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Originally published In Press as doi:10.1074/jbc.M603599200 on April 20, 2006

J. Biol. Chem., Vol. 281, Issue 25, 17034-17043, June 23, 2006
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An Asymmetric Contribution to {gamma}-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of {alpha}1, beta2, and {gamma}2 Subunits*

Tim G. Hales{ddagger}§1, Tarek Z. Deeb{ddagger}2, Haiyan Tang2, Karen A. Bollan, Dale P. King, Sara J. Johnson, and Christopher N. Connolly

From the {ddagger}Department of Pharmacology & Physiology and the §Department of Anesthesiology & Critical Care Medicine, The George Washington University, Washington, D. C. 20037 and the Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom

Received for publication, April 13, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations that impair the expression and/or function of {gamma}-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy {gamma}2(K289M) mutation affects a basic residue conserved in the TM2–3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys -> Met mutation in mouse {alpha}1(K278M), beta2(K274M), and {gamma}2(K289M) subunits. Compared with cells expressing wild-type and {alpha}1beta2{gamma}2(K289M) receptors, cells expressing {alpha}1(K278M)beta2{gamma}2 and {alpha}1beta2(K274M){gamma}2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of {alpha}1beta2(K274M){gamma}2 receptors coincided with reduced surface expression. By contrast the surface expression of {alpha}1(K278M)beta2{gamma}2 receptors was similar to wild-type and {alpha}1beta2{gamma}2(K289M) receptors suggesting that the {alpha}1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by {alpha}1(K278M)beta2{gamma}2 receptors had brief open times. To a lesser extent {gamma}2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of {alpha}1(K278M)beta2{gamma}2 receptors. By contrast, neither beta2(K274M) nor {gamma}2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the {alpha}1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2–3 loop lysine has an asymmetric function in different GABAA subunits.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid type A (GABAA)3 receptors belong to the homologous Cys-loop superfamily of ion channels that includes the nicotinic acetylcholine, 5-hydroxytryptamine type-3 and glycine receptors, and the Zn2+-activated ion channel (1, 2). GABAA receptors are pentameric, composed from distinct subunit classes, including {alpha} (1–6), beta (1–3), {gamma} (1–3), {delta}, {epsilon}, {theta}, and {pi}.

GABAA receptors mediate rapid synaptic inhibition. Impairment of GABAergic inhibition is associated with anxiety (3) and is the basis of several models of epilepsy (4). Several recently discovered GABAA mutations that reduce inhibition accompany hereditary forms of epilepsy (5–14). To date, the most thoroughly investigated of these are {gamma}2(R43Q) and {gamma}2(K289M), both associated with febrile seizures. Two mechanisms could account for reduced inhibition caused by these mutations: impaired receptor expression and/or function. The {gamma}2(R43Q) mutation alters receptor kinetics (13, 14), and this may contribute to inhibitory deficits. The mutation also reduces receptor biogenesis (15–18). Likewise {gamma}2(K289M) also impairs receptor function and expression (5, 14, 18).

The N-terminal {gamma}2 subunit Arg-43 residue is conserved across GABAA receptor subunits and systematic Arg -> Gln mutation in {alpha}1, beta2, and {gamma}2 uncovered a general role for the arginine in receptor assembly (17). Likewise the {gamma}2 subunit Lys-289 residue is conserved in the extracellular TM2–3 loops of GABAA and glycine receptors, suggesting that information can be revealed about its role by a similar comparative mutagenesis approach.

Mutation of the equivalent glycine receptor {alpha}1 subunit lysine is associated with hereditary hyperekplexia (19). Moreover, equivalent and/or nearby residues within the TM2–3 loops of the {alpha}7 and the beta2 nicotinic acetylcholine receptor subunits (20–22), {alpha}1 (23) and beta2 (24) GABAA receptor subunits, the glycine receptor {alpha}1 subunit (25, 26), and 5-hydroxytryptamine type 3A receptor (27), participate in channel gating.

The mechanism by which {gamma}2(K289M) reduces channel function is controversial. Baulac and colleagues (5) reported reduced current amplitude when compared with wild-type receptors upon expression of {alpha}1beta2{gamma}2(K289M) receptors in Xenopus oocytes. By contrast, Bianchi and colleagues (14) described faster GABA-evoked current deactivation without altered {alpha}1beta3{gamma}2(K289M)-mediated peak current or activation rate when compared with wild-type receptors expressed in human embryonic kidney (HEK293) cells. Using the same cells, examining the rate of current activation following laser initiated release of caged GABA onto {alpha}1beta2{gamma}2(K289M) and wild-type receptors, Ramakrishnan and Hess (28) concluded that the mutation reduced current activation rate. Homology modeling of the {alpha}1beta2{gamma}2 receptor onto the structural model of the Torpedo marmorata nicotinic acetylcholine receptor (29) revealed an additional possibility: that the K289M mutation may reduce single channel conductance (30).

The TM2–3 region may participate in the transduction of GABA binding to channel gating by coming into close proximity with N-terminal residues. Indeed an electrostatic interaction between the lysine residue on the {alpha}1 subunit and acidic residues in loops 2 and 7 may be responsible for intramolecular transduction, coupling GABA binding to channel opening (23).

To address the role of Lys-289 and equivalent lysine residues in the most common ({alpha}1beta2{gamma}2) GABAA receptor, we explored the effects of {alpha}1(K278M), beta2(K274M), or {gamma}2L(K289M) on receptor-surface expression and function. Each of the subunits exhibited distinct phenotypes when mutated, indicating an important but asymmetric contribution of this site to GABAA receptor function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—COS7 cells (ATCC CRL 1651) and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 units/ml penicillin in an atmosphere of 5% CO2. Exponentially growing cells were transfected by electroporation (400 V, infinity resistance, 125 mF, Bio-Rad Gene Electropulser II) in the case of COS7 cells and calcium phosphate precipitation, in the case of HEK293 cells (17). Cells were transfected with equimolar ratios of GABAA subunit cDNAs. Cells were analyzed 12–18 h and 24–96 h after transfection for biochemical and electrophysiological experiments, respectively.

DNA Constructions—Murine {alpha}1, beta2, and {gamma}2L subunit cDNAs containing the myc or FLAG tag (between amino acids 4 and 5 of the mature polypeptide) have been described previously (31) and shown to be functionally silent with respect to receptor pharmacology and physiology. The mutant expression constructs {alpha}1(K278M)Myc, beta2(K274M)Myc and {gamma}2L(K289M)Myc were generated by PCR. The fidelity of the final expression constructs was verified by DNA sequencing.

Antibodies—The 9E10 antibody was obtained from 9E10 hybridoma cells (32) and used directly as supernatant without purification. Antibodies to the FLAG epitope were purchased from Sigma. The secondary antibodies, goat anti-mouse Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488, were purchased from Molecular Probes (UK), and goat anti-mouse horseradish peroxidase from Amersham Biosciences.

Immunofluorescence—COS7 cells were fixed in 3% paraformaldehyde (in PBS) and washed twice in 50 mM NH4Cl (in PBS) and blocked (10% fetal bovine serum, 0.5% bovine serum albumin in PBS) for 30 min. Subsequent washes and antibody dilutions were performed in PBS containing 10% fetal bovine serum and 0.5% bovine serum albumin. Following surface labeling, cells were permeabilized by the addition of 0.5% Triton X-100 (10 min), and the immunofluorescence protocol was repeated from the NH4Cl step. Cells were examined using a wide-field imaging system (Improvision).

Quantification of Cell-Surface Expression—COS7 cells were plated into 96-well dishes. Eight transfections were used per dish (12 wells per transfection, with nine determinants for each condition). Cells were fixed in 3% paraformaldehyde (in PBS). Cell-surface detection was performed in the absence of detergent, and total expression levels were determined following Triton X-100 (0.5%, 15 min) treatment. Cells were washed twice in 50 mM NH4Cl (in PBS) and blocked (10% fetal bovine serum, 0.5% bovine serum albumin in PBS) for 1 h. Subsequent washes were performed in block. Receptor expression was determined using an horseradish peroxidase-conjugated secondary antibody and assayed using 3,3',5,5'-tetramethylenebenzidine (Sigma) as the substrate, with detection at 450 nm after 30 min, following the addition of 0.5 M H2SO4. The reaction rate was determined to remain linear for up to 1 h.

Electrophysiology—The whole cell patch clamp technique was used to record GABA-activated currents from HEK293 cells voltage-clamped at –60 mV. GABA (100 µM) was applied by local pressure ejection from low resistance micropipettes (33). In experiments investigating the modulation of GABA-evoked currents by bath applied flunitrazepam, GABA was applied for 1 s at ~EC10 concentrations. Data for concentration-response relationships were recorded by applying GABA or propofol for 4 s. The recording chamber was continuously perfused (5 ml/min) with an extracellular solution comprised of (in mM) NaCl, 140; KCl, 4.7; MgCl2, 1.2; CaCl2, 2.5; glucose, 10; and HEPES-NaOH, 10 (pH 7.4). The electrode solution contained (in mM): CsCl, 140; MgCl2, 2.0; EGTA, 11; ATP (Mg2+ salt) 3; and HEPES-CsOH, 10 (pH 7.4). Junction potentials were nulled with an open electrode in the recording chamber prior to each experiment. The liquid junction potential was trivial (~2 mV), and its inappropriate compensation was ignored. Experiments were performed at room temperature (20–24 °C). Macroscopic GABA-evoked currents were monitored by an Axopatch-200B amplifier, low pass filtered with a cut-off frequency of 2 KHz, and then recorded and digitized using a Digidata 1320A interface (Axon Instruments, Union City, CA) for acquisition at 10 kHz onto the hard drive of a personal computer. Currents were averaged and measured using pCLAMP 8.0 software (Axon Instruments).

Single Channel Recording—Single channel currents recorded from cell-attached and outside-out patches were low-pass filtered at 2 and 1 KHz, respectively (digitized at 10 KHz). Data were acquired as described previously (34). GABA was either applied to outside-out patches at 1 µM or 1 mM; there was no significant difference in the observed single channel conductances. GABA (1 mM) was applied to cell-attached patches through the recording electrode, which contained extracellular solution. Patches were voltage-clamped using electrode potentials provided in the figure legends. Sections of digitized data in which unitary events predominated were selected for analysis and were leak subtracted using Clampfit for the creation of all-points amplitude histograms and event lists using Fetchan (pCLAMP 8.0, Axon Instruments).

Analysis of Whole Cell Data—Graphs of GABA concentration-response relationships were fitted using the Hill equation as described previously (33). For fitting propofol concentration-response relationships (normalized to maximum GABA-evoked current) the Hill equation was modified as in Equation 1.

Formula 1(Eq. 1)
In this equation the whole cell current amplitude activated by propofol (IProp) is normalized to that activated by 10 mM GABA (IGMax). IPMax is the maximum amplitude of the propofol activated current relative to IGMax. EC50 is the concentration of propofol required to activate half of the maximum IProp, and H is the slope factor of the concentration-response relationship.

Current density measurements were calculated from each cell by dividing the peak GABA- or propofol-activated current amplitude (measured in picoamps (pA)) by the cell's capacitance (measured in picofarads (pF)).

Analysis of Single Channel Data—All-points amplitude histograms for single channel recordings were fitted with multiple Gaussians (least squares minimization) to amplitude histograms using the Simplex method within pSTAT (pCLAMP 8.0). The amplitude of the single channel current recorded from each patch was determined from the difference between the mean current amplitudes determined from the Gaussians fitted to the closed- and unitary open-state currents. Single channel conductances are reported as the chord conductance derived as {gamma} = i/(Vm–Erev), where i is unitary current amplitude, Vm is the holding potential, and Erev is the mean reversal potential of GABA-evoked single channels derived from linear fits to current-voltage relationships. In several outside-out patch recordings two unitary conductances were evident. Our analysis of single channels was restricted to the main state.


Figure 1
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  		FIGURE 1.
Alignment of TM2–3 extracellular loop of GABAA and glycine receptor subunits. Sequence alignment of {alpha}, beta, and {gamma} GABAA and glycine receptor subunits showing (boxed regions) conservation within each subunit class. An asterisk denotes residues conserved in most subunits. Amino acid numbering represents the mature proteins of the {alpha}1, beta2, and {gamma}2 subunits used in this study and the glycine {alpha}1 subunit.

 
To quantify channel open times event lists were generated from single channel data obtained from cell-attached patches. Fetchan (pCLAMP 8.0) was used to create event lists of unitary events recorded from cell-attached patches using the 50% threshold detection method (35), having determined baseline and open current amplitudes from all points amplitude histograms. Event lists were analyzed using pSTAT yielding values of mean channel open time. Event lists were also pooled from multiple patches to obtain a representative sample of events (≥4 patches). All such data were included in the open time histogram (10 bins per decade) plotted with a square root ordinate and logarithmic abscissa (36). The maximum likelihood method was used to fit the sum of three exponentials to open time histograms (pSTAT), omitting those events from the fit that were briefer than 0.15 ms that were compromised by the system dead-time (37).

Statistics—All data are expressed as the arithmetic mean ± S.E. Unless otherwise stated, statistical analysis involved analysis of variance (ANOVA) with the posthoc Tukey's test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Conservation of a TM2–3 Loop Lysine between GABAA and Glycine Receptor Subunits—A comparison of the primary sequence of GABAA receptor {alpha}, beta, and {gamma} subunits within the extracellular loop, between the transmembrane domains TM2–3, reveals a high level of conservation between subunits of the same class (Fig. 1). A consensus sequence R-LPK-Y exists between all {alpha}beta{gamma} subunits, with the proline residue being conserved in all members of the Cys-loop receptor superfamily, including receptors for acetylcholine, 5-hydroxytryptamine, and glycine. Interestingly, the lysine (Lys-289) residue, associated with epilepsy when mutated in the {gamma}2 subunit (5), is conserved in {alpha} and beta subunits (Arg in {gamma}3, {delta}, and {epsilon}, Asn in {pi}, and His in {theta}) as well as in glycine receptors, where it has been implicated in hyperekplexia (19). Intriguingly, this lysine residue present in the {alpha}1 (Lys-278) and beta2 (Lys-274) subunits has been implicated in the gating of GABAA receptors and may play a conserved and essential role in all subunits in receptor function (23, 24). We undertook, therefore, to examine the effect of the mutation in {alpha}1(K278M), beta2(K274M), and {gamma}2L(K289M) subunits.

The Role of Lysine at the Homologous Position in the {alpha}1 Subunit on Transport to the Cell Surface—To determine, qualitatively, the ability of the {alpha}1(K278M) subunit to access the cell surface, we examined its cellular distribution when expressed in COS7 cells. COS7 cells were used in these studies due to their clear definition of intracellular compartments (38). The existence of surface receptors was determined in the absence of detergent using anti-Myc antibodies and Alexa Fluor 488 secondary antibodies. Following permeabilization, cells were re-probed as above, using Alexa Fluor 568 secondary antibodies. As observed previously for wild-type {alpha}1Myc (31), the {alpha}1(K278M)Myc subunit could not access the cell surface when expressed alone (data not shown). When {alpha}1Myc was co-expressed with the beta2 subunit, there was robust cell-surface staining (Fig. 2A, upper right panel). Likewise, the co-expression of {alpha}1(K278M)Myc with the beta2 subunit produced robust cell-surface labeling (Fig. 2A, {alpha}*beta, lower right panel) as well as strong intracellular labeling. Identical results were observed when {alpha}1(K278M)Myc was co-expressed with beta2 and {gamma}2LFLAG subunits and immunofluorescence was performed via the FLAG epitope on the {gamma}2L subunit (Fig. 2B). Because the {gamma}2L subunit cannot access the surface in the absence of either the {alpha}1 or beta2 subunits (31), its surface expression is a faithful indicator of the presence of {alpha}1beta2{gamma}2L receptors. From hereafter `{gamma}2' refers to the {gamma}2L subunit, which was used throughout this study.

To quantify our observations, we used the cell ELISA technique to compare surface and total expression levels. Cell-surface expression (in the absence of detergent) of the {alpha}1(K278M)Myc is presented as a percentage of total (in the presence of detergent) levels and normalized to wild-type controls ({alpha}1Myc) performed in parallel. Using this approach (Fig. 2C), the cell-surface level for {alpha}1(K278M)Mycbeta2 receptors was 66 ± 11%, compared with the normalized wild-type {alpha}1Mycbeta2 receptor level of 100 ± 11%. Similarly, the cell-surface level for {alpha}1(K278M)Mycbeta2{gamma}2FLAG receptors was determined (via FLAG epitope) to be 87 ± 34%, compared with the normalized wild-type {alpha}1Mycbeta2{gamma}2FLAG receptors at 100 ± 13%. Thus, the presence of K278M in the {alpha}1 subunit does not have a major impact on biogenesis or the surface transport of {alpha}1(K278M)beta2{gamma}2 receptors.

The Role of Lysine at the Homologous Position in the beta2 Subunit on Transport to the Cell Surface—To determine, qualitatively, the ability of the beta2(K274M) subunit to access the cell surface, we examined its cellular distribution when expressed in COS7 cells. As observed previously for wild-type beta2Myc (31), the beta2(K274M)Myc subunit could not access the cell surface when expressed alone (data not shown). When beta2Myc was co-expressed with the {alpha}1 subunit, there was robust cell-surface staining (Fig. 3A, upper right panel). In contrast, when beta2(K274M)Myc was co-expressed with the {alpha}1 subunit there was no cell-surface labeling (Fig. 3A, {alpha}beta*; lower right panel). Instead, strong intracellular labeling within the endoplasmic reticulum was observed, as evidenced by the characteristic reticular pattern, typically observed in COS7 cells (38). We obtained identical results when co-expressing beta2(K274M)Myc with the {alpha}1 and {gamma}2FLAG subunits, performing immunofluorescence via the FLAG epitope on the {gamma}2 subunit to detect the presence of all three subunits (Fig. 3B). Quantification of these findings, using cell ELISA (Fig. 3C), revealed the cell-surface level for {alpha}1beta2(K274M)Myc receptors to be 4.2 ± 18.9%, compared with the normalized wild-type {alpha}1beta2Myc receptor at 100 ± 11%. Similarly, the cell-surface level of the {alpha}1beta2(K274M)Myc{gamma}2FLAG receptor was determined (via FLAG epitope) to be 12.7 ± 12.6%, compared with the normalized wild-type {alpha}1Mycbeta2{gamma}2FLAG receptor level of 100 ± 31%. Thus, the presence of K274M in the beta2 subunit perturbs receptor distribution, preventing the significant expression of cell-surface {alpha}1beta2(K274M){gamma}2 receptors.


Figure 2
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  		FIGURE 2.
Cell-surface expression of {alpha}1(K278M)beta2{gamma}2 receptors. A, immunofluorescence detection (via extracellular Myc epitope) of receptor ({alpha}1Mycbeta2 and {alpha}1(K278M)Mycbeta2) expression in COS7 cells, in the presence (TOTAL) or absence (SURFACE) of cell permeabilization. B, immunofluorescence detection (via extracellular FLAG epitope) of receptor ({alpha}1Mycbeta2{gamma}2FLAG and {alpha}1(K278M)Mycbeta2{gamma}2FLAG) expression in COS7 cells. Scale bar = 25 µm. C, quantification of cell-surface expression by ELISA. GABAA receptor ({alpha}1(K278M)Mycbeta2or {alpha}1(K278M)Mycbeta2{gamma}2FLAG) expression, detected in the presence or absence of cell permeabilization, was normalized to that of wild-type {alpha}1Mycbeta2 (via Myc) or {alpha}1Mycbeta2{gamma}2FLAG (via FLAG) receptors. Each value represents the mean ± S.E. of at least nine determinants in at least three independent experiments. The asterisk above the bar represents significant difference from control (p < 0.001, t test). The asterisk following a subunit denotes the presence of the K278M mutation.

 
The Role of Lysine in the {gamma}2 Subunit on Transport to the Cell Surface—To determine, qualitatively, the ability of the {gamma}2(K289M) to access the cell surface, we examined its cellular distribution when expressed in COS7 cells. As observed previously for wild-type {gamma}2Myc (31), the {gamma}2(K289M)Myc subunit could not access the cell surface when expressed alone, or in the presence of {alpha}1 or beta2 subunits (data not shown). Co-expression of {gamma}2Myc with {alpha}1 and beta2 subunits produced robust cell-surface staining (Fig. 4A, upper right panel). Likewise, co-expression of {gamma}2(K289M)Myc with {alpha}1 and beta2 subunits also produced robust cell-surface labeling (Fig. 4A, {alpha}beta{gamma}*; lower right panel) as well as strong intracellular labeling. To quantify these observations, we used the cell ELISA technique to compare surface and total expression levels. Cell-surface expression (in the absence of detergent) of the {alpha}1beta2{gamma}2(K289M)Myc receptor is presented as a percentage of total (in the presence of detergent) levels and normalized to wild-type controls ({alpha}1beta2{gamma}2Myc) performed in parallel. Using this approach (Fig. 4C), the cell-surface level for {alpha}1beta2{gamma}2(K289M)Myc receptors was 108 ± 26%, compared with the normalized wild-type {alpha}1beta2{gamma}2Myc receptor level of 100 ± 31%. Thus, the presence of the epilepsy mutation K289M in {gamma}2 did not perturb the biogenesis or the surface transport of {alpha}1beta2{gamma}2(K289M) receptors.


Figure 3
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  		FIGURE 3.
Cell-surface expression of {alpha}1beta2(K274M){gamma}2 receptors. A, immunofluorescence detection (via extracellular Myc epitope) of receptor ({alpha}1beta2Myc and {alpha}1beta2(K274M)Myc) expression in COS7 cells, in the presence (TOTAL) or absence (SURFACE) of cell permeabilization. B, immunofluorescence detection (via extracellular FLAG epitope) of receptor ({alpha}1beta2Myc{gamma}2FLAG and {alpha}1beta2(K274M)Myc{gamma}2FLAG) expression in COS7 cells. Scale bar = 25 µm. C, quantification of cell-surface expression by ELISA. GABAA receptor ({alpha}1beta2(K274M)Myc or {alpha}1beta2(K274M)Myc{gamma}2FLAG) expression, detected in the presence or absence of cell permeabilization, was normalized to that of wild-type {alpha}1beta2Myc (via Myc) or {alpha}1beta2Myc{gamma}2FLAG (via FLAG) receptors. Each value represents the mean ± S.E. of at least nine determinants in at least three independent experiments. The asterisks above the bars represent significant differences from control (p < 0.0005, t test). The asterisk following a subunit denotes the presence of the K274M mutation.

 
Asymmetric Effects of Mutant {alpha}1(K278M), beta2(K274M), and {gamma}2(K289M) Subunits on the GABA Concentration-Response Relationship—We transiently expressed wild-type or Lys -> Met mutant {alpha}1, beta2, and {gamma}2 GABAA receptor subunits and used the whole cell patch clamp technique to characterize their functional properties in HEK293 cells voltage-clamped at –60 mV. Receptors containing the mutant {gamma}2(K289M) subunit combined with wild-type {alpha}1 and beta2 subunits exhibited a similar GABA concentration-response relationship compared with wild-type {alpha}1beta2{gamma}2 receptors (Fig. 5A). The EC50 values were 10.5 ± 0.3 µM (n = 6) for the wild-type and 8.2 ± 1.0 µM (n = 5) for the {alpha}1beta2{gamma}2(K289M) mutant receptor. Next we compared peak GABA-activated current densities (calculated by normalizing maximum current amplitudes to cell membrane capacitances) in HEK293 cells expressing {alpha}1beta2{gamma}2 and {alpha}1beta2{gamma}2(K289M) receptors. Inclusion of the {gamma}2K289M mutant had no significant effect on the GABA-evoked current density (Fig. 5B). These data support our immunocytochemical demonstration of unaltered surface expression of receptors in the presence of the {gamma}2(K289M) subunit (Fig. 4C). Furthermore, flunitrazepam (100 nM) caused a similar potentiation of GABA-evoked currents mediated by {alpha}1beta2{gamma}2(K289M) and wild-type {alpha}1beta2{gamma}2 receptors (Fig. 5C).


Figure 4
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  		FIGURE 4.
Cell-surface expression of{alpha}1beta2{gamma}2(K289M) receptors. A, immunofluorescence detection (via extracellular Myc epitope) of receptor ({alpha}1beta2{gamma}2Myc and {alpha}1beta2{gamma}2(K289M)Myc) expression in COS7 cells, in the presence (TOTAL) or absence (SURFACE) of cell permeabilization. Scale bar = 25µm. B, quantification of cell-surface expression by ELISA. GABAA receptor ({alpha}1beta2{gamma}2(K289M)Myc) expression, detected in the presence or absence of cell permeabilization, was normalized to that of wild-type {alpha}1beta2{gamma}2Myc receptors. Each value represents the mean ± S.E. of at least nine determinants in at least three independent experiments. The asterisk following a subunit denotes the presence of the K289M mutation.

 
We next examined the effect of the equivalent Lys -> Met mutation in the {alpha}1 and beta2 subunits. Unlike the {gamma}2(K289M) mutant, both the {alpha}1(K278M) andbeta2(K274M) mutants caused a significant rightward shift in the GABA concentration-response relationships of {alpha}1(K278M)beta2{gamma}2 and {alpha}1beta2(K274M){gamma}2 receptors, respectively (Fig. 5A). The EC50 values were 42 ± 6 µM (n = 5) for the {alpha}1(K278M)beta2{gamma}2 receptor and 55 ± 3 µM (n = 5) for the {alpha}1beta2(K274M){gamma}2 mutant receptor. In both cases the mutant subunits caused a substantial decline in the current density, once again the beta2(K274M) mutant had the most deleterious effect (Fig. 5B). Neither the {alpha}1(K278M) nor the beta2(K274M) mutant subunit altered the potentiation of GABA-evoked currents by flunitrazepam (100 nM) (Fig. 5C).


Figure 5
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  		FIGURE 5.
The effect of K289M mutations on the functional expression of {alpha}1beta2{gamma}2 receptors. A, GABA concentration-response relationships recorded from wild-type {alpha}1beta2{gamma}2 (circles), {alpha}1beta2{gamma}2(K289M) (diamonds), {alpha}1(K278M)beta2{gamma}2 (triangles), and {alpha}1beta2(K274M){gamma}2 (squares) receptors. Current amplitudes are expressed as a fraction of the maximum current amplitude recorded from each cell (I/Imax). Data points (n ≥ 5) were fitted using a logistic equation yielding the following EC50 and Hill slope (h) values:{alpha}1beta2{gamma}2, EC50 = 10.5 ± 0.3µM, h = 1.1 ± 0.1; {alpha}1beta2{gamma}2(K289M), EC50 = 8.2 ± 1.0 µM, h = 1.2 ± 0.2; {alpha}1(K278M)beta2{gamma}2, EC50 = 42 ± 6 µM, h = 1.4 ± 0.2; {alpha}1beta2(K274M){gamma}2, EC50 = 55 ± 3 µM, h = 1.4 ± 0.1. B, maximal GABA-activated current densities expressed as current amplitude (picoamps (pA)) normalized to cell membrane capacitance (picofarads (pF)). Statistical analyses by ANOVA with posthoc Tukey's test: *, current density significantly below that of the wild-type{alpha}1beta2{gamma}2(p < 0.05). C, currents activated by GABA (applied at ~EC10 concentrations) mediated by wild-type {alpha}1beta2{gamma}2, {alpha}1beta2{gamma}2(K289M), {alpha}1(K278M)beta2{gamma}2, and {alpha}1beta2(K274M){gamma}2 receptors were enhanced to a similar percentage of control by flunitrazepam (100 nM). No significant difference (p > 0.05) was found using ANOVA with posthoc Tukey's test.

 
{alpha}1(K278M), beta2(K274M), and {gamma}2(K289M) Mutations Have No Effect on Single Channel Conductance—Structural models of the {alpha}1beta2{gamma}2 GABAA receptor place the conserved TM2–3 loop lysine residue in the outer vestibule where it could contribute to the conduction pathway (30). Such a location could imply a role for the basic residue in ion conduction in which case its replacement by the uncharged methionine would be expected to cause a reduction in single channel conductance. Assuming a stoichiometry of 2{alpha}:2beta:1{gamma} one would expect such an effect to be greatest when the Lys -> Met mutation is present in either {alpha} or beta subunits (39). We tested this hypothesis by recording single GABA-activated channels from outside-out patches excised from cells expressing {alpha}1beta2{gamma}2, {alpha}1(K278M)beta2{gamma}2, {alpha}1beta2(K274M){gamma}2, and {alpha}1beta2{gamma}2(K289M) receptors. In all cases there was a linear relationship between single channel current amplitude and voltage, with no difference in their equilibrium potentials (Fig. 6E). In all cases GABA-activated single channels with similar amplitudes were recorded from patches at –60 mV (Fig. 6, A–D). The chord conductances for unitary events mediated by {alpha}1beta2{gamma}2, {alpha}1(K278M)beta2{gamma}2, {alpha}1beta2(K274M){gamma}2, and {alpha}1beta2{gamma}2(K289M) receptors were 25.0 ± 0.5, 23.8 ± 0.4, 25.9 ± 0.4, and 23.8 ± 0.8 pS, respectively (Fig. 6F).


Figure 6
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  		FIGURE 6.
The effect on single channel conductance of mutating the conserved TM2–3 loop lysine to methionine. A–D, examples of channels activated by GABA (1 mM) applied transiently to outside-out patches excised from cells expressing {alpha}1beta2{gamma}2, {alpha}1(K278M)beta2{gamma}2, {alpha}1beta2(K274M){gamma}2, and {alpha}1beta2{gamma}2(K289M) receptors, respectively. Patches were voltage-clamped at –60 mV. All-points amplitude histograms for sections of data containing predominantly single channel openings were fitted with the sum of two Gaussians, representing closed and open state current distributions. Current amplitudes in the examples shown in A–D were –1.6, –1.7, –1.8, and –1.5 pA, respectively. E, single channel currents mediated by {alpha}1beta2{gamma}2 (circles), {alpha}1(K278M)beta2{gamma}2 (triangles), {alpha}1beta2(K274M){gamma}2(squares), and {alpha}1beta2{gamma}2(K289M) (diamonds) receptors exhibited a linear relationship to voltage and reversed near the Cl– equilibrium potential. F, cord conductances determined from data (n = 4), including those illustrated in A–D were not significantly different for any of the wild-type or mutant receptors. No significant difference (p > 0.05) was found using ANOVA with posthoc Tukey's test.

 
Asymmetric Effects of Mutant {alpha}1(K278M), beta2(K274M), and {gamma}2(K289M) Subunits on Channel Open Time—The Lys -> Met mutation in the {gamma}2 subunit increases the deactivation rate of macroscopic GABA-activated currents and shortens single channel mean open time in outside-out patch recordings (14). We examined the effect of the Lys -> Met mutation in {alpha}1, beta2, and {gamma}2 subunits on open time of single channels activated by GABA (1 mM) in cell-attached patches (Fig. 7). This approach ensures steady-state exposure of receptors to a saturating concentration of GABA. Stretches of data were analyzed in which unitary events occurred >90% of the time. Three methods for comparing channel open time of wild-type and mutant receptors revealed that both {alpha}1(K278M) and {gamma}2(K289M) made openings briefer, whereas beta2(K274M) had no effect (Fig. 7). The open time histogram generated from events mediated by wild-type {alpha}1beta2{gamma}2 receptors, when fitted with the sum of three exponentials (14, 37), revealed three time constants ({tau}1–3) representing open times of brief, medium duration, and long lasting events (Fig. 7A). The average open time of {alpha}1beta2{gamma}2 receptors was 3.9 ± 0.3 ms (n = 4) corresponding to the midpoint of the cumulative distribution curve (Fig. 7, E and F) a less conventional method of displaying the data that nevertheless provides a convenient comparison of the full data range (35). By contrast to wild-type receptors, {alpha}1(K278M)beta2{gamma}2 receptors exhibited brief openings with only a minor contribution of long lasting events represented by the {tau}3 component (Fig. 7B). The mean open time of {alpha}1(K278M)beta2{gamma}2 receptors (1.4 ± 0.4 ms, n = 7) was significantly briefer (p < 0.001) than the mean open times of both wild-type {alpha}1beta2{gamma}2 receptors and mutant {alpha}1beta2(K274M){gamma}2 receptors (3.5 ± 0.4 ms, n = 5) (Fig. 7F). The open times of {alpha}1beta2(K274M){gamma}2 receptors were similar to those of wild-type receptors suggesting that the conserved lysine in the beta2 subunit does not participate in gating kinetics. By contrast, but in agreement with a previous report (14), {alpha}1beta2{gamma}2(K289M) receptors have disrupted gating kinetics (Fig. 7D) with a briefer mean open time (2.4 ± 0.2 ms, n = 8) compared with both wild-type {alpha}1beta2{gamma}2 receptors and mutant {alpha}1beta2(K274M){gamma}2 receptors (p < 0.05, Fig. 7F). These data demonstrate that the Lys -> Met mutation in either the {alpha}1 or {gamma}2 subunits disrupts gating kinetics culminating in a reduction in mean GABA-gated channel open time.


Figure 7
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  		FIGURE 7.
The effect on channel open time of mutating the conserved TM2–3 loop lysine to methionine. A–D, examples of channels activated by GABA (1 mM) recorded in the cell-attached patch configuration from cells expressing {alpha}1beta2{gamma}2, {alpha}1(K278M)beta2{gamma}2, {alpha}1beta2(K274M){gamma}2, and {alpha}1beta2{gamma}2(K289M) receptors, respectively. The electrode potential was –60 mV; downward deflections represent outward currents caused by Cl– entering the cell. Open time histograms are illustrated for events pooled from multiple patches expressing each of the subunit combinations. Histograms were fitted with the sum of three exponentials (dark line). Individual exponential components are also included (gray lines). Open times derived from the fits ({tau}1, {tau}2, and {tau}3) and the relative area of each exponential are listed. The calibration bars in D also serve single channel current traces in A–C. E, an alternative method for visualizing the effect of the Lys -> Met mutation in each subunit on open time is the cumulative distribution plot of the same data used for histograms in A–D (35). F, the histogram indicates the effect of the Lys -> Met mutation on mean open time. Statistical significance was determined using ANOVA with posthoc Tukey's test: *, p < 0.05 compared with WT {alpha}1beta2{gamma}2; **, p < 0.001 compared with both WT {alpha}1beta2{gamma}2 and {alpha}1beta2(K274M){gamma}2. Data were obtained from between four and eight cell-attached patches.

 


Figure 8
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  		FIGURE 8.
The effect on direct activation by propofol of mutating the conserved TM2–3 loop lysine to methionine. A, currents activated by propofol (3, 10, 30, 100, and 300 µM) applied to HEK293 cells expressing {alpha}1beta2{gamma}2(top) and {alpha}1(K278M)beta2{gamma}2 receptors (bottom). Arrows indicate surge currents seen upon cessation of propofol (300 µM) administration. B, concentration-dependent activation and block of {alpha}1beta2{gamma}2 (filled circles) and {alpha}1(K278M)beta2{gamma}2 (open circles) receptors by propofol normalized to GABA (10 mM)-evoked current amplitudes recorded from the same cells. Data points (excluding 300 µM) were fitted with the Hill equation ("Experimental Procedures") and values of EC50, slope, and efficacy relative to GABA are provided in the text. C, concentration-dependent activation and block of {alpha}1beta2(K274M){gamma}2 (filled triangles) and {alpha}1beta2{gamma}2(K289M) (open triangles) receptors by propofol normalized to GABA (10 mM)-evoked current amplitudes recorded from the same cells. The gray line represents the Hill equation fit to the wild-type {alpha}1beta2{gamma}2 data in B, provided here for comparison. Data were fitted as described in B, and values are provided in the text. D, maximal propofol-activated current densities expressed as current amplitude (picoamps (pA)) normalized to cell membrane capacitance (picofarads (pF)). Statistical analyses by ANOVA with posthoc Tukey's test: *, current density significantly below that of the wild-type {alpha}1beta2{gamma}2(p < 0.05).

 
Asymmetric Effects of Mutant {alpha}1(K278M), beta2(K274M), and {gamma}2(K289M) Subunits on the Propofol Concentration-Response Relationship—Mutation of Lys-278 and Lys-274 residues in {alpha}1 and beta2 subunits, respectively, may disrupt the intramolecular transduction of GABA binding to channel gating (23). If this is the case, such mutations may not affect the ability of anesthetics, such as propofol, to directly activate the GABAA receptor (40) through sites distinct from the GABA binding site (41). We examined the ability of propofol relative to GABA to activate {alpha}1beta2{gamma}2, {alpha}1(K278M)beta2{gamma}2, {alpha}1beta2(K274M){gamma}2, and {alpha}1beta2{gamma}2(K289M) receptors. Propofol activates GABAA receptors at concentrations between 3 and 100 µM. The blocking effect of propofol (33, 40, 41) reduces the amplitude of the propofol (300 µM)-activated current and is associated with a pronounced surge current upon cessation of application (Fig. 8A). The blocking effect complicates attempts to determine the EC50 of propofol (Fig. 8, B and C). Surge currents were negligible following the application of 100 µM propofol suggesting that there is minimal blockade with this concentration. Therefore we fitted data points between 3 and 100 µM propofol with the modified Hill equation to obtain estimates of EC50 values and the efficacy of propofol relative to GABA as an activator of current. We excluded the final data point obtained with 300 µM propofol due to blockade associated with this concentration. Using this approach the EC50 value for propofol as an activator of {alpha}1beta2{gamma}2 receptors was 32 ± 3 µM (slope factor = 2.0 ± 0.3) (Fig. 8B). The apparent maximum efficacy of propofol, relative to GABA (10 mM), was 56 ± 3%. The mutant {alpha}1(K278M) subunit reduced the apparent potency of propofol, shifting the concentration-response relationship to the right (Fig. 8B) without altering the maximum current amplitude activated by propofol (100 µM) relative to GABA (10 mM). Assuming an unaltered maximum efficacy of propofol relative to GABA (56%) the fit to the propofol concentration-response relationship provided an estimate of the EC50 of propofol as an agonist of the {alpha}1(K278M)beta2{gamma}2 receptor of 68 ± 2 µM (slope factor = 3.7 ± 0.2).

The EC50 values for propofol as an activator of {alpha}1beta2(K274M){gamma}2 and {alpha}1beta2{gamma}2(K289M) receptors were similar to that of the wild-type receptor: 31 ± 4 (slope factor = 3 ± 1) and 31 ± 2 µM (slope factor = 3 ± 1), respectively (Fig. 8C). The fits yielded the following values for the efficacy of propofol relative to GABA as an agonist at {alpha}1beta2(K274M){gamma}2 and {alpha}1beta2{gamma}2(K289M) receptors: 25 ± 2% and 62 ± 4%, respectively. Interestingly, although the Lys -> Met mutation in the beta2 subunit had no effect on the potency of propofol as an agonist, it caused a significant reduction in the efficacy of propofol (100 µM) as an agonist relative to GABA (10 mM) compared with the wild-type receptor (p < 0.05).

We determined the propofol current density by expressing peak propofol-activated current amplitudes as a function of cell capacitance (Fig. 8D). The pattern of propofol current densities for wild-type and mutant receptors resembles that seen for GABA current densities (Fig. 5B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We examined the impact of replacing a TM2–3 loop lysine residue by methionine in {alpha}1(K278M), beta2(K274M), and {gamma}2(K289M) subunits on GABAA receptor surface expression and function. The {gamma}2(K289M) mutation is associated with hereditary epilepsy characterized by febrile seizures (5). By introducing the Lys -> Met mutation into each of the three major GABAA subunits expressed in the brain we examined whether the role of the homologous lysine was similar at each position in the heteropentamer.

We assayed surface expression of epitope-tagged receptors qualitatively and quantitatively using epifluorescence microscopy and ELISA approaches, respectively. Surface expression was compared with functional receptor expression by measuring GABA-activated current density. We used outside-out and cell-attached patch recording configurations to evaluate the effects of the Lys -> Met mutation on GABA-activated single channel conductance and open times, respectively. Finally we examined whether deficits in channel function associated with mutant subunits were specific to GABA activation or generalized to activation by propofol, a GABAA receptor agonist that acts through a site distinct from that of GABA (41).

Using these varied approaches we found that the conserved lysine within the extracellular TM2–3 loop plays an asymmetric role in GABAA receptor function and expression that is dependent on the subunit in which it is located.

The {alpha}1(K278M) and {gamma}2(K289M) constructs had no effect on the level of receptor surface expression, whereas beta2(K274M) caused a marked reduction in cell-surface receptors. A recent report demonstrates that {gamma}2S(K289M) has a temperature-dependent effect on GABAA receptor expression. Elevating the temperature of HEK293 cells from 37 to 40 °C reduced cell-surface expression of {alpha}1beta2{gamma}2S receptors, an effect that wasmorepronouncedinthepresenceof{gamma}2S(K289M). This temperature-dependent effect may participate in the deficit in inhibitory signaling underlying febrile seizures in individuals harboring the mutation (18). Our functional studies were performed at room temperature, and cell-surface expression assays were performed on cells cultured at 37 °C prior to fixation. Furthermore it is worth noting that we used the {gamma}2L subunit variant, which may have different trafficking properties compared with those of {gamma}2S.

Unaltered surface expression of {alpha}1beta2{gamma}2(K289M) receptors compared with wild-type receptors coincided with unaltered GABA-evoked current density. By contrast, despite having no effect on surface expression, {alpha}1(K278M) reduced GABA-evoked current density. Two mechanisms contribute to current density: expression levels of cell-surface receptors and GABAA receptor function. Because there was no difference in the surface expression of {alpha}1(K278M)beta2{gamma}2 receptors compared with wild-type receptors, reduced current density must result from a functional deficit. Consistent with this assertion GABA had a substantially reduced apparent potency as an activator of {alpha}1(K278M)beta2{gamma}2 receptors compared with wild-type receptors; the GABA concentration-response relationship was shifted to the right, reflecting a 4-fold increase in the GABA EC50. Such a reduction in the potency of GABA could be caused by reduced binding affinity, impaired transduction of GABA-binding to channel activation, or both (42). Analysis of single channels activated in cell-attached patches identified a deficit in channel function caused by {alpha}1(K278M): the durations of channel openings were substantially reduced compared with those of wild-type receptors. This functional deficit presumably reduces the efficacy of GABA leading to a diminished current density.

In contrast to wild-type {alpha}1beta2{gamma}2, {alpha}1beta2{gamma}2(K289M), and {alpha}1(K278M)-beta2{gamma}2 receptors, {alpha}1beta2(K274M){gamma}2 receptors had substantially reduced surface expression, suggesting that the beta2 TM2–3 region may be important in receptor biogenesis and/or transport. The consequence of reduced surface expression of {alpha}1beta2(K274M){gamma}2 receptors is a dramatic reduction in current density. GABA-activated channels mediated by {alpha}1beta2(K274M){gamma}2 receptors were indistinguishable from those mediated by wild-type receptors in terms of their open times, suggesting a lack of a role for the beta2 Lys-274 in gating by GABA. However, the GABA concentration-response relationship was shifted to the right by beta2(K274M) consistent with a reduced GABA potency. It seems likely that this shift is caused by a reduction in GABA affinity for the mutant receptor.

Replacement of the conserved TM2–3 loop lysine by either alanine (43) or aspartate (23, 24) in either the {alpha}1orthe beta2 subunit also increases the GABA EC50. In the case of the human {alpha}1(K279D) subunit (equivalent to mouse {alpha}1(K278)) the deficit in receptor function could be rescued by simultaneously introducing either D57K or D149K mutations into the subunit's N-terminal domain loops 2 and 7, respectively (23). These data support the hypothesis that, upon activation of wild-type receptors by GABA, there is an interaction between acidic residues in the N-terminal domain of the {alpha}1 subunit and the lysine in the TM2–3 loop. This mechanism appears to be unique to the {alpha}1 subunit of the GABAA receptor, because similar simultaneous charge reversals in the beta2 subunit fail to recover the reduced GABA potency induced by the beta2(K274D) mutation (24). This is an intriguing difference that again demonstrates asymmetry in the role of the conserved TM2–3 lysine in GABAA receptor function. The failure of charge reversal to recover the potency of activation of {alpha}1beta2(K274D){gamma}2 receptors for GABA may also point to a role of beta2(K274) in GABA binding affinity rather than receptor activation.

The {gamma}2(K289M) mutant reduced GABA-gated channel open time albeit to a lesser extent than did {alpha}1(K278M). The attenuation of open time induced by {gamma}2(K289M) was not sufficient to significantly impact current density. Our data examining {alpha}1beta2{gamma}2(K289M) receptors in cell-attached patches agree with those previously reported for {alpha}1beta3{gamma}2(K289M) receptors in outside-out patches (14) suggesting that decreased inhibition underlying epilepsy associated with the mutation could be caused in part by briefer channel openings.

Interestingly, none of the mutant subunits significantly altered potentiation by flunitrazepam. Similarly there was no difference in the potentiation by a maximal concentration of diazepam of GABA-evoked currents mediated by wild-type {alpha}1beta3{gamma}2 and mutant {alpha}1beta3{gamma}2(K289M) receptors (14). These data suggest that the conserved lysine is not responsible for transducing the potentiating effects of benzodiazepines. However, this needs to be tested over a broader range of benzodiazepine concentrations in future studies.

Febrile seizures, the most common of childhood seizures, are linked to several environmental factors and a host of mutations, several of which affect genes encoding ion channels (44). A more complete understanding of the molecular mechanisms underlying each ion channel deficit associated with familial febrile seizures may lead to the development of individualized pharmacotherapies. The observation that {alpha}1beta2{gamma}2(K289M) and {alpha}1beta3{gamma}2(K289M) receptors (14) have reduced GABA-evoked channel open times, compared with their respective wild-type receptors, suggests that drugs that prolong open time may help rectify the functional deficit. Benzodiazepines increase the amplitude of sub-maximal GABA-evoked currents by increasing the frequency of GABA-gated channel bursts (45). This contrasts with the mechanism of enhancement by barbiturates and other general anesthetics that predominantly increase channel burst duration (45, 46). It is possible that the anesthetic potentiation of GABA-evoked responses mediated by mutant {alpha}1(K278M)beta2{gamma}2 and {alpha}1beta2{gamma}2(K289M) receptors will be enhanced through "correction" of the functional deficit of brief open time. This hypothesis will be tested in future single channel studies.

Deficits in the function of GABAA receptors containing {alpha}1(K278M) or {gamma}2(K289M) may indicate a role for the conserved lysine residue in the transduction of GABA binding to channel gating. Indeed this residue appears ideally situated for such a mechanism. If so, is this intramolecular transduction mechanism specific to GABA binding or can it be generalized to agonists that act through different sites on the GABAA receptor? Several general anesthetics, including the intravenous agent propofol, directly activate GABAA receptors when applied in the mid to high micromolar concentration range in the absence of GABA (40). Propofol also potentiates GABA-evoked currents starting in the low micromolar range and at concentrations >100 µM causes a concentration-dependent receptor blockade (33). Direct activation of GABAA receptors by propofol occurs through a distinct mechanism from that of GABA-evoked activation as demonstrated by the observation that propofol activates homomeric beta3 receptors that are resistant to activation by GABA (41). Furthermore, activation of recombinant {alpha}1beta2{gamma}2 receptors4 by propofol is resistant to blockade by the competitive GABA antagonist SR95531 (gabazine). Despite having different sites on the GABAA receptor, activation by both propofol and GABA is adversely affected by the {alpha}1(K278M) mutation. The disruption of propofol activation by {alpha}1(K278M) demonstrates that intramolecular transduction of binding to gating by this residue is not specific to GABA, instead the {alpha}1(Lys-278) plays a role in gating by diverse agonists.

Propofol has a similar apparent potency as an activator of {alpha}1beta2{gamma}2, {alpha}1beta2(K274M){gamma}2, or {alpha}1beta2{gamma}2(K289M) receptors. However, mutant {alpha}1beta2(K274M){gamma}2 receptors had reduced efficacy of activation by propofol relative to GABA, a property that distinguishes them from {alpha}1(K278M)beta2{gamma}2 and {alpha}1beta2{gamma}2(K289M) receptors. This is yet another example of an asymmetric role for the lysine.

The subunit-dependent effect of the Lys -> Met mutation on GABAA receptor activation by GABA and propofol is reminiscent of the asymmetric effects of equivalent mutations in glycine receptors (25, 26, 47). Incorporation of either the human startle disease {alpha}1(K276E) mutation or other non-conservative mutations at the same locus (including K276C) into recombinant homomeric {alpha}1 glycine receptors causes a substantial rightward shift in the glycine concentration-response relationship (25, 47). By contrast, the equivalent Lys -> Cys mutation in the beta subunit has no effect on the glycine concentration-response relationship (26). It appears that the functional roles of homologous lysines in the TM2–3 loops of subunits of both GABAA and glycine receptors are different.

A structural model of the {alpha}1beta2{gamma}2 GABAA receptor, produced by homology modeling its amino acid sequences onto the 4-Å resolution cryoelectron microscopic structure of the Torpedo marmorata nicotinic acetylcholine receptor (29), suggests that the conserved TM2–3 lysine lies in the conduction pathway within the channel's outer vestibule (30). On the basis of such a model it was proposed that removal of the basic residue from the mutant {gamma}2(K289M) subunit may reduce the number of Cl– ions within the outer vestibule thus reducing single channel conductance. Such a deficit in GABAA receptor function may contribute to the epileptic phenotype. However, there was no difference in the conductance of GABA-activated channels mediated by wild-type {alpha}1beta2{gamma}2 and {alpha}1beta2{gamma}2(K289M) receptors in this study in agreement with a previous report (14). There is likely to be only one {gamma}2 subunit per receptor perhaps minimizing the contribution of the {gamma}2(K289) to single channel conductance. Assuming a stoichiometry of 2{alpha}1:2beta2:1{gamma}2 (39) a greater contribution of {alpha}1(K278) and beta2(K274) residues to single channel conductance may be expected. However, neither the {alpha}1(K278M) nor the beta2(K274M) mutations had any effect on single channel conductance.

In conclusion, the conserved TM2–3 loop lysine plays an asymmetric role in GABAA receptor membrane expression and function. In the {gamma}2 subunit, under conditions of unaltered cell-surface expression, the K289M mutation reduces channel open time. In the beta2 subunit the K274M mutation impairs surface expression and reduces both the apparent potency of GABA and the efficacy of propofol as an agonist relative to GABA. In the {alpha}1 subunit the K278M mutation reduces the apparent potency of gating by both GABA and propofol without altering surface expression. Furthermore, the {alpha}1(K278M) mutation substantially reduced GABA-evoked channel open times.

Our data suggest that conserved TM2–3 lysine residues in the {alpha}1 and {gamma}2 subunits stabilize the open state of the receptor perhaps through a previously described electrostatic interaction with N-terminal acidic residues (23). The Lys-278 residue in the {alpha}1 subunit appears to be required for normal activation by both GABA and propofol suggesting that its putative role in initiating gating is not dependent on GABA binding.


   FOOTNOTES
 
* This work has been supported by the Biotechnology and Biological Sciences and Research Council (Grant 94/C17336 awarded to C. N. C.), Tenovus Scotland (to C. N. C.), Anonymous Trust (to C. N. C.), and the National Institutes of Health (Grant GM058037 awarded to T. G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Both authors contributed equally to this work. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, Medical Center, The George Washington University, 2300 Eye St. NW, Washington, D. C. 20037. Tel.: 202-994-3546; Fax: 202-994-2870; E-mail: phmtgh{at}gwumc.edu.

3 The abbreviations used are: GABAA, {gamma}-aminobutyric type A; PBS, phosphate-buffered saline; pA, picoamp(s); pF, picofarad(s); ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay. Back

4 M. R. McCartney, T. Z. Deeb, and T. G. Hales, unpublished observation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Davies, P. A., Wang, W., Hales, T. G., and Kirkness, E. F. (2003) J. Biol. Chem. 278, 712–717[Abstract/Free Full Text]
  2. Connolly, C. N., and Wafford, K. A. (2004) Biochem. Soc. Trans. 32, 529–534[CrossRef][Medline] [Order article via Infotrieve]
  3. Crestani, F., Lorez, M., Baer, K., Essrich, C., Benke, D., Laurent, J. P., Belzung, C., Fritschy, J. M., Luscher, B., and Mohler, H. (1999) Nat. Neurosci. 2, 833–839[CrossRef][Medline] [Order article via Infotrieve]
  4. Morimoto, K., Fahnestock, M., and Racine, R. J. (2004) Prog. Neurobiol. 73, 1–60[CrossRef][Medline] [Order article via Infotrieve]
  5. Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46–48[CrossRef][Medline] [Order article via Infotrieve]
  6. Wallace, R. H., Marini, C., Petrou, S., Harkin, L. A., Bowser, D. N., Panchal, R. G., Williams, D. A., Sutherland, G. R., Mulley, J. C., Scheffer, I. E., and Berkovic, S. F. (2001) Nat. Genet. 28, 49–52[CrossRef][Medline] [Order article via Infotrieve]
  7. Cossette, P., Liu, L., Brisebois, K., Dong, H., Lortie, A., Vanasse, M., Saint-Hilaire, J. M., Carmant, L., Verner, A., Lu, W. Y., Wang, Y. T., and Rouleau, G. A. (2002) Nat. Genet. 31, 184–189[CrossRef][Medline] [Order article via Infotrieve]
  8. Harkin, L. A., Bowser, D. N., Dibbens, L. M., Singh, R., Phillips, F., Wallace, R. H., Richards, M. C., Williams, D. A., Mulley, J. C., Berkovic, S. F., Scheffer, I. E., and Petrou, S. (2002) Am. J. Hum. Genet. 70, 530–536[CrossRef][Medline] [Order article via Infotrieve]
  9. Marini, C., Harkin, L. A., Wallace, R. H., Mulley, J. C., Scheffer, I. E., and Berkovic, S. F. (2003) Brain 126, 230–240[Abstract/Free Full Text]
  10. Dibbens, L. M., Feng, H. J., Richards, M. C., Harkin, L. A., Hodgson, B. L., Scott, D., Jenkins, M., Petrou, S., Sutherland, G. R., Scheffer, I. E., Berkovic, S. F., Macdonald, R. L., and Mulley, J. C. (2004) Hum. Mol. Genet. 13, 1315–1319[Abstract/Free Full Text]
  11. Fisher, J. L. (2004) Neuropharmacology 46, 629–637[CrossRef][Medline] [Order article via Infotrieve]
  12. Gallagher, M. J., Song, L., Arain, F., and Macdonald, R. L. (2004) J. Neurosci. 24, 5570–5578[Abstract/Free Full Text]
  13. Bowser, D. N., Wagner, D. A., Czajkowski, C., Cromer, B. A., Parker, M. W., Wallace, R. H., Harkin, L. A., Mulley, J. C., Marini, C., Berkovic, S. F., Williams, D. A., Jones, M. V., and Petrou, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15170–15175[Abstract/Free Full Text]
  14. Bianchi, M. T., Song, L., Zhang, H., and Macdonald, R. L. (2002) J. Neurosci. 22, 5321–5327[Abstract/Free Full Text]
  15. Sancar, F., and Czajkowski, C. (2004) J. Biol. Chem. 279, 47034–47039[Abstract/Free Full Text]
  16. Kang, J., and Macdonald, R. L. (2004) J. Neuroscience 24, 8672–8677[Abstract/Free Full Text]
  17. Hales, T. G., Tang, H., Bollan, K., Johnson, S., King, D., and Connolly, C. N. (2005) Mol. Cell Neurosci. 29, 120–127[CrossRef][Medline] [Order article via Infotrieve]
  18. Kang, J. Q., Shen, W., and Macdonald, R. L. (2006) J. Neurosci. 26, 2590–2597[Abstract/Free Full Text]
  19. Elmslie F. V., Hutchings S. M., Spencer V., Curtis A., Covanis T., Gardiner R. M., and Rees M. (1996) J. Med. Genet. 33, 435–436[Abstract/Free Full Text]
  20. Campos-Caro, A., Sala, S., Ballesta, J. J., Vicente-Agullo, F., Criado, M., and Sala F. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6118–6123[Abstract/Free Full Text]
  21. Sala, F., Mulet, J., Sala, S., Gerber, S., and Criado, M. (2005) J. Biol. Chem. 280, 6642–6647[Abstract/Free Full Text]
  22. Bouzat, C., Gumilar, F., Spitzmaul, G., Wang, H. L., Rayes, D., Hansen, S. B., Taylor, P., and Sine, S. M. (2004) Nature 430, 896–900[CrossRef][Medline] [Order article via Infotrieve]
  23. Kash, T. L., Jenkins, A., Kelley, J. C., Trudell, J. R., and Harrison, N. L. (2003) Nature 421, 272–275[CrossRef][Medline] [Order article via Infotrieve]
  24. Kash, T. L., Dizon, M. J., Trudell, J. R., and Harrison, N. L. (2004) J. Biol. Chem. 279, 4887–4893[Abstract/Free Full Text]
  25. Absalom, N. L., Lewis, T. M., Kaplan W., Pierce, K. D., and Schofield, P. R. (2003) J. Biol. Chem. 278, 50151–50157[Abstract/Free Full Text]
  26. Shan, Q., Nevin, S. T., Haddrill, J. L., and Lynch, J. W. (2003) J. Neurochem. 86, 498–507[CrossRef][Medline] [Order article via Infotrieve]
  27. Lummis, S. C., Beene, D. L., Lee, L. W., Lester, H. A., Broadhurst, R. W., and Dougherty, D. A. (2005) Nature 438, 248–252[CrossRef][Medline] [Order article via Infotrieve]
  28. Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534–7540[CrossRef][Medline] [Order article via Infotrieve]
  29. Miyazawa, A., Fujiyoshi, Y., and Unwin, N. (2003) Nature 423, 949–955[CrossRef][Medline] [Order article via Infotrieve]
  30. O'Mara M., Cromer B., Parker M., and Chung, S. H. (2005) Biophys. J. 88, 3286–3299[CrossRef][Medline] [Order article via Infotrieve]
  31. Connolly, C. N., Krishek, B. J., McDonald, B. J. Smart, T. G., and Moss, S. J. (1996) J. Biol. Chem. 271, 89–96[Abstract/Free Full Text]
  32. Evan, G. I., Lewis, G., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell Biol. 5, 3610–3616[Abstract/Free Full Text]
  33. Adodra, S., and Hales, T. G. (1995) Br. J. Pharmacol. 115, 953–960[Medline] [Order article via Infotrieve]
  34. Davies, P. A., Kirkness, E. F., and Hales, T. G. (2001) J. Physiol. 537, 101–113[Abstract/Free Full Text]
  35. Colquhoun, D., and Sigworth, F. J. (1983) in Single Channel Recording (Sakmann, B., and Neher, E., eds) pp. 191–263, Plenum Press, New York
  36. Sigworth, F. J., and Sine, S. M. (1987) Biophys. J., 52, 1047–1054[Medline] [Order article via Infotrieve]
  37. Haas, K. F., and Macdonald, R. J. (1999) J. Physiol. 514, 27–45[Abstract/Free Full Text]
  38. Bollan, K., King, D., Robertson, L. A., Brown, K., Taylor, P. M., Moss, S. J., and Connolly, C. N. (2003) J. Biol. Chem. 278, 4747–4755[Abstract/Free Full Text]
  39. Tretter, V., Ehya, N., Fuchs, K., and Sieghart, W. (1997) J. Neurosci. 17, 2728–2737[Abstract/Free Full Text]
  40. Hales, T. G., and Lambert, J. J. (1991) Br. J. Pharmacol. 104, 619–628[Medline] [Order article via Infotrieve]
  41. Davies, P. A., Kirkness, E. F., and Hales, T. G. (1997) Br. J. Pharmacol. 120, 899–909[CrossRef][Medline] [Order article via Infotrieve]
  42. Colquhoun, D. (1998) Br. J. Pharmacol. 125, 923–948[CrossRef]
  43. Sigel, E., Buhr, A., and Baur, R. (1999) J. Neurochem. 73, 1758–1764[CrossRef][Medline] [Order article via Infotrieve]
  44. Waruiru, C., and Appleton, R. (2004) Arch. Dis. Child. 89, 751–756[Abstract/Free Full Text]
  45. Twyman, R. E., Rogers, C. J., and Macdonald, R. L. (1989) Ann. Neurol. 25, 213–220[CrossRef][Medline] [Order article via Infotrieve]
  46. Akk, G., Bracamontes, J. R., Covey, D. F., Evers, A., Dao, T., and Steinbach, J. H. (2004) J. Physiol. 558, 59–74[Abstract/Free Full Text]
  47. Lynch J. W., Rajendra S., Pierce K. D., Handford C. A., Barry P. H., and Schofield P. R. (1997) EMBO J. 16, 110–120[CrossRef][Medline] [Order article via Infotrieve]

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