A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs

Abstract

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 μM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert–Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype (“L-type”) of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP’s mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1–10 μM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1–1 mM) antagonist activity. We also showed that 1.5-μM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-μM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-μM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

Presynaptic Paraneoplastic Disorders of the Neuromuscular Junction: An Update

“…More recently, a further mechanism of action, a direct agonistic effect of 3,4-DAP on Cav1 type channels, has been reported, although its relevance at therapeutic concentrations is debated [89][90][91]. Several randomized controlled trials demonstrated the superiority of 3,4-DAP compared to placebo in improving motor and autonomic symptoms [92][93][94][95][96][97][98][99], and 3,4-DAP use in LEMS has been approved in 2009 in Europe and 2018 in the US.…”

Neuromuscular Active Zone Structure and Function in Healthy and Lambert-Eaton Myasthenic Syndrome States

“…This is primarily due to the fact that presynaptic nerve terminals at most synapses are too small to probe with an electrode (with a few notable exceptions). Regarding the mammalian motor nerve terminal, recently, Ojala et al [ 26 ] used voltage imaging to characterize the presynaptic AP waveform. In these studies, they reported that motor-nerve-terminal APs are exceptionally brief, with a full width of 250–350 μs at half maximal amplitude ( Figure 1 ).…”

Antidotal treatment of botulism in rats by continuous infusion with 3,4-diaminopyridine

“…LEMS is an autoimmune disease caused by reduced acetylcholine release and neuromuscular weakness (McEvoy et al 1989 ). Mechanistically, 3,4-diaminopyridine (3,4-DAP) prolongs action potential duration by reversibly blocking voltage-gated potassium channels, facilitating presynaptic Ca 2+ influx and increasing acetylcholine release (Thomsen and Wilson 1983 ; Kirsch and Narahashi 1978 ; Ojala et al 2021 ). In LEMS patients, 3,4-DAP partially reverses neuromuscular weakness by restoring threshold release of acetylcholine, with a median effective serum level of 30 ng/mL (Thakkar et al 2017 ).…”