Mutating the Highly Conserved Second Membrane-Spanning Region 99 Leucine Residue in the a1 or b1 Subunit Produces Subunit-Specific Changes in the Function of Human a1b1 g-Aminobutyric AcidA Receptors

The properties of the human a1b1 g-aminobutyric acid (GABA)A receptors were investigated after mutation of a highly conserved leucine residue at the 99 position in the second membrane-spanning region (TM2). The role of this residue in a1 and b1 subunits was examined by mutating the 99 leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the a1 subunit (apb), the b1 subunit (abp), or in both subunits (apbp), and the receptors were expressed in Sf9 cells. Our results show that the rate of desensitization is increased as the size and hydrophobicity of the 99 residue in the a1 subunit is increased: Y, F . L . A, T. Mutation of L99 in only the b1 subunit (abp) to either phenylalanine or tyrosine increased the EC50 value for GABA at least 100 times, but the EC50 was unchanged in abp alanine mutants. In the 99 a1 mutants (apb, apbp) the GABA EC50 was minimally affected. In apb and apbp, but not abp, the peak currents evoked by millimolar concentrations of GABA were greatly reduced. The reduction in currents could only be partially accounted for by decreased expression of the receptors These findings suggest different roles for the two types of subunits in GABA activation and later desensitization of a1b1 receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalanine and tyrosine mutants, indicating that the side chain size at the 99 position is a major determinant of current flow in the closed conformation. g-Aminobutyric acid (GABA) binds at an extracellular site on the GABAA receptor and activates an integral chloride ion channel. How GABA binding is coupled to channel opening is not well understood. The receptors are thought to be heterooligomeric pentamers with each subunit contributing a transmembrane segment to line the pore. Residues in the second membrane-spanning region (TM2) contribute to the ion permeation pathway. A leucine residue at the 99 position (L99) in the middle of the TM2 region is highly conserved in GABAA receptor subunits and across other members of the C-C loop receptor family. Mutation of L99 in rat a1b2g2 GABAA (Chang et al., 1996), 5-hydroxytryptamine type 3 (Yakel et al., 1993), and nicotinic acetylcholine (nACh) (Revah et al., 1991, 1995; Filatov and White, 1995; Labarca et al., 1995) receptors can alter the agonist EC50 value and the rate of desensitization, suggesting functional roles for L99 in desensitization and gating. Mutation of L99 to threonine (L99T) in the a1 subunit of the human a1b1 GABAA receptors slows receptor activation and desensitization (Tierney et al., 1996). When the L99T mutation is in either the b1 subunit or both a1 and b1 subunits together, the response to GABA is abolished and the channel becomes constitutively open. The differential effects of mutating the 99 residue on the response to GABA suggest that there are functional differences between subunits in the response of the receptor to GABA, despite the high conservation of L99 across subunits and the high sequence homology of the TM2 region. Mutation of L99 to serine in subunits of the nACh receptor resulted in a reduction in the EC50 that was approximately proportional to the number of mutated subunits in the receptor complex (Filatov and White, 1995; Labarca et al., 1995). In contrast, mutation of L99 to serine in rat a1b2g2 GABAA subunits showed differences in the degree of shift in EC50 depending on which subunit was mutated (Chang et al., 1996). It is possible that unlike in nACh receptors, GABAA subunits do not contribute in an equivalent manner to the mechanisms 1 Present address: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, CA. 2 Present address: Department of Cell and Molecular Physiology, Institute of Physiological Sciences, Lund University, Lund S-223 62, Sweden. ABBREVIATIONS: GABA, g-aminobutyric acid; TM2, second membrane-spanning region; nACh, nicotinic acetylcholine; MES, 2-(N-morpholino)ethanesulfonic acid; Sf9, Spodoptera frugiperda; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. 0026-895X/00/050875-08$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:875–882, 2000 /13049/822098 875 at A PE T Jornals on M ay 0, 2017 m oharm .aspeurnals.org D ow nladed from involved in receptor activation. An additional effect of mutating L99 to smaller or more hydrophilic residues in GABAA and nACh receptors is current flow in the absence of agonist (Labarca et al., 1995; Tierney et al., 1996; Bertrand et al., 1997; Mihic et al., 1997; Pan et al., 1997; Chang and Weiss, 1998, 1999). Given the apparent importance of TM2 99 position in GABAA receptor function, we investigated what effects different amino acids at this location had on the properties of the receptors and whether any subunit specificity could be detected. Materials and Methods Construction and Expression of Mutated Receptors. Double-stranded mutagenesis (Pharmacia Biotech, Piscataway, NJ) was used to introduce site-directed mutations to either a1 (L99: amino acid 264) or b1 (L99: amino acid 259) human GABAA cDNA in the dual promoter baculovirus transfer vector pAcUW31 (ClONTECH, Palo Alto, CA). Plasmids with mutations in both a1 and b1 subunits were produced by restriction enzyme digestion with Bgl2 and NheI, gel purification, and ligation of mutated fragments. Alternatively, a plasmid previously mutated in the a1 subunit cDNA was used as a template in a subsequent mutagenesis reaction to mutate the homologous residue in the b1 subunit . Recombinant ab(L99A) and a(L99A)b(L99A) baculoviruses for L99A mutated sequences were generated using the Bac-to-Bac expression system (Life Technologies, Grand Island, NY). The presence of mutations was confirmed by DNA sequencing across the mutated regions. Although an a(L99A)b mutant plasmid was generated, we could not isolate a recombinant a(L99A)b baculovirus. Techniques for general handling of Sf9 (Spodoptera frugiperda) cells, production of high titer viral stock, and infection procedures have been described previously (Birnir et al., 1995). Muscimol Binding. The method used to measure high-affinity muscimol binding in cells infected with recombinant baculovirus was as described previously (Tierney et al., 1996). The concentrations of muscimol used consisted of 10% radioactively labeled [H]muscimol and 90% cold muscimol (Sigma Chemical Co., St. Louis, MO) measured over a concentration range of 1 to 512 nM. Scintillation count values were multiplied by 10 to account for the 1:10 dilution of [H]muscimol. Flow Cytometry. Antibody labeling methods to detect a1 subunit expression were similar to that described previously (Tierney et al., 1996). To determine the level of a1 subunit present in the plasma membrane, Sf9 cells were infected with recombinant baculovirus 40 to 48 h before use in experiments. Nonpermeabilized cells were labeled with primary monoclonal antibody bd24 (1:50 dilution), fixed in Zamboni’s solution (2% formaldehyde, 15% picric acid, 0.1 M phosphate buffer, pH 7.4) for 90 min, and then labeled with secondary antibody using a fluorescein isothiocyanate-conjugated sheep anti-mouse Ig antibody (1:40 dilution; Silenus Laboratories, Hawthorn, Australia). To detect the total level of a1 subunit present in the plasma membrane and within the cell, cells were permeabilized in PBS buffer containing 0.1% SDS plus 1% BSA (Boehringer-Mannheim Biochemica, Mannheim, Germany) for 10 min before labeling with the primary antibody. The level of fluorescence was detected using a FACStar Plus flow cytometer (Becton Dickinson, Mountain View, CA), and results were analyzed with the WinMDI 2.7 computer program (courtesy of Joseph Trotter, Scripps Cancer Research Institute, La Jolla, CA). The level of background fluorescence was determined from cells infected with the wildtype parent baculovirus (AcNPV) and subtracted from all other values. These were then calculated as a percent of wild-type fluorescence. Electrophysiology. Cells were infected with virus when growing at a density of 1 to 3 3 10 cells/ml and incubated at 25 6 1C for 33 to 45 h before use in electrophysiological experiments. Whole-cell currents were recorded from voltage-clamped cells with a pipette potential of 240 mV. At this potential background, chloride current was minimized (Birnir et al., 1995). Cells were perfused with bath solution (14 ml/min) containing 180 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM MES adjusted to pH 6.2 with NaOH (330 mOsmol/liter). pH 6.2 is the normal pH for growth and maintenance of Sf9 cells. Pipettes were made from borosilicate glass with resistances of 3 to 10 MV and filled with a solution containing 178 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, 4 mM ATP, and 10 mM TES, adjusted to pH 7.2 with NaOH. GABA (Sigma Chemical Co.) was dissolved in bath solution, serially diluted, and rapidly applied to cells by gravity feed through tubes aimed at cells. The rate of solution exchange across the cell surface was examined by monitoring the whole-cell current when the Cl concentration was changed around the cell. When the bath solution contained 184 mM Cl and a jet of solution containing 34 mM Cl was switched through the tubes, an inward current that reached a plateau in less than 1 ms was evoked (Birnir et al., 1995). The current increased from 10 to 90% of the final steady-state current in approximately 0.5 ms. Currents were monitored with a current-to-voltage converter (Axopatch1D; Axon Instruments, Foster City, CA) using series resistance compensation. Data were digitized using an analog-to-digital converter (TL-1, DMA interface; Axon Instruments) and the Capture data acquisition program and then analyzed using the Channel 2 data analysis program (M. Smith, John Curtin School of Medical Research). To account for rundown of whole-cell currents over successive agonist applications, a control concentration of GABA was applied before and after a test concentration of GABA. Results were only used from cells in which the two control concentrations gave currents that differed by less than 20% in amplitude. Responses were calculated as a fraction of the averaged control currents. Equations. Concentration-response data were averaged for each concentration and fitted using a Hill-type equation (nonlinear least-