br The MIR The MIR is defined

The MIR The MIR is defined by the ability of a single rat mAb to muscle or electric organ AChRs, typically mAb 35, to block binding of half or more of autoantibodies in human MG or EAMG [44], [45], [46]. Antibodies to the MIR are pathologically significant because they exhibit all the major pathological activities of autoantibodies from MG patients. mAbs to the MIR can passively transfer EAMG into experimental animals [39], [42]. These mAbs exhibit the primary pathological activities of serum antibodies: complement-dependent focal lysis of the postsynaptic membrane and antigenic modulation. AChRs are formed by five homologous subunits organized around a central channel (Fig. 1) [47]. Mammalian muscle AChR exists in fetal and adult forms, whose expression is developmentally regulated. In adult muscle, postsynaptic AChRs have their subunits organized in the order α1ϵα1δβ1 to form the central cation channel. In fetal muscle, or after denervation, ϵ is replaced by γ. Each subunit has a conformationally complex N-terminal extracellular domain of about 210 MK-571 sodium salt hydrate receptor followed by 4 transmembrane domains and a short extracellular C-terminal sequence. Conformation-dependent epitopes on the extracellular domain, especially the MIR, are the targets of the pathological autoimmune response. Between transmembrane domains M3 and M4 is a loosely structured large cytoplasmic domain of 112–151 amino acids, which is inaccessible to antibodies in vivo and becomes immunodominant by default in denatured AChR [48]. The MIR is located at the extracellular apex of α1 subunits of muscle AChR [49]. All AChR subunits have structures homologous to the MIR, but the MIR on muscle AChR α1 subunits is uniquely immunogenic and myasthenogenic [3], [50], [51]. The MIR is not a single epitope, but a region of closely spaced overlapping epitopes, several of which can be obscured by a single bound mAb [15]. The MIR loop (α1 66–76) is critical for many of these epitopes [52]. Amino acids 68 and 71 are particularly important, since mutations of either prevent binding of many mAbs to the MIR [53]. Many mAbs and serum autoantibodies bind only to conformationally mature α1 subunits [54]. By making chimeras of human muscle AChR α1 subunits with human AChR α7 subunits and Aplysia acetylcholine binding protein (AChBP), we proved that the native conformation of the MIR results from interaction between the MIR loop (α1 66–76) and the N-terminal α helix (α1 1–14) [52], [55]. The chimeras, incorporating both structural elements, exhibit high affinity for mAbs to the MIR. A MIR/AChBP chimera, human AChR α1(1–30, 60–81)/AChBP, is bound with high affinity by mAbs to the MIR (including a MG patient-derived mAb to the MIR) and reacts with autoantibodies from cat, dog and human MG patients [52]. This chimera is also potent at inducing EAMG [34], [56]. This proves that the chimera contains pathologically significant epitopes that are sufficient to initiate a pathological autoimmune response. Surprisingly, wild-type AChBP also induces EAMG, but is less potent. The fact that proteins quite distantly related to muscle AChRs can induce EAMG suggests the possibility that MG may be triggered by exogenous antigens that carry structural features which are similar to regions on AChR. Rats with EAMG induced by the chimera have concentrations of antibodies to muscle AChR cytoplasmic domains as high as those developed after immunization with Torpedo AChR [34], [56]. Antibodies to the MIR on the extracellular surface of rat muscle AChRs trigger complement-mediated focal lysis of the postsynaptic membrane, resulting in shedding of membrane fragments into the synaptic cleft [57], [58], [59], [60]. An autoimmune response stimulated by endogenous muscle AChR in these fragments must account for the antibodies to cytoplasmic domains in rats immunized with the chimera because the chimera lacks cytoplasmic domains. This suggests that pathogenic autoantibody attack on the extracellular surface of muscle AChR in MG and EAMG drives a vicious cycle that sustains the autoimmune response. Other evidence for this concept is: (1) rats immunized with bacterially-expressed extracellular domain of human AChR α1 subunits first developed antibodies to the extracellular domain but later developed antibody to AChR cytoplasmic domain when they began to develop EAMG [61]; and (2) a continued increase in the concentration of autoantibodies to rat muscle AChR after 30 days following the initial immunization with electric organ AChR, a moment when the concentration of antibodies to electric organ AChRs decreases [38].