Department of Neuroscience, Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Departments of Chemistry and Molecular & Cell Biology, University of California, Berkeley, Berkeley, California, USA.
J Biol Chem. 2021 Jan-Jun;296:100302. doi: 10.1016/j.jbc.2021.100302. Epub 2021 Jan 17.
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.
3,4-二氨基吡啶（3,4-DAP）能增加神经肌肉接头（NMJ）的递质释放，小剂量的 3,4-DAP（估计在血清中达到约 1 μM）是美国食品和药物管理局（FDA）批准的治疗 Lambert-Eaton 肌无力综合征引起的神经肌肉无力的药物。通常认为，3,4-DAP 可阻断电压门控钾（Kv）通道，从而延长突触前动作电位（AP）。然而，最近的报告显示，低毫摩尔浓度的 3,4-DAP 对电压门控钙（Cav）通道的 Cav1 亚型（“L 型”）具有非靶向激动剂作用，并推测这种激动剂作用可能有助于 3,4-DAP 对 NMJ 递质释放的影响。为了确定 3,4-DAP 的作用机制，我们首先使用膜片钳电生理学技术来描述 3,4-DAP 对哺乳动物 NMJ 上发现的主要突触前 Kv 通道亚型（Kv3.3 和 Kv3.4）的浓度依赖性阻断作用。我们发现了一种以前未报道的 3,4-DAP 的高亲和力（1-10 μM）部分拮抗剂作用，除了众所周知的低亲和力（0.1-1 mM）拮抗剂活性。我们还表明，1.5 μM 的 DAP 对 Cav1.2 或 Cav2.1 电流没有影响。接下来，我们使用电压成像显示，1.5-或 100-μM 的 3,4-DAP 以剂量依赖的方式扩宽 AP 波形，与 Cav1 钙通道无关。最后，我们证明 1.5-或 100-μM 的 3,4-DAP 以剂量依赖的方式增强递质释放，这种作用也与 Cav1 通道无关。根据这些结果，我们得出结论，低微摩尔浓度的 3,4-DAP 仅作用于 Kv 通道以介导 AP 扩宽并增强 NMJ 处的递质释放。
