Complete reversal of Lambert-Eaton myasthenic syndrome synaptic impairment by the combined use of a K+ channel blocker and a Ca2+ channel agonist

Abstract

Lambert-Eaton myasthenic syndrome (LEMS) is characterized by an autoimmune-mediated attack on presynaptic P/Q-type Ca2+ channels at the neuromuscular junction (NMJ). The current common symptomatic treatment option is 3,4-diaminopyridine (3,4-DAP), a potassium channel blocker that widens the presynaptic action potential, causing an increase in the amount of neurotransmitter release. This approach, however, does not completely reverse symptoms and can have dose-limiting side-effects. Thus, there is a need for additional treatment options. We show that GV-58, a Ca2+ channel agonist developed from the cyclin-dependent kinase inhibitor (R)-roscovitine, does not significantly inhibit cell division at physiological levels of ATP. We further show that GV-58 has a greater agonist effect when more Ca2+ channels are open, and combining GV-58 and 3,4-DAP elicits a supra-additive effect that completely restores the magnitude of neurotransmitter release in LEMS model NMJs. These results suggest that a combination of GV-58 and 3,4-DAP is promising as a possible alternative treatment approach to LEMS and other neuromuscular diseases. Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca2+ channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are thought to be removed. There is no cure for LEMS, and the current most commonly used symptomatic treatment option is a potassium channel blocker [3,4-diaminopyridine (3,4-DAP)] that does not completely reverse symptoms and can have dose-limiting side-effects. We previously reported the development of a novel Ca2+ channel agonist, GV-58, as a possible alternative treatment strategy for LEMS. In this study, we tested the hypothesis that the combination of GV-58 and 3,4-DAP will elicit a supra-additive increase in neurotransmitter release at LEMS model NMJs. First, we tested GV-58 in a cell survival assay to assess potential effects on cyclin-dependent kinases (Cdks) and showed that GV-58 did not affect cell survival at the relevant concentrations for Ca2+ channel effects. Then, we examined the voltage dependence of GV-58 effects on Ca2+ channels using patch clamp techniques; this showed the effects of GV-58 to be dependent upon Ca2+ channel opening. Based on this mechanism, we predicted an interaction between 3,4-DAP and GV-58. We tested this hypothesis using a mouse passive transfer model of LEMS. Using intracellular electrophysiological ex vivo recordings, we demonstrated that a combined application of 3,4-DAP plus GV-58 had a supra-additive effect that completely reversed the deficit in neurotransmitter release magnitude at LEMS model NMJs. This reversal contrasts with the less significant improvement observed with either compound alone. Our data indicate that a combination of 3,4-DAP and GV-58 represents a promising treatment option for LEMS and potentially for other disorders of the NMJ. © 2014 The Authors.