The use of the FLIPR in the study of the heterodimeric GABA receptor

Introduction• Recently the GABA B has been cloned and shownto be a heterodimer of 2 similar subunits• Here we describe studies which have used theFLIPR to:– pharmacologically characterise theheterodimeric receptor– study the role of the subunits in trafficking thereceptor to the cell membrane

Pharmacological characterisationEvidence for GABA B receptor subtypes:• Phaclofen reverses baclofen-mediated inhibition of GABArelease, but does not reverse baclofen-mediated inhibitionof glutamate release• GHB (γ-hydroxybutyrate) discriminates GHB -sensitiveand -insensitive GABA B receptors• Gabapentin has been claimed to be a GABA B receptoragonist with subunit selectivity

• Recently the GABA B receptor was cloned - shown to consist ofa heterodimer of two subunits – GABA B1 (which exists as twosplice variants GABA B1a and GABA B1b ) and GABA B2• We have studied the pharmacological profile of theserecombinant receptors using radioligand binding (3H-CGP-54626A) and functional studies

• We have co-expressed the GABA B1a /GABA B2 andGABA B1b /GABA B2 subunits in cells co-expressing theadapter G-protein, G qi5• This system couples GABA B receptor activation tochanges in intracellular Ca 2+• Changes in intracellular Ca 2+ were measured using theCa 2+ -sensitive dye Fluo-4 using the FLIPR (see Wood etal, 2000, Br.J.Pharmacol. 131:1050-1054)

Antagonism of GABA stimulation on intracellular calciumat the GABA B1b/B2 dimer% Maximal response10080604020010 -11 10 -1010 -910 -810 -710 -610 -510 -410 -3CGP-46381CGP-35348CGP-54626CGP-62349CGP-55845SCH-50911PhaclofenSaclofen[Anta gonist].M

Antagonist profileGABAB1a/B2GABAB1b/B2Compound pKB Vs pKi [3H]CGP- pKB VsGABA 54626 GABA BaclofenPhaclofen

Studies with GHB and GABAPENTIN• No agonist activity or antagonist activityat either GABA B1a/B2 dimer or GABA B1b/B2 dimerfor GHB at concentrations up to 10 mMfor Gabapentin at concentrations up to 1 mM

Summary• The human recombinant GABA B heterodimeric receptordisplays a low sensitivity to the antagonists phaclofen, saclofenand CGP 35348 and was not modulated by GHB• This is consistent with a phaclofen- and GHB-insensitivesubtype of the GABA B receptor• Gabapentin did not act as an agonist at either the GABA B1a/2 orGABA B1a/2 heterodimers

Summary cont• But, in-vivo and in-vitro, the majority of GABA B -mediatedresponses are sensitive to phaclofen• Are there other GABA B sub-units?• Are there other components which are missing in therecombinant system e.g. accessory proteins• Is the signalling mechanism faithful?• Need to study distinct GABA B receptor subunitcombinations in physiological conditions- cell line withnative GABA B receptor ?

GABA B1 and GABA B2 deletion and chimera constructsGABA B1bc-mycTMD 1-7C-CGABA B1b∆806GABA B1b∆771GABA B1b∆747GABA B2GABA B2∆748GABA B2/1bCGABA B1b/2C

Expression of GABA B1 deletion constructsGABA B1bGABA B1b∆806GABA B1b∆771- TX-100 + TX-100GABA B1b∆747

Expression of GABA B2 deletion & chimera constructs- TX-100 + TX-100GABA B2GABA B2∆748GABA B2/1bC

GABA B2 takes GABA B2/1bc to the cell surface- TX-100GABA B2/1bC+GABA B2

Antagonist binding to GABA B1D747 is displaced by GABAGABAB1b+ GABAB2GABA B1bGABA B1b∆747% Specific [ 3 H]CGP-54626 binding100806040200Untransfected HEK293-6-4log [GABA] (M)-2

Functional evaluation of GABA B deletion and chimera constructs6000600012000100004000Untransfected cells40008000FIUGABA B1bGABA B1b∆747FIUGABA B1b + GABA B2FIU60002000GABA B1b/2C20004000GABA B1b∆747+ GABA B2∆74800-8-6-4-8GABA B1b + GABA B2-4-6-8GABA B1b∆747 + GABA B2-420000-6Log [GABA] (M)Log [GABA] (M)Log [GABA] (M)

Novel sites of interaction between the two GABA B receptor subunitsNH 2NH 2??GABA B1GABA B2CC

Summary• The C-terminal domain of GABA B1 mediates theintracellular retention of this subunit• Interaction between GABA B1 and GABA B2 alsooccurs at sites other than the C-terminal coilcoildomain• Cell surface GABA B1 is only functional when coexpressedwith the GABA B2 subunit

Conclusions• The FLIPR is a versatile tool which can be usedfor a variety of purposes• Pharmacological characterisation of expressedreceptors• Probe for receptor function

AcknowledgementsNeuroimagingKelly CharlesGuillame HervieuMartyn EvansBiotechnology and GeneticsMelanie StammersIsro GlogerChemistryMervyn ThompsonGene Expression SciencesSimon RiceOwen Jenkins