Desensitization of mutant acetylcholine receptors in transgenic mice reduces the amplitude of neuromuscular synaptic currents

Abstract

While the slow onset of desensitization of nicotinic acetylcholine receptors (AChRs), relative to the rate of acetylcholine removal, excludes this kinetic state from shaping synaptic responses in normal neuromuscular transmission, its role in neuromuscular disorders has not been examined. The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder caused by point mutations in the AChR subunit-encoding genes leading to kinetically abnormal (slow) channels, reduced miniature endplate current amplitudes (MEPCs), and degeneration of the postsynaptic membrane. Because of this complicated picture of kinetic and structural change in the neuromuscular junction, it is difficult to assess the importance of the multiple factors that may be responsible for the reduced endplate current amplitudes, and ultimately the clinical syndrome. In order to address this we have used a transgenic mouse model for the SCCMS that has slow AChR ion channels and reduced endplate responsiveness in the absence of any of the degenerative changes. We found that the reduction in MEPC amplitudes in these mice could not be explained by either reduced AChR number or by reduced AChR channel conductance. Rather, we found that the mutant AChRs in situ manifested an activity-dependent reduction in sensitivity that caused diminished MEPC and endplate current amplitude with nerve stimulation. This observation demonstrates that the basis for the reduction in MEPC amplitudes in the SCCMS may be multifactorial. Moreover, these findings demonstrate that, under conditions that alter their rate of desensitization, the kinetic properties of nicotinic AChRs can control the strength of synaptic responses.

Genetic Manipulation of AChR Responses Suggests Multiple Causes of Weakness in Slow‐Channel Syndromea

“…We investigated the basis for the reduction in MEPC amplitude in δS262T‐transgenic mice and showed that diminished MEPC amplitudes were not due to a decrease in ion channel conductance. Patch‐clamp recordings of single channels recorded from acutely dissociated muscle fibers of the flexor digitorum brevis demonstrated that AChRs of δS262T‐transgenic mice have a channel conductance of 61.8 pS, 21 which was identical to that of control end plate AChRs.…”

“…Figure 2 is an electron micrograph of a neuromuscular junction from a forelimb muscle of a 6‐month‐old δS262T‐transgenic mouse, showing normal synaptic cleft, postsynaptic folds, and junctional sarcoplasm. 21…”

“…The number of αBT binding sites in transgenic muscle was normal or slightly increased relative to control (0.32 ± 0.04 pmol/g, n = 30 versus 0.22 ± 0.03 pmol/g, n = 11, p > 0.1). 21…”

“…FIGURE 2 is an electron micrograph of a neuromuscular junction from a forelimb muscle of a 6-month-old ␦S262T-transgenic mouse, showing normal synaptic cleft, postsynaptic folds, and junctional sarcoplasm. 21 Finally, as determined by immunoprecipitation of ␣-bungarotoxin (␣BT) binding sites from solubilized muscle, there is no reduction in skeletal muscle AChR content in ␦S262T-transgenic mice to account for the reduced MEPC amplitudes. The number of ␣BT binding sites in transgenic muscle was normal or slightly increased relative to control (0.32 ± 0.04 pmol/g, n = 30 versus 0.22 ± 0.03 pmol/g, n = 11, p > 0.1).…”

Macroscopic properties of spontaneous mutations in slow‐channel syndrome: Correlation by domain and disease severity

“…For example, mutations in the neuronal nicotinic AChR in nocturnal frontal lobe epilepsy lead to a decrease in Ca 2+ permeability and overactive presynaptic terminals in inhibitory neurons (Kuryatov et al, 1997). Also in both the congenital slow-and fastchannel syndromes changes in the rates of desensitization are believed to be partially responsible for both impaired neuromuscular transmission and progressive muscle atrophy (Bhattacharyya et al, 1997;Milone et al, 1997;Sine et al, 1995). Finally, differences in the decay time of macroscopic postsynaptic currents observed in this syndrome may explain in part the dif-ferent levels of disease severity in SCS, presumably by through different degrees of cation overload.…”