18s Biochemical Society Transactions ( 1 99 1) 20 Cloning and Expresslon of a cDNA Encoding the Human GABA-A Receptor a5 Subunit
HUMAN
BAT
cons
P Wlngrove, K Hadlngham, K Wafford, J A Kemp, C I Ragan and P Whiting
The y-aminobutyric acid (GABA-A) receptor is the site of action of one of the most widely prescribed group of drugs, benzodiazepines. This receptor is abundant at inhibitory synapses throughout the central nervous system; binding of GABA to the GABA-A receptor results in the opening of a chloride channel which is intrinsic to the GABA-A receptor macromolecular complex, leading to an inhibition of synaptic transmission (1). Heterogeneity of the GABA-A receptorlbenzodiazepine binding sites was originally recognised by ligand binding pharmacology (2). Two benzodiazepine binding site (BZ1 and 822) were defined according to their afinity for certain ligands. More recently, using molecular genetic approaches, the diversity of GABA-A receptors has been shown to be much greater than originally thought. To date, 6a,38,37, 16 and 1o subunits have been identified by cDNA cloning (3). Transient expression in transfected cells or Xenopus oocytes of various subunit comblnatlons has shown that although an a and a p subunit can form a GABA gated channel, a y 2 subunit is required for benzodiazepine binding (4). The a subunit present appears to define the benzodiazepine pharmacology of the expressed receptor: a1 containing receptors have a BZ1 pharmacology, while a2. a3 and a5 containing receptors have pharmacologiescorresponding more to 822 sites (5,6). As a prelude to understanding the structure, function and pharmacology of human GABA-A receptor subtypes we have begun cloning cDNAs encoding the various human receptor subunits. Here we report the cloning and sequencing of a cDNA encoding the human a5 subunit, and its expression together with 61 and72 subunit, in Xenopus oocytes and transfected cells. A human hippocampal Z A P cDNA library (Stratagene) was screened at moderate stringency with a rat a5 subunit (6,7) cDNBA probe derived by polymerase chain reaction. Several human a5 cDNA clones were isolated, one of which was shown to contain the complete coding region of the human a5 subunit. Figure 1 shows the deduced primary amino acid sequence of this subunit aligned with the published rat a5 sequence. Analysis of the human a5 ssquence indicates that it contains a putative 31 amino acid signal peptide, 4 putative transmembrane (TM) domains and the two conserved Cys residues (Cys 173 and Cys 187) which are a motif of the ligand gated ion channel super gene family. The a5 subunit also contains 4 putative Nglycosylation sites and 4 protein kinase C consensus phosphorylation sites in the large cytoplasmlc loop between TM regions 3 and 4. Overall human a5 has 94% amino acid sequence identity with the rat a5 subunit (6,7),with most of the changes occurring in the signal peptide and the putative large cytoplasmic loop. Human a5 has 57% sequence identity with human a l , again most of the amino acid differences being in the signal peptide and the putative large cytoplasmic loop domain.
The function and pharmacology of a5 containing human GABA-A receptors was investigated by expression studies. Xenopus oocytes were injected with a6 81 and 'y2L cRNAs, and the expressed receptors subsequently characterised. Activation of a5 containing GABA-A receptors by application of GABA resulted in an inward current, and the response
IIDNM-S-FI--K-LL-FCISM-LSSHFGFSOEIPTSSV-DETNDNITIFTRILDGLWGYD 62 N R L R P G L G E R I T Q V R T D I W T S F G P V S D T ~ Y T I D V T F R Q S ~ ~ ~ K G P M Q R ~ ~ N
IlIIIIIIIIIIIlIIlIIIIIlIIIlIIlIIIIIIIIIIIIlIIIlIIIIIIIIIlIlII 62 NRLRPGLGERITQVRTDIWTS~CP\'SDTCI(EYTIDVIFRQSVI(DERWVKGPIX)RLPLNN
cons
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwlck Road, Harlow, Essex, CM20 2QR, UK
1 IIDNGHfS~FIm~KnLLlFCISHnlSSHFGF~OEIPTSSVkDETND~ITIFTRI~GLLDGYD IIIII I II I I 1 IIIII IIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIII 1 IIDNMlSrFIntKtLLvFCISlltlSSHFGFS~TSSVqDETNDNITIFTRILDGLWGYD
NRLRPGLGCRI~VRTDIWTSFGPVSDT~YTlDVTFRQS~-KGP~~LNN 123 LLASKIWTPDTFFHNGKKSIAH~TPNKLLRLEDDGTL~YWATYTMRLTISAECPMQLEDFPMD
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
123 LLWKIWTPDTFFHNGRI(SIAHNElTTPNKLLRLCDDGTLLYTMRLT1SAECPMQLEDFPMD
cons
LLASKIWTPDTFFHNGKKSIAHNElTTPNKLLRLCDDGTLLYTURLTISAECPMQLEDFPMD 184 A H A C P L K P G S Y A Y P N S E V ~ G S T K S ~ A E D G S ~ Q Y H ~ ~ ~ G T E ~ I S T S T G E X
IIIIIIIIlIIIIIIIIIIIIIIIlIIIIIIIlIIIIIIIIIIIIIIIIIllIlIIIIIII
184 A H A C P L K P G S Y A Y P N S E V N G S T K S V W A E D C S R G T E N I S T S T G E Y
cons
AHACPLKPGSYAYPNSEVNGSTKS~AEDGSRLNQYH~~TVGTENISTSTGEY I . . . .
245 T l M T A H F H L K R K I G ~ W I Q T Y L P c I M T ~ R E S ~ ~ ~ ~ ~
IIIIIlIIIIIIIIIIlIIllIlIIlIIlIIIIIIllIIllIIIIIIIllIIIIIIIIlIl 245 T I H T ~ F H L K R K I G Y ~ I Q T Y L P C I n T V I L S Q V S ~ ~ R E S W ~ ~ G ~ T V L T M T T L S I cons TIMTAHFHLKRKIGY~IQTYLPCIMTVILSQVS~~~SW~~~~L~TLSI
366 RevlLNKSTNAFTTGKmJHPPNIPKeQtPaGT rnttavSvkpSEEKTSESKTYNSlSKI II IIIIIIIIIIIII IIIIIIIII I II I IIIIIIIIIIIIIIIIII 361 RelILNKSTKAPTTGKltHPPNIP~QlPgGT~nav~taS1raSO~KTS&SIV(TYNSISKI
cons
Re-ILNKSTNMTTGK--nPPNIP)(EP-P-GTg------S---S~KTSCSKTXNSlSKl
TI44 426 D K ~ ~ R X V T P V ~ ~ C T ~ N R E P V I K ~ S P K
IIIIIIIII IIIIIIIIIIIIIIIIIIIIIII I l l 428 DKHSRIVFPILFGTFNLVXLNREPVIKGAtSPK cons DKHSRIVFP-LFCTFNLVYWATYLNREPVIKGA-SPK
Figure 1 Alignment of the deducad primary m i n o acid requbncer 0 5 the human and rat alpha5 GABA-A rbceptoc rubunita. Ovbrlined raquencba indicate putative transmembrane domainr. I l l indicates putativb ri9n.l Ibj indicatea putativb N - 9 l y c o a y l a t i o n SitbS peptlde cleavage f l t e
could be modulated by nanomolar concentrations of the benzodiazepine flunitrazepam. but not by micromolar concentrations of the BZ1 selective compound zolpidem. This i s in agreement with previous radioligand binding data determined for receptors expressed by cells transiently transfected with rat a5. and $2 subunit cDNAs (6).Thus a5 containing human GABA-A receptor subtypes, like their rat equivalent, have a unique benzodiazepinepharmacology.
-
1.
Olsen RW and Venter JC (1986), Benzodlazeplne GABA Receptors and Chloride Channels: Structural and Functional Propertles, Alan Liss. New Yo&.
2.
Squires RF, Benson DI, Braestrup C, Coupet J, Klepner CA, Myres V and Beer B (1979), Pharmacol. Blochem. Behav. 10,825830.
3. Luddens H and Wisden W (1991). Trends Pharmacol. Scl. 12. 49-51. 4.
Pritchett DB, Sonthelmer H. Shivers B. Ymer S . Kettenmann H, Schofield PR and Seeburg PH (1989), Nature 338,582-585.
5.
Pritchett DB, Luddens H and Seeburg PH (1989), Science 245, 1389-1391.
6.
Pritchett DB and Seeburg PH (1990), J Neurochem 54, 1802-1804.
7.
Khrestchatisky M, MacLennan AJ, Chiang MY, Xu W, Jackson MB, Brecha N, Sternini C, Olsen RW and Tobin AJ (1989), Neuron 3, 745-753.
HUMAN
BAT
cons
P Wlngrove, K Hadlngham, K Wafford, J A Kemp, C I Ragan and P Whiting
The y-aminobutyric acid (GABA-A) receptor is the site of action of one of the most widely prescribed group of drugs, benzodiazepines. This receptor is abundant at inhibitory synapses throughout the central nervous system; binding of GABA to the GABA-A receptor results in the opening of a chloride channel which is intrinsic to the GABA-A receptor macromolecular complex, leading to an inhibition of synaptic transmission (1). Heterogeneity of the GABA-A receptorlbenzodiazepine binding sites was originally recognised by ligand binding pharmacology (2). Two benzodiazepine binding site (BZ1 and 822) were defined according to their afinity for certain ligands. More recently, using molecular genetic approaches, the diversity of GABA-A receptors has been shown to be much greater than originally thought. To date, 6a,38,37, 16 and 1o subunits have been identified by cDNA cloning (3). Transient expression in transfected cells or Xenopus oocytes of various subunit comblnatlons has shown that although an a and a p subunit can form a GABA gated channel, a y 2 subunit is required for benzodiazepine binding (4). The a subunit present appears to define the benzodiazepine pharmacology of the expressed receptor: a1 containing receptors have a BZ1 pharmacology, while a2. a3 and a5 containing receptors have pharmacologiescorresponding more to 822 sites (5,6). As a prelude to understanding the structure, function and pharmacology of human GABA-A receptor subtypes we have begun cloning cDNAs encoding the various human receptor subunits. Here we report the cloning and sequencing of a cDNA encoding the human a5 subunit, and its expression together with 61 and72 subunit, in Xenopus oocytes and transfected cells. A human hippocampal Z A P cDNA library (Stratagene) was screened at moderate stringency with a rat a5 subunit (6,7) cDNBA probe derived by polymerase chain reaction. Several human a5 cDNA clones were isolated, one of which was shown to contain the complete coding region of the human a5 subunit. Figure 1 shows the deduced primary amino acid sequence of this subunit aligned with the published rat a5 sequence. Analysis of the human a5 ssquence indicates that it contains a putative 31 amino acid signal peptide, 4 putative transmembrane (TM) domains and the two conserved Cys residues (Cys 173 and Cys 187) which are a motif of the ligand gated ion channel super gene family. The a5 subunit also contains 4 putative Nglycosylation sites and 4 protein kinase C consensus phosphorylation sites in the large cytoplasmlc loop between TM regions 3 and 4. Overall human a5 has 94% amino acid sequence identity with the rat a5 subunit (6,7),with most of the changes occurring in the signal peptide and the putative large cytoplasmic loop. Human a5 has 57% sequence identity with human a l , again most of the amino acid differences being in the signal peptide and the putative large cytoplasmic loop domain.
The function and pharmacology of a5 containing human GABA-A receptors was investigated by expression studies. Xenopus oocytes were injected with a6 81 and 'y2L cRNAs, and the expressed receptors subsequently characterised. Activation of a5 containing GABA-A receptors by application of GABA resulted in an inward current, and the response
IIDNM-S-FI--K-LL-FCISM-LSSHFGFSOEIPTSSV-DETNDNITIFTRILDGLWGYD 62 N R L R P G L G E R I T Q V R T D I W T S F G P V S D T ~ Y T I D V T F R Q S ~ ~ ~ K G P M Q R ~ ~ N
IlIIIIIIIIIIIlIIlIIIIIlIIIlIIlIIIIIIIIIIIIlIIIlIIIIIIIIIlIlII 62 NRLRPGLGERITQVRTDIWTS~CP\'SDTCI(EYTIDVIFRQSVI(DERWVKGPIX)RLPLNN
cons
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwlck Road, Harlow, Essex, CM20 2QR, UK
1 IIDNGHfS~FIm~KnLLlFCISHnlSSHFGF~OEIPTSSVkDETND~ITIFTRI~GLLDGYD IIIII I II I I 1 IIIII IIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIII 1 IIDNMlSrFIntKtLLvFCISlltlSSHFGFS~TSSVqDETNDNITIFTRILDGLWGYD
NRLRPGLGCRI~VRTDIWTSFGPVSDT~YTlDVTFRQS~-KGP~~LNN 123 LLASKIWTPDTFFHNGKKSIAH~TPNKLLRLEDDGTL~YWATYTMRLTISAECPMQLEDFPMD
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
123 LLWKIWTPDTFFHNGRI(SIAHNElTTPNKLLRLCDDGTLLYTMRLT1SAECPMQLEDFPMD
cons
LLASKIWTPDTFFHNGKKSIAHNElTTPNKLLRLCDDGTLLYTURLTISAECPMQLEDFPMD 184 A H A C P L K P G S Y A Y P N S E V ~ G S T K S ~ A E D G S ~ Q Y H ~ ~ ~ G T E ~ I S T S T G E X
IIIIIIIIlIIIIIIIIIIIIIIIlIIIIIIIlIIIIIIIIIIIIIIIIIllIlIIIIIII
184 A H A C P L K P G S Y A Y P N S E V N G S T K S V W A E D C S R G T E N I S T S T G E Y
cons
AHACPLKPGSYAYPNSEVNGSTKS~AEDGSRLNQYH~~TVGTENISTSTGEY I . . . .
245 T l M T A H F H L K R K I G ~ W I Q T Y L P c I M T ~ R E S ~ ~ ~ ~ ~
IIIIIlIIIIIIIIIIlIIllIlIIlIIlIIIIIIllIIllIIIIIIIllIIIIIIIIlIl 245 T I H T ~ F H L K R K I G Y ~ I Q T Y L P C I n T V I L S Q V S ~ ~ R E S W ~ ~ G ~ T V L T M T T L S I cons TIMTAHFHLKRKIGY~IQTYLPCIMTVILSQVS~~~SW~~~~L~TLSI
366 RevlLNKSTNAFTTGKmJHPPNIPKeQtPaGT rnttavSvkpSEEKTSESKTYNSlSKI II IIIIIIIIIIIII IIIIIIIII I II I IIIIIIIIIIIIIIIIII 361 RelILNKSTKAPTTGKltHPPNIP~QlPgGT~nav~taS1raSO~KTS&SIV(TYNSISKI
cons
Re-ILNKSTNMTTGK--nPPNIP)(EP-P-GTg------S---S~KTSCSKTXNSlSKl
TI44 426 D K ~ ~ R X V T P V ~ ~ C T ~ N R E P V I K ~ S P K
IIIIIIIII IIIIIIIIIIIIIIIIIIIIIII I l l 428 DKHSRIVFPILFGTFNLVXLNREPVIKGAtSPK cons DKHSRIVFP-LFCTFNLVYWATYLNREPVIKGA-SPK
Figure 1 Alignment of the deducad primary m i n o acid requbncer 0 5 the human and rat alpha5 GABA-A rbceptoc rubunita. Ovbrlined raquencba indicate putative transmembrane domainr. I l l indicates putativb ri9n.l Ibj indicatea putativb N - 9 l y c o a y l a t i o n SitbS peptlde cleavage f l t e
could be modulated by nanomolar concentrations of the benzodiazepine flunitrazepam. but not by micromolar concentrations of the BZ1 selective compound zolpidem. This i s in agreement with previous radioligand binding data determined for receptors expressed by cells transiently transfected with rat a5. and $2 subunit cDNAs (6).Thus a5 containing human GABA-A receptor subtypes, like their rat equivalent, have a unique benzodiazepinepharmacology.
-
1.
Olsen RW and Venter JC (1986), Benzodlazeplne GABA Receptors and Chloride Channels: Structural and Functional Propertles, Alan Liss. New Yo&.
2.
Squires RF, Benson DI, Braestrup C, Coupet J, Klepner CA, Myres V and Beer B (1979), Pharmacol. Blochem. Behav. 10,825830.
3. Luddens H and Wisden W (1991). Trends Pharmacol. Scl. 12. 49-51. 4.
Pritchett DB, Sonthelmer H. Shivers B. Ymer S . Kettenmann H, Schofield PR and Seeburg PH (1989), Nature 338,582-585.
5.
Pritchett DB, Luddens H and Seeburg PH (1989), Science 245, 1389-1391.
6.
Pritchett DB and Seeburg PH (1990), J Neurochem 54, 1802-1804.
7.
Khrestchatisky M, MacLennan AJ, Chiang MY, Xu W, Jackson MB, Brecha N, Sternini C, Olsen RW and Tobin AJ (1989), Neuron 3, 745-753.
