Future Directions

To achieve cell surface targeting of y2L G FP fusion protein in cultured neurones it was necessary to introduce cD N A s for the a l and P2 subunits at the sam e tim e. Triple transfection of neurones is both difficult and time consum ing. It is perhaps surprising that the y2L G FP fusion protein could not assem ble w ith en d ogen ou s G A B A ^ receptor.

subunits when expressed in neurones. H ow ever, it could be explained by low levels of endogenous subunits available in the E R for olig om erisation , com p ared to very high levels o f the o verexp ressed y2L G FP su bu nit achieved by m icro in jectio n . T his may preclude oligom erisation o f y2L G FP w ith endogenous subunits into functional receptors. Similarly, recom binant a l or p2 receptor subunits expressed in superior cervical ganglion neurones are also unable to access the cell surface on hom om eric expression (G orrie et al., 1997). To avoid these problem s it w ould be advantageous to develop a system that w ould allow oligom erisation o f the y2L subunit G FPN fusion protein with endogenous subunits

To facilitate the study o f G A B A ^ receptor trafficking using the y2L -G F P N , a transgenic m ouse constitutively expressing this fusion protein under a neuronal prom oter was produced. In theory, since this co n stru ct w ill only be in teg rate d once into the transgenic m ouse genom e, it should have an expression level closer to that o f endogenous subunits and hopefully will therefore assem ble w ith endogenous receptor subunits and be targeted to synaptic sites. U nfortunately, there w as insufficient tim e to fully characterise this m ouse strain. H ow ever, this m ouse strain should be very useful for the study of G A B Aa receptor clustering. It should be possible to culture neurones from this m ouse that

w ould express G FP labelled inhibitory synapses. This w ould provide a very pow erful tool to study the ontogeny and dynam ics of G A B A ergic synaptogenesis and the m olecular m echanism s of GABA^^ receptor targeting in live cells.

This thesis also describes a m echanism s for GABA^ receptor rem oval via clathrin m ediated endocytosis. H ow ever, it w ill be im portant to identify signals in the CNS that could m odify the internalisation rate o f receptors. W ith respect to the association o f GABA^ recep to rs w ith A P2, it w ould be in terestin g to d eterm in e the p o ly p ep tid e sequence in rece p to r in tracellu lar dom ain s im p o rtan t fo r the in teractio n w ith A P2 com plexes. It is know n that both Y x x 0 and L L signals in teract d irectly w ith A P2

the \x2 chain o f AP2. B oth Y x x 0 and LL m otifs can be found in G A BA ^ receptor intracellular dom ains (Barnes, 1996). Im portantly, co-crystallisation studies o f \l2 w ith Y x x 0 peptides indicate that phospho-tyrosine can not bind in the |li2 hydrophobic binding pocket (M arsh and M cM ahon, 1999). Therefore, phosphorylation could sw itch o ff some tyro sin e based signals to regulate endocytosis (S hiratori et al., 1997; S tephens and B anting, 1997; M arsh and M cM ahon, 1999). Interestingly, a Y x x 0 m o tif in the GABA^ rece p to r y2 su b u n in t fo rm s p art o f th e m ajor site o f G A B A ^ re c e p to r ty ro sin e phosphorylation (M oss et al., 1995). This could therefore provide a m echanism to regulate the association o f G A BA ^ receptors with A P2 to regulate recep to r in tern alisation . It w ould therefore be o f interest to determ ine if this m o tif is im portant for the association of G ABA ^ receptors with A P2 and the consequence o f phosphorylation at this site.

It w ould also be helpful to better understand the functional role o f som e GABA^^ recep to r protein association s. W ork p resented in this thesis p ro vides evid en ce that G A B A R A P is not acting as a bridging m olecule betw een G A BA ^ receptors and gephyrin nor is it a key com ponent o f the inhibitory postsynaptic scaffold. G A B A R A P does bind N SF suggesting a role in GABA^^ receptor trafficking how ever, it w ould be useful to define further the functional consequences o f the association betw een G A B A R A P and N SF in GABA^ receptor trafficking.