As discussed in the introduction, the large num ber o f G ABA ^ receptor subtypes provides the p o te n tia l fo r c o n s id e ra b le stru c tu ra l d iv e rsity . H o w e v e r, b io c h e m ic a l and im m unological analysis of neuronal receptors has revealed the ex istence o f receptors contain in g only a lim ited num ber o f subunit co m bin ation s. T here m ust th erefore be m e c h a n ism s to c o n tro l su b u n it o lig o m e risa tio n a llo w in g o n ly c e rta in su b u n it com binations to form . F urtherm ore, G ABA^ recepto rs are clu stered and anchored at specific postsynaptic sites w ithin the neurone. These processes as well as the m echanism s im portant for the cell surface targeting o f G ABA^ receptors are essential for the correct function o f inhibitory synaptic transm ission (Essrich et al., 1998; C restani et al., 1999). H ow ever, the m o lecu lar m echanism s th at govern the assem bly, cell su rface sorting, p o stsy n ap tic m em b ran e in sertion and ancho rin g o f th ese ch an n els are still poorly understood. To gain further insight into these processes it w ould be useful to be able to study receptor assem bly and clustering in live cells. This is particularly im portant because it has been show n that the levels of G ABA ^ receptors can be regulated by a num ber of processes including kinase activation and kindling (Barnes, 1996; C onnolly et al., 1999b; N u sser et al., 1997). Shi et al., recently used fusion proteins o f G PP and the G lu R l subunit o f A M P A type glutam ate receptors to study the translocation o f these receptors into spines upon induction o f LT P (Shi et al., 1999). N um erous other groups have also used G PP fusions to study receptor processes (C arter and Sorkin, 1998; D oherty et al.,
1999; D avid-W atine et al., 1999).
To date the study of GABA^ receptor assem bly (C onnolly et al., 1996a; G orrie et al., 1997, T ay lo r et al., 1999, 2000), targeting (C onnolly et al., 1996b) and endocytosis (C onnolly et al., 1999a,b) in m am m alian cell lines and neurones has been lim ited to antibody lab elling m ethodologies. H ow ever these m ethodologies are not adequate for
visualising receptors in live cells and cannot be used to label intracellu lar receptors in n o n -p e rm e a b ilise d cells. F u rth erm o re , fix atio n and sam p le p re p a ra tio n fo r lig h t m icroscopy can lead to re-d istribution o f proteins and changes in the m orphology o f endosom al com partm ents (Carter and Sorkin, 1998). In addition to this, although antibody feeding can be used to study receptor in ternalisatio n, a n um ber o f lim itations to this approach can arise including non-stoichiom etric labelling o f receptors, partial dissociation of antibody-receptor com plexes in acidic organelles and sorting o f antibody and receptor to different com partm ents (C arter and Sorkin, 1998; C onnolly et al., 1999a). G PP from the jelly fish A eq u o ria victoria has been used as a fluorescent reporter m olecule in the localisation o f an increasing num ber o f proteins (Tsien, 1998). Im portantly, the fusion of G PP to the recep tor allow s the study of receptor localisation and surface stability to be investigated in live cells. To facilitate the study o f G ABA ^ receptor assem bly, m em brane targeting and trafficking, y2 subunit green fluorescent protein (GPP) fusion proteins were constructed that contain G PP either at the am ino or carboxyl term inus.
G ABA ^ receptors com prise several receptor subunit classes the m ajority of which contain m ultiple m em bers. It is how ever, generally believed that the m ajority o f receptors in the CNS contain at least an a , (3 and y subunit (M acdonald and O lsen, 1994; Rabow , 1995). O f these subunit classes, the y2 subunit is im portant in m ultiple aspects o f GABA^ receptor function and therefore appeared to be a particularly good candidate for tagging w ith G PP. F irstly , although pro du ction o f G A B A -g ated chann els is ach iev ed upon expression o f recep to r a a n d (3 subunits, the co -ex p ressio n o f a y su bu nit isoform confers benzodiazepine sensitivity (G unther et al., 1995). The G ABA ^ receptor y2 subunit is also the m ajor site o f tyrosine phosphorylation o f G ABA ^ receptors (M oss et al., 1995). Im portantly, the GABA^ receptor y2 subunit has also been show n to be essential for the cell surface targ eting and p ostsynaptic clusterin g of m ajor GABA^^ sub ty pes in vivo
interaction w ith the cytoskeletal linker m olecule G A B A R A P, a sm all 17 kD a m icrotubule binding m o lecule th at m ay be im portant for recep to r anchoring (W ang et al., 1999). Finally, the protein kinase C m ediated internalisation o f G ABA^ receptors has been found to be dependent on the presence of a yZ subunit (Connolly 1999b). It is therefore clear that the y2 su b u n it plays a key role in G ABA ^ recep to r function and w ould be a good candidate subunit to use to m ake a flu orescen t G A BA ^ receptor su bu nit G FP fusion protein.
A lternative splicing generates 2 form s o f the y2 subunit, y2L and y2S that differ by the presence ^ f ^ ^ m in o acids in y2L (W hiting et al., 1990; Kofuji et al., 1991). Recently it has been show n that these two subunits differ in their ability to access the cell surface. W hereas the y2S subunit is able to access the cell surface as a non-functional m onom er, the y2L sub un it can only access the cell surface w hen co-ex pressed w ith an a and p subunit (C onnolly et al 1996a, 1999a). It was therefo re decided to m ake G FP fusion proteins of the y2L subunit. This w ould ensure that when co-expressed with an a and p su b u n it any y2L G F P cell su rface flu o re sc e n c e w ould be re p re s e n ta tiv e o f fully oligom erised receptors containing a , p and y2L subunits.
A ddition of the green fluorescent protein to the y2L subunit w ould result in an increase in 30 k D a to the m olecular w eight o f this subunit polypeptide. T his could in theory affect the folding, subunit assem bly and / or function o f the subunit. It is also p o ssib le th a t fu sin g the tw o p o ly p e p tid es co u ld have a n eg ativ e in flu en ce on the fluorescence o f GFP.
E xperim ents w ere carried out to determ ine a num ber o f the criteria listed below that w ould be essential for the use of the y2L subunit G FP fusion proteins.
1) the expressed y2L subunit G FP fusion proteins w ere o f the correct size and that the G FP m oiety was not subject to proteolysis
2) the fusion proteins produced a green fluorescent signal.
3) upon heterologous expression the subunit could be correctly targeted to cell surface in a sim ilar fashion to the wild type subunit and that it behaved sim ilarly to the wild type subunit with respect to it’s cell biology.
4) cell surface receptors containing y2L subunit G FP fusion proteins w ere functional and had a sim ilar pharm acology to wild type receptors.
5) the y2L subunit G FP fusion proteins could be introduced into neurones and targeted to inhibitory postsynaptic sites at the cell surface.
The exp ressio n, assem bly and function o f G A B A ^ receptors co n tain in g the y2L subunit G FP am ino and carboxyl term inal fusion proteins was tested in A 293 cells using both biochem ical and im m unocytochem ical techniques. The correct function o f these channels was also tested in A293 cells using electrophysiological m ethodologies. Finally, to study the expression and synaptic targeting of these constructs in neurones they w ere introduced into cultured hippocam pal neurones by nuclear m icroinjection.