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

• Published in: The Journal of biological chemistry Vol. 296; p. 100302
• Main Authors: Ojala, Kristine S., Ginebaugh, Scott P., Wu, Man, Miller, Evan W., Ortiz, Gloria, Covarrubias, Manuel, Meriney, Stephen D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021; American Society for Biochemistry and Molecular Biology
• Subjects: Acetylcholine - metabolism; action potential (AP); Action Potentials - drug effects; Action Potentials - physiology; Amifampridine - pharmacology; Animals; Calcium Channels, L-Type - genetics; Calcium Channels, L-Type - metabolism; Calcium Channels, N-Type - genetics; Calcium Channels, N-Type - metabolism; Dose-Response Relationship, Drug; Female; Gene Expression; Lambert–Eaton myasthenic syndrome (LEMS); Male; Mice; Microelectrodes; neurological disease; Neuromuscular Agents - pharmacology; neuromuscular junction (NMJ); Neuromuscular Junction - drug effects; Neuromuscular Junction - metabolism; neurotransmitter release; Potassium Channel Blockers - pharmacology; Presynaptic Terminals - drug effects; Presynaptic Terminals - metabolism; Rana pipiens; Shaw Potassium Channels - antagonists & inhibitors; Shaw Potassium Channels - genetics; Shaw Potassium Channels - metabolism; Tissue Culture Techniques; voltage-gated calcium (Cav) channel; voltage-gated potassium (Kv) channel; BeRST; 3,4-DAP; EPP; neuromuscular junction (NMJ); neurological disease; BTX; mEPP; NMJ; Kv; action potential (AP); neurotransmitter release; ROI; AP; QC; voltage-gated potassium (Kv) channel; FWHM; Cav; LEMS; Lambert–Eaton myasthenic syndrome (LEMS); voltage-gated calcium (Cav) channel
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2021.100302

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.