PROPUESTAS ACERCA DE LOS MUSEOS PARA DESARROLLAR EN LA ETAPA DE EDUCACIÓN INFANTIL.

Quality control of protein folding and assembly is performed in the ER of the cell and attempts to stop misfolded proteins, incompletely assembled receptors, or abnormal products being transported to their final cellular destination where their imperfect structure could have a deleterious effect on the cell. The ER retains these structures where they are degraded before they can be passed to the Golgi and exported to their final subcellular location (Lippincott-Schwartz et a l, 1988). Degradation of some m isfolded and unassem bled products is blocked by inhibitors of lysosom al degradation suggesting that it takes place in lysosomes or autophagic vacuoles (Davis and Hunter, 1987) and there is evidence for degradation by an autophagosomal route (Masaki et a l , 1987). The time and rate of protein maturation varies from minutes to several hours and depends on rate of folding and rate of oligomer assembly. This can vary in different cells due to physiological differences and oligomerisation depends on concentration levels of the individual proteins that make up the oligomer and this in turn depends on expression levels. There must be a fine balance between having enough unassembled subunits to oligomerise to form mature proteins and degradation of unassembled proteins.

Misfolded proteins are often retained in the ER as aggregates, frequently found to be associated with BiP, these complexes are not transported to the cell surface (Sitia et al., 1990). The identity of the enzymes involved in degradation are still not known, some degradation is blocked by inhibitors of lysosomal degradation which suggests that degradation takes place in lysosomes or autophagosomal vacuoles (Davis and Hunter, 1987) and some degradation occurs via the autophagasomal route (Masaki et a l, 1987).

The control o f transport of proteins from the ER and the retention of incompletely assembled proteins and of resident proteins such as the chaperones involve several mechanisms. Many of the resident ER proteins have C-terminal KDEL or HDEL sequences which serve as retrieval signals (Pelham, 1991), these and other retention determinants may interact with receptors in the ER and the Golgi. Unassembled subunits and assembly intermediates may be retained by structural properties such as hydrophobicity or flexibility of exposed peptide groups. These are unlike misfolded proteins which form stable aggregates and associate with BiP. Unassembled subunits and assembly intermediates may be retained by weak interactions with a protein scaffold in the ER which may restrict diffusion to retain the proteins in the ER and allow enough freedom for subunit mixing for oligomerisation (Pfeffer and Rothman, 1987). Finally, misfolded proteins seem to be retained by aggregate formation, often in association with BiP. Aggregation occurs soon after synthesis and may be due to poor solubility of the incorrectly folded proteins. The association with BiP in the aggregates may be important for retention since BiP has a KDEL sequence which is how it is retained in the ER (Hurtley, 1989).

1.10.5 Glycosylation of nAChRs

The subunits of ion channels are synthesised in the ER and then are transferred to various intracellular locations via the Golgi body. Tunicamycin is an inhibitor of protein glycosylation and has been used to study in vivo glycosylation of ion channels.

Tunicamycin can be added directly to cell growth medium to inhibit glycosylation of asparagine residues and changes in apparent molecular weight determ ined by polyacrylamide gel electrophoresis. Endoglycosidases H and F have been used to study carbohydrate side chains added to proteins in the ER, sensitive to both endoglycosidases, and more complex carbohydrates modified in the Golgi apparatus are insensitive to endoglycosidase H. This has been a useful technique to study the subcellular location of assembled and unassembled nAChR subunits (Ross et a l ,

1991). Site-directed mutagenesis has also been used to study the importance of glycosylation in assembly, cell surface expression and function of nAChRs (Mishina

et al., 1985; Blount and Merlie, 1990; Gehle and Sumikawa, 1991).

A conunon structural feature that may be involved in post-translational modification has been identified in muscle nAChR subunits (Noda et a l, 1983d), neuronal nAChRs (Boulter, 1986; Goldman, 1987) and G A B Aa receptors (Schofield et a l, 1987) located at asparagine 141 in the nAChR a subunit there is a conserved N-linked glycosylation site. Site-directed mutagenesis of this site produces muscle a subunits that are not glycosylated and that do not achieve a mature conformation (Blount and Merlie, 1990). When expressed in oocytes, the mutated subunits do retain the ability to assemble with the ô subunit. Complexes containing the mutated a subunit do not bind a-B T X and are rapidly degraded (Blount and Merlie, 1990). The results suggest that incorrectly assembled receptor complexes are rapidly degraded and preclude surface expression of non-functional nAChRs in oocytes.

1.10.6 Phosphorylation of nAChRs

Protein phosphorylation can modulate the functional properties of many cellular proteins. Phosphorylation has been studied by expressing cloned ion channels in m am m alian cell lines by m etabolic labelling with [32p]orthophosphate and immunoprécipitation (Miles et a l, 1989). The sites of phosphorylation have been determined by site-directed mutagenesis, for example, serine or threonine are altered

to alanine residues, tyrosine to phenylalanine residues. The functional effects of such mutations can be determined by comparing wild type and mutated receptors.

Protein phosphorylation has been implicated in modulating synaptic transmission and regulating the function of ion channels such as the nAChRs (Huganir and Greengard, 1987). The Torpedo nAChR is phosphorylated in vitro by three different protein kinases. Protein kinase C phosphorylates the a and 5 subunits (Huganir et a l, 1984; Safran et a l, 1987), cyclic AMP dependent protein kinase phosphorylates the y and ô subunits (Huganir and Greengard, 1983) and an endogenous protein tyrosine kinase phosphorylates the p, y and ô subunits (Huganir et a l, 1984). Phosphorylation of the

Torpedo nAChR increases the rate of desensitisation of the receptor (Huganir et a l,

1986).

Protein phosphorylation of the Torpedo nAChR is also thought to be involved in the assembly of the receptor. Torpedo nAChR subunits expressed in mouse fibroblasts were incubated with forskolin, which increases the levels of cAMP, which in turn increases receptor expression 2-3 fold by stimulating subunit assembly. The increased assembly and increased cell surface expression was shown to be due to phosphorylation of the y subunit but not the ô subunit (Green et a l, 1991). This was later shown not to be due to direct phosphorylation (Jayawickreme et a l, 1994). The phosphorylation of the y subunit is thought to increase the association with the a subunit (Claudio, 1989). The fact that unassembled subunits are phosphorylated indicates that phosphorylation occurs in the ER, possibly by protein kinase A. The kinase(s) that act in the ER may have a modulatory role in the signal transduction whereby changes in cAMP levels could regulate protein assembly and protein release from the ER. An increase in cAMP levels has been shown to cause an increase in surface expression of neuronal nAChRs such as the a l receptor in SH-SY5Y cell (Cooper and Millar, 1997).

The nature of the host cell is thought to be important for the expression of nAChRs. There are many processes involved in protein folding, assembly and trafficking to the final subcellular destination such as glycosylation, phosphorylation and the presence of chaperone proteins. These are critical events that lead to the formation of functional nAChRs and disruption or absence of any of these can result in failure to express a functional receptor. Once a suitable host cell has been established and functional receptors can be expressed then nAChRs can be investigated in vitro to study the action of nicotinic ligands at these receptors and to screen potentially useful therapeutic compounds.