Rendimiento académico

D espite th e fact th a t m an y com ponent su b u n its of LGICs have b een cloned, in m ost cases it is still u n c le ar w hich of th ese su b u n its assem ble to form channels in vivo, an d how th is process occurs.

T h e n e u r o m u s c u la r n A C h R (fo u n d a t n e rv e - m u s c le ju n c tio n s ) c o n sists of fo u r d is tin c t b u t h o m ologous s u b u n its d e s ig n a te d a,p,6 a n d e ith e r y or e, a rra n g e d in a p e n ta m e r as d e sc rib e d above, in th e sto c h io m e try «2» P» S, y. B ecau se lig a n d

b in d in g is associated w ith th e a -su b u n its, two lig an d b in d in g sites ex ist, a t th e side of th e two a -su b u n its w hich a re n o t ad jacen t. T he two b in d in g sites differ in b inding ch arac teristics because b in d in g is affected by th e s u b u n its a d ja ce n t to th e a su b u n its (B lo u n t a n d M erlie, 1989).

S ev eral subtypes of n e u ro n a l nA ChRs h av e b een described. T h ese a re found on n e u ro n s of th e PN S a n d CNS, a n d co n sist of sim ila r b u t d istin ct sub u n its, of w hich about te n have been discovered to d a te (review;Role, 1992). The su b u n its have been subdivided on th e b a sis of th e p resence or absence of th e a d ja c e n t cysteine re sid u e s described above (a-type contain th e p a ire d cysteines, p-type do not), b ecau se th e y are n o t sim ila r enough to th e v ario u s n e u ro m u sc u la r su b u n its to be classified as homologous to p, 6, y or e su b u n its. Indeed, it is n o t y et clear if th e re are n eu ro n al homologues to all th e different n e u ro m u s c u la r su b u n its, or how m an y d ifferen t ty p es of su b u n it assem ble to produce n eu ro n al nAChRs.

T h is pro b lem of su b u n it function b a s b een a d d re ssed by th e use of ex p ressio n system s, m o st n o tab ly th e u se of oocytes from th e A frican claw ed to ad , X en o p u s laevis (Sigel, 1990, B e rtra n d e t al,

1991). In je c tio n of m RN A, e ith e r e x tra c te d fro m n e u ro n s or tr a n s c rib e d from cloned c h an n el s u b u n it genes, can r e s u lt in th e encoded p ro te in s b ein g sy n th e sise d a n d in s e rte d in to th e oocyte m e m b ra n e a s com plexes w h ich m a y fu n c tio n a s c h a n n e ls in response to ag o n ist (B oulter et al, 1987, W ada e t al, 1988, D e n e n s e t al, 1989, C o u tu rier e t al, 1990).

By e x a m in in g th e s u b u n it c o m b in atio n s t h a t r e s u l t in fu n ctio n al channels, a s ta r t h a s been m ade in d eterm in in g th e role of th e cloned s u b u n its . F or n e u ro n a l nA C hR s for in s ta n c e , a f ir s t w orking ru le w as t h a t a t lea st one type of a-su b u n it an d one type of p- s u b u n it w a s re q u ire d to m ak e a fu n c tio n a l re c e p to r, w ith th e exception of th e a 7 su b u n it w hich is able to form a hom o-oligom er (C o u tu rie r e t al, 1990). An e le g a n t stu d y u sin g m u ta te d ch an n e l s u b u n its w ith su b tly a lte re d p ro p e rtie s h a s d e m o n s tra te d t h a t a c h an n e l c o n stru c te d by com bining th e pro d u cts of th e n e u ro n a l a4 an d p2 s u b u n it genes is a p e n ta m e r consisting of two a4 a n d th re e p2 su b u n its (Cooper e t al, 1991).

In f u rth e r oocyte expression ex p erim en ts, it h a s b een show n t h a t som e of th e n e u ro n a l a s u b u n its c an s u b s titu te for th e n e u r o m u s c u la r a s u b u n it a n d form fu n c tio n a l c h a n n e ls if co­ in je c te d w ith th e o th e r su b u n its of th e n e u ro m u s c u la r rec ep to r. S im ila rly som e of th e n e u ro n a l p su b u n its can s u b s titu te for th e n e u ro m u s c u la r p su b u n it.

O f th e n e u ro n a l su b u n its functionally te ste d in oocytes, th e a5, a 6 an d p3 su b u n its (W ada e t al, 1990, D eneris e t al, 1989) hav e y et to be d e m o n s tra te d to function in a p airw ise fash io n w ith a n y of th e o th e r s u b u n its , a lth o u g h th e a 5 does assem b le to g e th e r w ith two o th e r s u b u n its ty p es (a3 an d p4) in chick ciliary ganglion n e u ro n s (Conroy e t al, 1992).

T h is asso ciatio n of th re e su b u n it types em p h asises t h a t i t is still n o t c le ar exactly how m an y of th e su b u n its a re assem b led in to n a tiv e recep to rs. I t is common for n e u ro n s to ex p ress four or m ore n e u ro n a l nA C hR su b u n its (for e.g., chick ciliary ganglion n e u ro n s, C o rriv eau a n d B erg, 1993; chick su p erio r cervical ganglion n eu ro n s, L iste ru d e t al, 1991, r a t dorsal root ganglion neurons, see c h ap ter 6).

I t m u st still be considered a po ssib ility t h a t th e fu n ctio n of p a irs of th e v ario u s LGIC su b u n its in oocytes could be fo rtu ito u s, or t h a t lac k of fu n ctio n in some in sta n c e s m ay be due to th e lack of m odifications or th e lack of accessory p ro tein s req u ire d for assem bly r a th e r th a n a genuine lack of functional com patibility.

A sim ila r problem exists w ith th e o th er m em bers of th e LGIC su p erfam ily . T he su b u n its of o th e r LG ICs h av e b een s tu d ie d in a sim ila r w ay b y expression in oocyte system s, an d fu n ctio n al hom o­ o lig o m ers a n d o th e r fu n c tio n a l s u b u n it c o m b in atio n s h a v e b e en described, b u t once again, th e relatio n of th ese com binations to in vivo c h a n n e ls is m o stly n o t know n (Som m er a n d S eeburg, 1992, B etz, 1990b, 1991, Seigel e t al, 1990).

T h e g lu ta m a te re c e p to r fa m ily of L G IC s is th e m o st a b u n d a n tly expressed receptor fam ily in th e CNS an d PN S (reviews: B etz, 1991, G asic an d H ollm an, 1992, Som m er a n d S eeburg, 1992). T he ch an n els can be divided on th e b asis of w h e th er th ey can be g ated by T i-m ethyl-d-aspartate (as NMDA or non-NDMA receptors).

Follow ing th e cloning of th e firs t NMDA recep to r su b u n it, NMDA- R1 or NR-1 (M oriyoshi e t al, 1991), th re e fu rth e r su b u n its h av e b een d escrib ed , NR-2A, NR-2B a n d NR-2C (M onyer e t al, 1992). T h ese sh a re b etw een 55 an d 70% p ro tein sim ilarity betw een each o th e r b u t show only ab o u t 20% homology to NR-1.

A lth o u g h NR-1 is th e only su b u n it w hich is ab le to form fu n ctio n al hom om eric ch an n els w hen expressed in X enopus oocytes, th e h etero m eric channels form ed by expressing NR-1 w ith a n y one of th e th re e o th e r su b u n its produces ch an n els w ith a m p litu d e s w hich a re a ro u n d 100 tim e s la rg e r. I t seem s lik e ly t h a t h e te ro m e ric ch an n els ex ist in vivo (Monyer e t al, 1992).

T h e non-NM DA recep to rs can be fu rth e r divided, a d is tin c t c lass of th e s e h a s a h ig h a ffin ity for k a in a te a n d a re k n o w n as k a in a te re c e p to rs, w hile th e re m a in d e r a re m ore se n sitiv e to a- am ino-3-hydroxy-5-m ethyl-isoxole-4-proprionate a n d a re k n o w n as AM PA rec e p to rs (Gasic an d H ollm an, 1992, Som m er a n d S eeburg,

1992). F u n c tio n a l expression screen in g w as u se d to clone th e firs t g lu ta m a te c h a n n e l s u b u n it, G lu R l, w h ic h fo rm ed a n A M PA re c e p to r (H o llm an e t al, 1989). Homology screen in g h a s re v e a le d sev eral o th e r re la te d sub u n its, know n as G luR l-G luR 7 (B e ttler e t al, 1990) or G luR -A etc (K ein an en e t al, 1990). T h ese s u b u n its can

fu nction in d ifferen t com binations to produce channels w ith differing p ro p erties (B oulter et al, 1990, N ak an ish i et al, 1990).

T he discovery t h a t two a lte rn a tiv e ly spliced v ersio n s of a n exon, te rm e d ‘flip ‘ a n d 'flop', ex isted for sev eral of th e se su b u n its in c re a s e d th e d iv ersity of th ese ch an n els (Som m er e t al, 1990). In a d d itio n to th is, i t ap p ears t h a t cell-specific RNA ed itin g m ay re s u lt in th e conversion of a single am ino acid in th e crucial p o rtion of th e p o re-lin in g TMD2 of some AMPA recep to r su b u n its, th u s affecting ion selection (Som m er e t al, 1991).

S u b u n its corresponding to th e h ig h -affin ity k a in a te -b in d in g non-N M D A re c e p to rs h av e b e en id e n tifie d as d is tin c tly s m a lle r p ro te in s th a n th e AMPA receptor su b u n its a fte r peptide p u rificatio n from recep to rs. Some of th ese have also been cloned (Egebjerg e t al, 1991, H e rb e t a l 1992), a n d w h ile som e c an form fu n c tio n a l h o m o m e ric c h a n n e ls in oocytes, o th e rs s u r p r is in g ly p ro d u c e re c e p to rs in oocytes only in com bination w ith m em bers of th e low affinity k ain ate-b in d in g class of su b u n its (H erb e t al 1992).

S ev eral com ponent su b u n its of th e lig an d -g ated chloride ion c h an n e ls h av e so fa r been described, a n d biochem ical an aly sis of th e p ro te in s p u rifie d from glycine recep to rs from d ifferen t p a rts of th e n e rv o u s sy s te m re v e a l only tw o or so m etim es th re e species of polypeptide su b u n it (Betz, 1990b). Two homologous polypeptides were o rig in a lly id e n tifie d in p u rified glycine recep to rs from m a m m a lia n s p in a l cord, a n d p h o to affin ity lab ellin g stu d ie s h av e m ap p e d th e lig an d b in d in g site to one of them , th e 48 kD a a l su b u n it (G rah am e t al, 1983). Since th e n , how ever, o th er v a ria n ts of th e a su b u n it have b een cloned, an d i t h as been d em o n strated th a t altern a tiv e splicing of th e m R N A s fro m th e s e gen es a d d s to th e d iv e rs ity of glycine receptors (Malosio e t al, 1991, review; Betz, 1991).

T he GABAa ch an n el su b u n its a re sim ila rly d e sig n ated w ith