| Chansu, which is isolated from the secretion of the Asiatic toad Bufo gargarizansa Cantor or Bufo melanostictus Schneider, possess both pharmacological and toxicological effects as shown by in vitro and in vivo studies such as cardiotoxic, cardiotonic, anesthesia, and anti-tumor effects. In the nervous system, the neurons function is closely related to neuron excitability. Changes of neuron excitability will lead to neurons dysfunction. Various ion channels on neuron membrane, which are molecular basis of neuron excitability, maintain basic life processes due to their structure and function. The genetic variation and dysfunction are related to the occurrence and development of many diseases. In addition, a large number of ion channels in the membranes of neurons are targets for many kinds of toxins and drugs, and many neuronal protections and damages of central nervous system (CNS) are caused by improving or disrupting the functions of ion channels.This study mainly aims to investigate the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG) isolated from Chansu, on neuron excitability and the underlying ion mechanisms. The main results are summarized as following.1. Effects of RBG/CBG on neuron excitability and voltage-gated sodium, potassium and calcium channels.RBG has distinct effects on the excitability of hippocampal CA1neurons. RBG influenced peak amplitude, overshoot and half-width of the evoked single action potential and the first action potential of repetitive firing, but simultaneously lessened the firing rate of evoked repetitive firing. CBG has the similar effects as RBG. Voltage-gated sodium channels, voltage-gated potassium channels and voltage-gated calcium channels play important roles in maintaining the excitability of hippocampal neurons. According to the results, RBG activated voltage-gated sodium channel current, inhibited voltage-gated potassium channel current, and hardly influenced voltage-gated calcium current.2. Influences of RBG/CBG on the outward transient potassium channel current (IA) and outward delayed rectifier potassium channel current (IK).The present study investigated the effects of RBG/CBG, on IK and IA in rat hippocampal neurons using whole-cell and single-channel patch recordings. The results indicated that RBG inhibited both IK and IA in concentration-dependent and voltage-dependent manners. RBG shifted the activation of IK to the positive potential and the inactivation of IK to the negative potential. RBG shifted inactivation of IA to more negative voltage without noticeable effect on activation of IA. CBG inhibited IK in a concentration-dependent manner without noticeable effect on IA, and CBG also could alter some channel kinetics of IK. Moreover, at1μM concentration both RBG and CBG could alter some gating properties of IK, such as open probability and time constants.3. The interaction of Kv1.2and Kv1.2-Kv2.1with various bufadienolides molecules by applying AutoDock software to predict the binding sites.In part of molecular docking calculation, we choose Kv1.2and Kv1.2-2.1as the receptors, and choose12bufadienolides (RBG/CBG/Bufalin etc.) and cardiac glycosides of parent nucleus as ligands in the docking experiments by Autodock, respectively. Blind docking results are high consistency, docking calculation instructions further trusted blind docking results. According these results, we determine the active sites of receptor and ligand compounds. Docking results also show that the ligand nucleus plays an important role. Polar and hydrophobic interactions are the most important mode of action.4. Effects of RBG on Kv2.1-WT and Kv2.1-ΔC mutants plasmids expressed in HEK293cells electrophysiological properties.In the present study, we investigated the action of RBG on the Kv2.1(the prevalent delayed-rectifier K+channel in neurons) channel using whole-cell patch voltage-clamp. RBG inhibited Kv2.1current in a concentration-dependent manner, with an IC50value of1.17μM. The inhibition of1μM RBG on Kv2.1current was similar as10mM TEA. The peak amplitude of Kv2.1current was decreased to55.29±7.01%in the presence of1μM RBG. RBG shifted both the voltage dependence of channel activation and inactivation to more negative voltage. Remarkably, this bufadienolide potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1when the internal K+replaced with Na+and external K+was decreased from4to1mM. In addition, the effects of this bufadienolide were dissipated by partial deletions of the C terminus.In conclusion, some bufadienolides have distinct effects on neuron excitability of rat hippocampal neurons. The binding sites and the interaction modes can interpret the inhibition of these bufadienolides drugs to some extent. The effects and the underlying mechanisms of RBG on the Kv2.1channel current might explain the modulation of bufadienolides on cell excitability. The present study might provide scientific foundation for bufadienolides to be exploited to be an agent for neuron diseases.
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