Tecnología de la información y de la comunicación (tic)

T he n e u ro m u s c u la r nA C hR c h a n n e l is com posed of fo u r ty p es of su b u n its, a, p, Ô a n d e ith e r y or e w ith two n o n -ad ja ce n t a

s u b u n its s e p a ra te d by th e y s u b u n it (K a rlin e t al, 1987). T he c o n serv e d f e a tu r e s of L G IC s a re o fte n m a p p e d a g a in s t th e corresponding nu m b ered resid u es on th e T.californica nA C hR a a n d

p su b u n its (Claudio, 1989, an d see Fig. 3.1, page 100 an d Fig. 3.2, page 101). T he m ost strik in g of th ese is th e four tra n sm e m b ra n e dom ains (TM D 1,2,3,4) w hich a re hydrophobic s tre tc h e s of re s id u e s w hich sp an th e m em brane. An am p h ip ath ic helix w as a t first th o u g h t to be a fifth TMD, b u t it is likely th a t th is helix, n o t p resen t in all channels.

lies along th e in sid e side of th e m em brane an d does n o t cross it (MA - Fig. 3.2, page 101).

T h is tra n s m e m b ra n e topology c o n tra sts w ith th e com m on p u ta tiv e topology of G protein-coupled receptors w hich h av e a single recep to r p ro tein w ith seven TMDs (review; D ohlm an e t al, 1991, Hille, 1992), a n d w ith th e p u ta tiv e topology an d sto ch io m etry of voltage- g a te d ch an n els w hich are e ith e r m onom ers w ith tw e n ty four p u ta tiv e TM Ds or te tra m e rs w ith six p u ta tiv e TMDs to each su b u n it (review; Betz, 1990a, C a tte ra ll, 1991). E ach receptor superfam ily th erefo re h a s c h arac teristic s tru c tu ra l design (S trange, 1988, Betz, 1990a).

E x p erim e n ts to try to m ap v arious regions of th e su b u n its to th e in tr a - , or e x tra c e llu la r side of th e m em b ran e h a v e p ro d u ced conflicting r e s u lts , b u t th e consensus locates b o th th e am ino- a n d carb o x y -term in als of th e p ro tein ex tracellu larly an d seem s to su p p o rt th e fo u r TM D schem e. T he p resen c e of a sig n a l p e p tid e on a ll su b u n its su p p o rts th e belief t h a t th e am ino term in a l is ex tracellu lar, a n d fu sio n p r o te in s tu d ie s h a v e confirm ed th e lo c a tio n of th e m e m b ran e -sp a n n in g regions (Chavez an d H all, 1992).

In w ork on th e n e u ro m u scu lar nAChR, w hich is a ssem b led from fo u r d isc re te ty p es of su b u n it, d o m ain s involved in s u b u n it i n te r a c tio n s in v o lv e d in a sse m b ly h a v e b e e n m a p p e d to th e e x tr a c e llu la r a m in o -te rm in a ls of s u b u n its (Yu a n d H a ll, 1991, V errall a n d H all, 1992).

T h ere is a h ig h degree of conservation betw een th e m em b ran e s p a n n in g d o m a in s of th e m e m b e rs of th e LG IC s u p e rfa m ily , especially in th e reg io n of T M D l a n d 3, w hich a re th o u g h t to flan k T M D 2, w ith TM D4 on th e o u tsid e of th e su b u n it. W h e th e r th e s e tra n s m e m b ra n e sp a n n in g reg io n s a re all a-h e lic es re m a in s to be d e m o n s tra te d . In a re c e n t a lte rn a tiv e m odel, TM D 2 is th e only m e m b ra n e -s p a n n in g a-h elix , w hile TMD 1,3 a n d 4 a re m em b ran e- s p a n n in g P -p le ated sh e ets w hich su rro u n d i t in th e lip id b ila y e r (Sansom , 1993, U nw in, 1993, Gom e-Tscheinokow e t al, 1994).

M u ta tio n stu d ies have in d icated th a t TM D2 is th e p a r t of th e s u b u n it m o lecu le t h a t faces in w a rd s to w a rd s th e p o re. S m a ll u n c h a rg e d re sid u e s w hich a p p e a r a t re g u la r four re sid u e in te rv a ls alo n g th is re g io n a re th o u g h t to lin e th e pore, a n d a conserved hydrophobic leu cin e resid u e is th o u g h t to m a rk th e p o sitio n of th e ^gate' t h a t re g u la te s th e pore. T his leucine is p re s e n t a t ,t h e m o st co n stricted p a r t of th e pore th ro u g h o u t th e AChR family.

I t is also p re s e n t in some g lu ta m a te rec ep to r su b u n its, b u t o th e rs have a la rg e r hydrophobic am ino acid, p h e n y la la n in e. In th e g lu ta m a te recep to rs, a resid u e p osition close to th is p h e n y la la n in e seem s to reg u la te th e p erm eab ility to d iv alen t cations (B u rn ash ev e t al, 1992).

G roups of charged resid u es a t th e ends of TMD2 a re th o u g h t to p lay a p a rt in ion selectivity, an d ag ain th e re are hig h ly conserved r e s id u e s a t s im ila r p o sitio n s b e tw e e n s u b u n its : fo r in s ta n c e c o n se rv e d a s p a ra g in e , g lu ta m a te a n d ly sin e r e s id u e s in th e c h a n n e ls p e rm e a n t to catio n s a n d a rg in in e re sid u e s in th e an io n p e rm e a n t channels (review; C hangeux e t al, 1992, K arlin, 1993).

O th e r fe a tu re s of th e su b u n its include a conserved p a ir of cy stein e re sid u e s t h a t a re th ir te e n re sid u e s a p a r t k n o w n a s th e lig an d -g ated channel motif, an d a n N -linked glycosylation site w ith in th is w hich is p re se n t in all n e u ro m u scu lar nA C hR su b u n its an d all G ABA recep to r P -subunits (review: S tro u d e t al, 1990, see Figs. 3.1 a n d 3.2, pages 100 & 101). O th e r glycosylation a n d p h o sp h o ry latio n sites v ary betw een subunits.

A p a ir of adjacent cysteine resid u es found a t positions 192 an d 193 in th e T orpedo a -s u b u n it a n d in all n e u ro m u sc u la r nA C hR a - su b u n its is involved in ag o n ist binding, an d is u se d to define a- an d P-type n eu ro n al nAChR su b u n its (see below).

T h ere a re several proline resid u es a t conserved positions in LGIC su b u n its, including one resid u e p re s e n t in all su b u n its cloned so fa r w hich lies b etw een tra n s m e m b ra n e dom ains tw o a n d th re e . T hese resid u es have been suggested to play a role in th e folding of th e s u b u n its , a n d th e re is evidence t h a t su ch folding m ay p la y a n im p o rta n t p a rt in th e assem bly of homo-oligomeric nAChR a n d SHTg channels (H elekar e t al, 1994).