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  • Comparatively cell based assays appear to be the most sensit

    2024-07-09

    Comparatively, cell-based assays appear to be the most sensitive method to detect patient autoantibodies directed at neurotransmitter receptors. Leite et al. demonstrated that many α-BTX RIA seronegative myasthenic patients do have autoantibodies against the AChR when a highly sensitive immunofluorescence-based assay was used for detection [8]. In this immunofluorescence-based assay, all four AChR subunits, along with rapsyn, are coexpressed in HEK cells to mimic the tightly clustered pentameric receptor found embedded in the plasma membrane at the neuromuscular junction. Although the specific antigenic epitopes on these other AChR chains have not been extensively investigated, it is possible that the β, δ and ε/γ subunits of AChR are direct targets of MG autoantibodies [21], [22]. Future LIPS studies using co-expression of other AChR subunits and/or additional proteins (e.g. rapsyn) may also be helpful for improving the sensitivity of detection of MG patient autoantibodies and understanding the relative contribution in immunoreactivity regarding these other subunits.
    Acknowledgments
    Introduction The nicotinic Cytarabine receptor (AChR) belongs to the family of Cys-loop ligand-gated ion channels (LGIC), which also includes the serotonin, glycine and γ-aminobutyric acid A (GABA) receptors [1]. All members of the LGIC family share a common topology; each subunit has an N-terminal extracellular domain (ECD), 4 transmembrane domains (M1–M4), an intracellular domain (ICD) between the M3 and M4, and a small extracellular tail after M4 [2], [3]. The members of the family also share a conserved loop at their N-terminal ligand binding domain, flanked by two disulfide-bound cysteine residues, the so-called Cys-loop. AChRs are divided into two groups, neuronal and muscle. The muscle AChRs are located at the post-synaptic membrane of the neuromuscular junction, where they mediate neuromuscular transmission in response to nerve stimulation. They are transmembrane glycoproteins (∼290kDa) composed of five homologous subunits that associate to form an ion channel [4]. The subunit composition of the muscle AChR varies depending on the developmental stage and it is (α1)2β1γδ for embryonic (or denervated) muscle and (α1)2β1ɛδ for adult muscle [5]. In addition to its physiological role, the AChR is of major importance in several pathological conditions, most notably the autoimmune disorder myasthenia gravis (MG). The muscle AChR is the main autoantigen in MG, since about 85% of MG patients have antibodies against at least one of the AChR subunits [6]. All the epitopes for pathogenic antibodies are located on the ECDs, while more than half of the autoantibodies are directed against the so-called main immunogenic region (MIR), a group of overlapping epitopes located on the ECD of the α1 subunit, whose central core lies between amino acids 67–76, though other segments contribute as well [7], [8], [9]. The pathophysiology of MG has been described in detail, making it a prototype autoimmune disease [10]. The importance of the AChR, in both the normal function of the neuromuscular junction, as well as the pathogenic mechanisms in MG, makes it an attractive target for structural studies. Because AChR is a large transmembrane protein, most efforts have focused on the globular ECD region of its subunits (∼210 amino acids long). The first high resolution data came from the solved crystal structure at 2.7Å of the acetylcholine binding protein (AChBP) from the snail Lymnaea stagnalis, a soluble protein, composed of five identical subunits, which share an overall 20–24% identity with the ECDs of the AChR [11], [12]. An insight on the structure of the AChR itself was provided by an electron microscopy image of the whole AChR from the Torpedo marmorata electric organ at 4Å resolution [2]. More recently, structural data were obtained from a mutant mouse α1 ECD in complex with the snake neurotoxin α-bungarotoxin (α-Btx) [13]. In this model, elements such as the location of the MIR, the Cys-loop, the glycosylation site of the α1 ECD and its role in α-Btx binding were clearly shown. Further success has come using chimeric proteins, as in the case of the published structure of an α7/AChBP chimera with a 64% sequence identity to the native α7 ECD [14]. However, studies on the human muscle AChRs are still missing.