During the past decade, the genetics of type 1 (insulin-dependent) diabetes mellitus (IDDM) has been studied extensively and the disorder has become a paradigm for genetically complex diseases. Previous genome screens and studies focused on candidate genes have provided evidence for genetic linkage between polymorphic DNA markers and 15 putative IDDM susceptibility loci, designated IDDM1-IDDM15. We have carried out a second-generation screen of the genome for linkage and analysed the data by multipoint linkage methods. An initial panel of 212 affected sibpairs (ASPs) was genotyped for 438 markers spanning all autosomes, and an additional 467 ASPs were used for follow-up genotyping. Other than the well-established linkage with the HLA region at chromosome 6p21.3, there was only one region, located on chromosome 1q and not previously reported, where the log likelihood ratio (lod) was greater than 3. Lods between 1.0 and 1.8 were found in six other regions, three of which have been reported in other studies. Another reported region, on chromosome 6q and loosely linked to HLA, also had an elevated lod. Little or no support was found for most reported IDDM loci (lods were less than 1), despite larger sample sizes in the present study.

Muscle nicotinic acetylcholine receptors (AChRs) are pentamers that contain two ␣ subunits a ␤, ␥ (or ⑀), and ␦ subunit. In this paper, we have characterized subunit processing and folding events leading to formation of the two AChR ligand binding sites. ␣ subunit residues, 187-199, which are part of overlapping ACh and ␣-bungarotoxin (Bgt) binding sites on AChRs, were assayed using a monoclonal antibody (mAb) specific for these residues. We found that this region was inaccessible to the mAb early during AChR assembly but became accessible as the first of two Bgt binding sites formed later during assembly, indicating that the region changes conformation as the Bgt binding site appears. Without previous reduction, 20% of the ␣ subunits could be alkylated by bromoacetylcholine bromide as the first ACh binding site formed, which further indicated that the disulfide bond between cysteines 192 and 193 does not form until the first ACh binding site appears soon after Bgt binding site formation. When ␣ subunits were mutated to add a glycosylation site at residue 187, the number of Bgt binding sites increased threefold, AChRs assembled more efficiently, and 2.5-fold more AChRs reached the cell surface. Our results indicate that binding site formation involves a rate-limiting rearrangement of the ␣ subunit that exposes the 187-199 region to the endoplasmic reticulum lumen and determines when cysteines 192 and 193 disulfide bond.Key words: nicotinic receptor; Torpedo; muscle; ␣-bungarotoxin; protein folding and assembly; acetylcholine binding site Signal transduction by members of a family of neurotransmittergated ion channels, which include the acetylcholine (ACh), GABA A , glycine, and 5-HT 3 receptors, is initiated by binding of neurotransmitters to specific sites on the receptors. Muscle-type nicotinic ACh receptors (AChRs), which include the receptor in fish electric organs, have long served as a model for the family (for review, see Changeux, 1995;Karlin and Akabas, 1995). AChRs are composed of four homologous subunits, ␣, ␤, ␥ (or ⑀), and ␦, that assemble into pentamers with a stoichiometry of ␣ 2 ␤␥␦. There are two ligand binding sites per receptor. Affinity labeling and mutagenesis of the ␣ subunit have identified six residues, Tyr-93, , in the vicinity of the binding site and have shown that the adjacent cysteines, , are disulfide-bonded. Residues on the ␥ and ␦ subunits have also been identified as contributing to the binding site, from which it was concluded that the two binding sites are found near the interfaces between an ␣ subunit and either the ␥ or ␦ subunits (for review, see Karlin and Akabas, 1995;Tsigelny et al., 1997;Changeux and Edelstein, 1998).Venom-derived ␣-peptide neurotoxins are potent AChR competitive antagonists and bind with high affinity to regions overlapping ACh binding sites. The best characterized of these neurotoxins is ␣-bungarotoxin (Bgt), for which the major region contributing to the binding site has been localized to ␣ subunit residues 173-204 (Wilson et al., 1985;Tzartos and Rem...