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

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach similar to 1 mu M 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 pre-synaptic 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 mu M) 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-mu M DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-mu M 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-mu M 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.

Automated summary

Research shows that the main mechanism of action of 3,4-DAP at the neuromuscular junction is through acting on Kv channels to mediate AP broadening and enhance transmitter release.