Clustered acetylcholine receptors and myasthenia gravis

The muscle nicotinic acetylcholine receptor (AChR) belongs to the ligand-gated ion channel family and is located at the postsynaptic membrane of the neuromuscular junction (NMJ). The ligand, acetylcholine, gets released from the nerve terminal and activates postsynaptic AChRs resulting in a depolarisation of the postsynaptic membrane. Under physiological conditions each action potential arriving at the presynaptic nerve terminal is adequate to initiate an action potential in the muscle cell. This is accomplished by AChR concentration at the postsynaptic endplate region, mainly due to AChR clustering. In myasthenia gravis (MG), an autoimmune disorder of the NMJ causing fatigue and weakness, antibodies to different antigens of the postsynaptic membrane impair neuromuscular transmission. In most MG cases antibodies are directed against AChRs. Other antibodies bind instead to AChRs just in the clustered form, to MuSK, LRP4 or as yet unidentified antigens (i.e. seronegative MG). Although AChRs are counted among the best-studied ion channels, no single study has focused on the effect of clustering on the electrophysiological properties of AChRs. However, this is of great interest since AChR clustering represents the relevant physiologic state and some MG forms predominantly affect AChR clustering. It has not been examined yet how the impairment of AChR clustering in those MG cases compromises the excitability of muscle cells leading to fatigue. Hypotheses: I)Clustered AChRs have different electrophysiological properties compared to unclustered II) MuSK-antibodies affect clustered AChR function III) MG sera without antibodies to known antigenic targets can alter the electrophysiological parameters of clustered AChRs IV) Some of the effects seen may be due to perturbation of second messengers Methods: Using two groups of TE671 and DB40 cell lines expressing clustered and unclustered AChRs, respectively, we will perform electrophysiological analyses applying the patch clamp technique. Activation of AChRs will be achieved using a fast perfusion system for the ACh application. We will quantify surface AChRs in order to exclude any detected electrophysiological difference being related to different AChR numbers. The same experiments will also be performed after incubation of the cells with the immunoglobulin fractions from MuSK-positive and seronegative MG patients, respectively. Moreover, the influence of second messengers and of phosphorylation will be evaluated by the intracellular application of specific inhibitors or by supplementing second messengers via the patch pipette. Preliminary data: In a pilot study we were able to show that AChR clustering results in higher peak current amplitudes and slower desensitisation kinetics. Thus, AChR clustering seems to improve neuromuscular transmission not only by the concentration of AChRs at the postsynaptic endplate region but also by changing their electrophysiological properties.

The adult form of the acetylcholine receptor recovers faster from inactive states There are two forms of the acetylcholine receptor in muscle with a developmental switch from fetal to adult in the last trimenon. Upon receptor activation by agonists both acetylcholine receptor forms undergo a conformational change into an inactive state and become activatable again only after agonist removal. We found that the adult form can recover faster from the inactive state and, consequently, might facilitate the robust and high frequent neuromuscular transmission in adult muscle. Our findings are relevant to patients with genetic and autoimmune myasthenic disorders causing loss of adult acetylcholine receptors and where the fetal form is sustained. Acetylcholine receptor clustering facilitates recovery from inactive states A clustering process concentrates acetylcholine receptors at the muscle membrane opposite the nerve terminal resulting in high receptor densities, which is important for the successful neuromuscular transmission. This process involves the interaction of an intracellular protein called rapsyn with adjacent acetylcholine receptors. However, it has not been examined whether this interaction has also an impact on acetylcholine receptor function. We now provide evidence that rapsyn effects in acetylcholine receptors recovering faster from inactive states. This finding is of relevance to myasthenia gravis patients with antibodies that predominantly disturb acetylcholine receptor clustering. Sera from patients with myasthenia gravis can inhibit recovery of acetylcholine receptors from inactive states Myasthenia gravis is a neuromuscular disorder associated with weakness and increased fatigability and with antibodies directed against different molecular targets at the neuromuscular junction. In about 10% of myasthenia gravis patients no antibodies can be detected using routine assays. We have shown that some of these patients have serological factors impairing recovery of acetylcholine receptors from inactive states.