An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

Abstract

Mutations that impair the expression and/or function of γ-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy γ2(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 α1(K278M), β2(K274M), and γ2(K289M) subunits. Compared with cells expressing wild-type and α1β2γ2(K289M) receptors, cells expressing α1(K278M)β2γ2 and α1β2(K274M)γ2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of α1β2(K274M)γ2 receptors coincided with reduced surface expression. By contrast the surface expression of α1(K278M)β2γ2 receptors was similar to wild-type and α1β2γ2(K289M) receptors suggesting that the α1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by α1(K278M)β2γ2 receptors had brief open times. To a lesser extent γ2(K289M) also reduced mean open time, whereas β2(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 α1(K278M)β2γ2 receptors. By contrast, neither β2(K274M) nor γ2(K289M) affected the potency of propofol. The β2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the α1 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. Mutations that impair the expression and/or function of γ-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy γ2(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 α1(K278M), β2(K274M), and γ2(K289M) subunits. Compared with cells expressing wild-type and α1β2γ2(K289M) receptors, cells expressing α1(K278M)β2γ2 and α1β2(K274M)γ2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of α1β2(K274M)γ2 receptors coincided with reduced surface expression. By contrast the surface expression of α1(K278M)β2γ2 receptors was similar to wild-type and α1β2γ2(K289M) receptors suggesting that the α1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by α1(K278M)β2γ2 receptors had brief open times. To a lesser extent γ2(K289M) also reduced mean open time, whereas β2(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 α1(K278M)β2γ2 receptors. By contrast, neither β2(K274M) nor γ2(K289M) affected the potency of propofol. The β2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the α1 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. γ-Aminobutyric acid type A (GABAA) 3The abbreviations used are: GABAA, γ-aminobutyric type A; PBS, phosphate-buffered saline; pA, picoamp(s); pF, picofarad(s); ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay. 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 (1Davies P.A. Wang W. Hales T.G. Kirkness E.F. J. Biol. Chem. 2003; 278: 712-717Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 2Connolly C.N. Wafford K.A. Biochem. Soc. Trans. 2004; 32: 529-534Crossref PubMed Scopus (160) Google Scholar). GABAA receptors are pentameric, composed from distinct subunit classes, including α (1Davies P.A. Wang W. Hales T.G. Kirkness E.F. J. Biol. 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Essrich C. Benke D. Laurent J.P. Belzung C. Fritschy J.M. Luscher B. Mohler H. Nat. Neurosci. 1999; 2: 833-839Crossref PubMed Scopus (422) Google Scholar) and is the basis of several models of epilepsy (4Morimoto K. Fahnestock M. Racine R.J. Prog. Neurobiol. 2004; 73: 1-60Crossref PubMed Scopus (688) Google Scholar). Several recently discovered GABAA mutations that reduce inhibition accompany hereditary forms of epilepsy (5Baulac S. Huberfeld G. Gourfinkel-An I. Mitropoulou G. Beranger A. Prud'homme J.F. Baulac M. Brice A. Bruzzone R. LeGuern E. Nat. Genet. 2001; 28: 46-48Crossref PubMed Scopus (708) Google Scholar, 6Wallace 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. Berkovic S.F. Nat. Genet. 2001; 28: 49-52Crossref PubMed Google Scholar, 7Cossette 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. Rouleau G.A. Nat. 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Wallace R.H. Harkin L.A. Mulley J.C. Marini C. Berkovic S.F. Williams D.A. Jones M.V. Petrou S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15170-15175Crossref PubMed Scopus (92) Google Scholar, 14Bianchi M.T. Song L. Zhang H. Macdonald R.L. J. Neurosci. 2002; 22: 5321-5327Crossref PubMed Google Scholar). To date, the most thoroughly investigated of these are γ2(R43Q) and γ2(K289M), both associated with febrile seizures. Two mechanisms could account for reduced inhibition caused by these mutations: impaired receptor expression and/or function. The γ2(R43Q) mutation alters receptor kinetics (13Bowser 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. Petrou S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15170-15175Crossref PubMed Scopus (92) Google Scholar, 14Bianchi M.T. Song L. Zhang H. Macdonald R.L. J. Neurosci. 2002; 22: 5321-5327Crossref PubMed Google Scholar), and this may contribute to inhibitory deficits. The mutation also reduces receptor biogenesis (15Sancar F. Czajkowski C. J. Biol. Chem. 2004; 279: 47034-47039Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16Kang J. Macdonald R.L. J. Neuroscience. 2004; 24: 8672-8677Crossref PubMed Scopus (110) Google Scholar, 17Hales T.G. Tang H. Bollan K. Johnson S. King D. Connolly C.N. Mol. Cell Neurosci. 2005; 29: 120-127Crossref PubMed Scopus (54) Google Scholar, 18Kang J.Q. Shen W. Macdonald R.L. J. Neurosci. 2006; 26: 2590-2597Crossref PubMed Scopus (119) Google Scholar). Likewise γ2(K289M) also impairs receptor function and expression (5Baulac S. Huberfeld G. Gourfinkel-An I. Mitropoulou G. Beranger A. Prud'homme J.F. Baulac M. Brice A. Bruzzone R. LeGuern E. Nat. Genet. 2001; 28: 46-48Crossref PubMed Scopus (708) Google Scholar, 14Bianchi M.T. Song L. Zhang H. Macdonald R.L. J. Neurosci. 2002; 22: 5321-5327Crossref PubMed Google Scholar, 18Kang J.Q. Shen W. Macdonald R.L. J. Neurosci. 2006; 26: 2590-2597Crossref PubMed Scopus (119) Google Scholar). The N-terminal γ2 subunit Arg-43 residue is conserved across GABAA receptor subunits and systematic Arg → Gln mutation in α1, β2, and γ2 uncovered a general role for the arginine in receptor assembly (17Hales T.G. Tang H. Bollan K. Johnson S. King D. Connolly C.N. Mol. Cell Neurosci. 2005; 29: 120-127Crossref PubMed Scopus (54) Google Scholar). Likewise the γ2 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 α1 subunit lysine is associated with hereditary hyperekplexia (19Elmslie F. V. Hutchings S.M. Spencer V. Curtis A. Gardiner R. M. Covanis T.Rees M. J. Med. Genet. 1996; 33: 435-436Crossref PubMed Scopus (68) Google Scholar). Moreover, equivalent and/or nearby residues within the TM2–3 loops of the α7 and the β2 nicotinic acetylcholine receptor subunits (20Campos-Caro A. Sala S. Ballesta J.J. Vicente-Agullo F. Sala F. CriadoM. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6118-6123Crossref PubMed Scopus (83) Google Scholar, 21Sala F. Mulet J. Sala S. Gerber S. Criado M. J. Biol. Chem. 2005; 280: 6642-6647Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 22Bouzat C. Gumilar F. Spitzmaul G. Wang H.L. Rayes D. Hansen S.B. Taylor P. Sine S.M. Nature. 2004; 430: 896-900Crossref PubMed Scopus (241) Google Scholar), α1 (23Kash T.L. Jenkins A. Kelley J.C. Trudell J.R. Harrison N.L. Nature. 2003; 421: 272-275Crossref PubMed Scopus (283) Google Scholar) and β2 (24Kash T.L. Dizon M.J. Trudell J.R. Harrison N.L. J. Biol. 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Genet. 2001; 28: 46-48Crossref PubMed Scopus (708) Google Scholar) reported reduced current amplitude when compared with wild-type receptors upon expression of α1β2γ2(K289M) receptors in Xenopus oocytes. By contrast, Bianchi and colleagues (14Bianchi M.T. Song L. Zhang H. Macdonald R.L. J. Neurosci. 2002; 22: 5321-5327Crossref PubMed Google Scholar) described faster GABA-evoked current deactivation without altered α1β3γ2(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 α1β2γ2(K289M) and wild-type receptors, Ramakrishnan and Hess (28Ramakrishnan L. Hess G.P. Biochemistry. 2004; 43: 7534-7540Crossref PubMed Scopus (22) Google Scholar) concluded that the mutation reduced current activation rate. Homology modeling of the α1β2γ2 receptor onto the structural model of the Torpedo marmorata nicotinic acetylcholine receptor (29Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1082) Google Scholar) revealed an additional possibility: that the K289M mutation may reduce single channel conductance (30O'Mara M. Cromer B. Chung Parker M.S.H. Biophys. J. 2005; 88: 3286-3299Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). 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 α1 subunit and acidic residues in loops 2 and 7 may be responsible for intramolecular transduction, coupling GABA binding to channel opening (23Kash T.L. Jenkins A. Kelley J.C. Trudell J.R. Harrison N.L. Nature. 2003; 421: 272-275Crossref PubMed Scopus (283) Google Scholar). To address the role of Lys-289 and equivalent lysine residues in the most common (α1β2γ2) GABAA receptor, we explored the effects of α1(K278M), β2(K274M), or γ2L(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. 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 (17Hales T.G. Tang H. Bollan K. Johnson S. King D. Connolly C.N. Mol. Cell Neurosci. 2005; 29: 120-127Crossref PubMed Scopus (54) Google Scholar). 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 α1, β2, and γ2L subunit cDNAs containing the myc or FLAG tag (between amino acids 4 and 5 of the mature polypeptide) have been described previously (31Connolly C.N. Krishek B.J. McDonald B.J. Smart Moss T. G.S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) and shown to be functionally silent with respect to receptor pharmacology and physiology. The mutant expression constructs α1(K278M)Myc, β2(K274M)Myc and γ2L(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 (32Evan G.I. Lewis G. Ramsay G. Bishop J.M. Mol. Cell Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2167) Google Scholar) 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 (33Adodra S. Hales T.G. Br. J. Pharmacol. 1995; 115: 953-960Crossref PubMed Scopus (86) Google Scholar). 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 (34Davies P.A. Kirkness E.F. Hales T.G. J. Physiol. 2001; 537: 101-113Crossref PubMed Scopus (35) Google Scholar). 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 (33Adodra S. Hales T.G. Br. J. Pharmacol. 1995; 115: 953-960Crossref PubMed Scopus (86) Google Scholar). For fitting propofol concentration-response relationships (normalized to maximum GABA-evoked current) the Hill equation was modified as in Equation 1. IPropIGMax=IPMax{1+[EC50[Prop]]H}−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 γ = 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. 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 (35Colquhoun D. Sigworth F.J. Single Channel Recording. 1983; (Plenum Press, New York): 191-263Crossref Google Scholar), 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 (36Sigworth F.J. Sine S.M. Biophys. J. 1987; 52: 1047-1054Abstract Full Text PDF PubMed Scopus (649) Google Scholar). 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 (37Haas K.F. Macdonald R.J. J. Physiol. 1999; 514: 27-45Crossref PubMed Scopus (234) Google Scholar). 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. Conservation of a TM2–3 Loop Lysine between GABAA and Glycine Receptor Subunits—A comparison of the primary sequence of GABAA receptor α, β, and γ 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 αβγ 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 γ2 subunit (5Baulac S. Huberfeld G. Gourfinkel-An I. Mitropoulou G. Beranger A. Prud'homme J.F. Baulac M. Brice A. Bruzzone R. LeGuern E. Nat. Genet. 2001; 28: 46-48Crossref PubMed Scopus (708) Google Scholar), is conserved in α and β subunits (Arg in γ3, δ, and ϵ, Asn in π, and His in θ) as well as in glycine receptors, where it has been implicated in hyperekplexia (19Elmslie F. V. Hutchings S.M. Spencer V. Curtis A. Gardiner R. M. Covanis T.Rees M. J. Med. Genet. 1996; 33: 435-436Crossref PubMed Scopus (68) Google Scholar). Intriguingly, this lysine residue present in the α1 (Lys-278) and β2 (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 (23Kash T.L. Jenkins A. Kelley J.C. Trudell J.R. Harrison N.L. Nature. 2003; 421: 272-275Crossref PubMed Scopus (283) Google Scholar, 24Kash T.L. Dizon M.J. Trudell J.R. Harrison N.L. J. Biol. Chem. 2004; 279: 4887-4893Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We undertook, therefore, to examine the effect of the mutation in α1(K278M), β2(K274M), and γ2L(K289M) subunits. The Role of Lysine at the Homologous Position in the α1 Subunit on Transport to the Cell Surface—To determine, qualitatively, the ability of the α1(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 (38Bollan K. King D. Robertson L.A. Brown K. Taylor P.M. Moss S.J. Connolly C.N. J. Biol. Chem. 2003; 278: 4747-4755Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). 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 α1Myc (31Connolly C.N. Krishek B.J. McDonald B.J. Smart Moss T. G.S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), the α1(K278M)Myc subunit could not access the cell surface when expressed alone (data not shown). When α1Myc was co-expressed with the β2 subunit, there was robust cell-surface staining (Fig. 2A, upper right panel). Likewise, the co-expression of α1(K278M)Myc with the β2 subunit produced robust cell-surface labeling (Fig. 2A, α*β, lower right panel) as well as strong intracellular labeling. Identical results were observed when α1(K278M)Myc was co-expressed with β2 and γ2LFLAG subunits and immunofluorescence was performed via the FLAG epitope on the γ2L subunit (Fig. 2B). Because the γ2L subunit cannot access the surface in the absence of either the α1 or β2 subunits (31Connolly C.N. Krishek B.J. McDonald B.J. Smart Moss T. G.S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), its surface expression is a faithful indicator of the presence of α1β2γ2L receptors. From hereafter 'γ2′ refers to the γ2L 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 α1(K278M)Myc is presented as a percentage of total (in the presence of detergent) levels and normalized to wild-type controls (α1Myc) performed in parallel. Using this approach (Fig. 2C), the cell-surface level for α1(K278M)Mycβ2 receptors was 66 ± 11%, compared with the normalized wild-type α1Mycβ2 receptor level of 100 ± 11%. Similarly, the cell-surface level for α1(K278M)Mycβ2γ2FLAG receptors was determined (via FLAG epitope) to be 87 ± 34%, compared with the normalized wild-type α1Mycβ2γ2FLAG receptors at 100 ± 13%. Thus, the presence of K278M in the α1 subunit does not have a major impact on biogenesis or the surface transport of α1(K278M)β2γ2 receptors. The Role of Lysine at the Homologous Position in the β2 Subunit on Transport to the Cell Surface—To determine, qualitatively, the ability of the β2(K274M) subunit to access the cell surface, we examined its cellular distribution when expressed in COS7 cells. As observed previously for wild-type β2Myc (31Connolly C.N. Krishek B.J. McDonald B.J. Smart Moss T. G.S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), the β2(K274M)Myc subunit could not access the cell surface when expressed alone (data not shown). When β2Myc was co-expressed with the α1 subunit, there was robust cell-surface staining (Fig. 3A, upper right panel). In contrast, when β2(K274M)Myc was co-expressed with the α1 subunit there was no cell-surface labeling (Fig. 3A, αβ*; lower right panel). Instead, strong intracellular labeling within the endoplasmic reticulum was observed, as evidenced by the characteristic reticular pattern, typically observed in