells; Fig. 12, B and C). We suspect that poor solubility of the steroid in aqueous solutions prevented stronger potentiation when nominally higher steroid concentrations were used. In any case, channel potentiation by a steroid that is devoid of groups on C3 that can form an H-bond indicates that an H-bond between the A ring of the steroid and the receptor is not required to produce channel potentiation.
We also examined the effect of the 1Q241L mutation on channel potentiation by 3deoxy5P. The summary of the data and sample currents are shown in Fig. 12, B and D. The data demonstrate that the steroid is unable to potentiate currents from the 1Q241L mutant receptor. Coapplication of 1 µM 3deoxy5P with 20 µM GABA (EC15) led to peak response of 97 ± 1% of control (n = 7 cells). This finding is not consistent with a model where the lack of steroid potentiation in a receptor containing the 1Q241L mutation is due to the inability of the leucine residue to form an H-bond with the C3-OH group.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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1 subunit M1 membrane-spanning region on channel modulation by neuroactive steroids. A previous report (Hosie et al., 2006) had implicated the 1Gln241 residue in the actions of neurosteroids by showing that when the glutamine residue was replaced with a tryptophan or leucine residue, potentiation by 35P and 35βP was greatly diminished. It was concluded that the 1Gln241 interacts with the steroid 3-hydroxyl group (Hosie et al., 2006). The goal of the present work was to provide a more detailed kinetic and pharmacological characterization of the effects of mutations on channel activation, and modulation by neurosteroids. Overall, our data are consistent with the 1Gln241 residue acting as a critical site for channel modulation by 35P. However, our data are most consistent with a model in which the 1Gln241 residue forms a crucial intraprotein contact rather than participates in direct interaction with the C3-OH group. We have identified an additional residue in the 1 subunit M1 domain (1Ser240) that is required for modulation by the steroid analog 3CH2OH5βP but not 35P and a residue (1Trp245) that may participate as a transduction element in channel potentiation by steroids but not by pentobarbital.
The 1Q241W and 1Q241L mutations affected the kinetic properties of channels activated by GABA. The tryptophan substitution enhanced channel opening efficacy, whereas the leucine substitution had a slightly deleterious effect on channel function. Our data confirm that the 1Q241W and 1Q241L mutations drastically diminish receptor potentiation by 35P, but we show that different mechanisms underlie the absence of potentiation in the mutant receptors. The 1Q241W mutation functionally mimics the presence of steroid, and receptors activated by GABA (or P4S) demonstrate single-channel kinetic properties found in wild-type receptors exposed to GABA (or P4S) in the presence of high concentrations of potentiating steroids. The coapplication of 35P with GABA had no further effect on mutant receptor activation.
The major effect of the 1Q241L mutation on channel activity was the loss of the longest-lived open-time component. The application of 35P did not modify single-channel or whole-cell currents elicited by GABA, consistent with the previous hypothesis (Hosie et al., 2006) that the leucine substitution interferes with the ability of 35P to interact with the steroid binding site.
As with all mutational studies, the possibility exists that the amino acid substitutions have a deleterious structural effect. Figure 13 shows a DOPE plot (Shen and Sali, 2006) of the M1 region, illustrating that there are no major energetic changes caused by the 1Q241W or 1Q241L mutations. DOPE provides a statistical potential for assessing the deviation of a particular structure from an idealized reference state, and the plots shown in Fig. 13 show the results of a sliding window of 13 residues through the models. Examining the M1 region, we see that the mutations studied produced small changes in this parameter, supporting the idea that no major structural consequences resulted from the mutations.
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1Q241S mutation on channel activation and 35P-induced potentiation. Hosie et al. (2006) had found that the 1Q241S mutation had little effect on steroid potentiation and concluded that the serine side-chain was able to replace glutamine as an H-bond acceptor and so retain potentiation. We confirm that 35P elicits potentiation of macroscopic currents (Fig. 2B) but find that the single-channel potentiation profile is distinct in the mutant and wild-type receptors. In the wild-type receptor, the presence of 35P results in increases in the duration and prevalence of the longest open-time component and a decrease in the prevalence of the activation-related closed-time component. In the 1Q241S mutant receptor, 35P has a strong effect on the mean duration of the longest-lived open-time component but is largely without effect on the prevalence of long openings. The presence of steroid has a complex effect on the intracluster closed-time distributions. However, the signature feature of steroid modulation in the wild-type receptor—a decrease in the prevalence of CT3—is absent in the mutant receptor.
In addition, we have shown that the 1Q241W mutation does not interfere with the actions of the inhibitory neurosteroid pregnenolone sulfate. This is in agreement with previous studies that have concluded that the sites of action for potentiating and inhibitory neuroactive steroids are distinct (Park-Chung et al., 1999; Akk et al., 2001).
It is noteworthy that the 1Q241L mutation did not affect channel potentiation by the steroid analog 3CH2OH5βP. Site-directed mutagenesis of residues in the vicinity of 1Gln241 led to the identification of two additional residues adjacent to (or within) the steroid binding pocket: 1Ser240 and 1Trp245. Mutation of the 1Ser240 residue to leucine disrupted the potentiating effect of 3CH2OH5βP but not 35P. The 1W245L mutation abolished potentiation by both steroids but left intact channel potentiation by pentobarbital and the marine cembranoid eupalmerin acetate (data not shown). In addition, the 1W245L mutation did not affect channel modulation by the inhibitory steroid pregnenolone sulfate (data not shown). We propose that the 1Trp245 residue participates as a transduction element in the actions of potentiating neuroactive steroids.
In single-channel recordings, some neuroactive steroids have three kinetically distinct effects on channel open- and closed-time distributions (Akk et al., 2004; Li et al., 2007a). Many of our previous findings could be interpreted as the effects being produced by steroid interactions with multiple nonoverlapping sites. The dose-response relationships for the effects on open and closed times can be different for a given steroid. For example, (3,5β,17β)-3-hydroxy-18-norandrostance-17-carbonitrile elicits an increase in the prevalence of OT3 with an EC50 of <100 nM, whereas the EC50 for the increase of the duration of OT3 is >10 µM (Akk et al., 2004). We have also found that the steroid etiocholanolone, which has a single kinetic effect (to increase the prevalence of OT3), does not compete with a steroid having three kinetic effects (35βP) suggesting that etiocholanolone is unable to bind to the sites that mediate the increase in the duration of OT3 and the decrease in the prevalence of CTβ (Li et al., 2007a). Additional evidence for multiple binding sites for steroids comes from the finding that potentiation by 5- but not 5β-reduced steroids is inhibited by a steroid analog 17-PA (Mennerick et al., 2004), and that the dose-response curves for steroid enhancement of muscimol-elicited 36Cl- uptake are biphasic (Morrow et al., 1990).
In contrast, the finding that the 1Q241L mutation abolishes all kinetically distinguishable effects that 35P exerts in the wild-type receptor suggests that the steroid interacts with a single site on the receptor to produce potentiation. The presence of all three types of effects in the receptor containing the 1Q241W mutation, which we believe mimics the presence of steroid, is similarly in favor of a single binding site for steroid.
We hypothesize that the steroid binding pocket presents a hydrophobic surface capable of accommodating steroid molecules of different structure. Different steroids, through interactions with nonoverlapping loci, elicit a particular combination of kinetic effects that lead to channel potentiation. The lack of competition between etiocholanolone and 35βP could be explained if the 1Gln241 residue were not a docking site for the steroid per se, but rather one of the necessary components to maintain the structure of the surface to which steroids bind. Mutations to the M1 domain differentially affect the structure of the binding surface so that 3CH2OH5βPremains capable of interacting with its binding site after the 1Q241L but not the 1S240L mutation, whereas 35βP requires both 1Ser240 and 1Gln241 intact to modulate receptor activity. In this model, differential sensitivity of 35P and 35βP to mutations in the M1 domain suggests that the two steroids bind to different loci, accounting for their different sensitivity to the steroid antagonist 17-PA.
Support for this hypothesis comes from the finding that the 1Q241S mutation alters the kinetic mode of action of 35P. The data showing that the 1Q241L mutation disrupts potentiation by 3deoxy5P is further indication that the glutamine-to-leucine mutation does not act by simply preventing H-bonding between the A ring of the steroid and the receptor. Instead, the mutation may alter the structure of the protein so that the binding site can no longer accommodate 3deoxy5P. By extension, the data suggest that the 1Gln241 residue does not constitute the docking site for neurosteroids.
In sum, the present study confirms the crucial role of the amino acid residues in the 1 subunit M1 domain in GABA-A receptor modulation by potentiating neurosteroids. The data demonstrate a critical role of the 1Gln241 residue in channel potentiation by 3-hydroxysteroids and 3deoxy5P. We identified two additional residues in the 1 subunit M1 membrane-spanning domain, 1Ser240 and 1Trp245, whose mutation interferes with the ability of potentiating neuroactive steroids to modulate the GABA-A receptor. Our data are most consistent with a model in which mutations to residues in the M1 membrane-spanning domain shape the binding surface on the GABA-A receptor to which multiple steroid molecules can bind.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: 35P, (3,5)-3-hydroxypregnan-20-one; 35βP, (3,5β)-3-hydroxypregnan-20-one; 3CH2OH5βP, (3,5)-3-hydroxymethylpregnan-20-one; DMSO, dimethyl sulfoxide; PDB, Protein Data Bank; DOPE, discrete optimized protein energy; 3deoxy5P, 5-pregnan-20-one; P4S, piperidine-4-sulfonic acid; 17-PA, (3,5)-17-phenylandrost-16-en-3-ol; PS, pregnenolone sulfate.
Address correspondence to: Gustav Akk, Department of Anesthesiology, Washington University School of Medicine, Campus Box 8054, 660 S. Euclid Ave, St. Louis MO 63110. E-mail: akk{at}morpheus.wustl.edu
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