ABSTRACT
BACKGROUND AND PURPOSE: Buthus martensi Karsch (BmK) AS is a scorpion polypeptide toxin, said to target the voltage-gated sodium channels (VGSCs). However, the mechanism of action of BmK AS on the VGSCs has yet to be defined. EXPERIMENTAL APPROACH: We examined the electrophysiological effects of BmK AS in a wide dose range on the rat brain-type VGSC alpha-subunit, rNav1.2a, heterologously expressed in Xenopus oocytes and on the VGSCs endogenously expressed in the dorsal root ganglion neuroblastoma ND7-23 cell line. KEY RESULTS: In the oocytes, BmK AS depolarized the voltage dependence of activation and inactivation of rNav1.2a at 0.1 and 500 nM whereas these parameters were hyperpolarized at 1 nM. In ND7-23 cells, BmK AS hyperpolarized the voltage dependence of activation and inactivation at 0.1, 1 and 100 nM but not 10 nM. BmK AS also hyperpolarized the voltage dependence of recovery from inactivation at 0.1 and 100 nM and slowed the recovery kinetics at all concentrations, but the effects of 1 and 10 nM were relatively smaller than those at 0.1 and 100 nM. Moreover, the inactivation of VGSCs was potentiated by 10 nM BmK AS in both systems, whereas it was inhibited by 0.1 or 100 nM BmK AS in the oocytes or ND7-23 cells respectively. CONCLUSIONS AND IMPLICATIONS: BmK AS modulated the VGSCs in a unique U-shaped dose-dependent manner, which could be due to the opposing effects of binding to two distinct receptor sites on the VGSCs.
Subject(s)
Neuroblastoma/metabolism , Peptides/pharmacology , Scorpion Venoms/pharmacology , Sodium Channels/drug effects , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Electrophysiology , Female , Ganglia, Spinal/metabolism , Mice , NAV1.2 Voltage-Gated Sodium Channel , Nerve Tissue Proteins , Oocytes , Peptides/administration & dosage , Protein Binding , Rats , Scorpion Venoms/administration & dosage , Sodium Channels/metabolism , Xenopus laevisABSTRACT
BmK I is classified as alpha-like scorpion toxin that specifically binds the voltage-gated sodium channels via receptor site-3. Previous results showed BmK I induced epileptiform responses in rats via intra-hippocampal injection, but the mechanism has yet to be clarified. In this study, using two-electrode voltage/current clamp technique, we determined the effects of BmK I on rNav1.2a expressed in Xenopus oocytes. The results showed that BmK I prevented the development of slow inactivation of rNav1.2a from the open-state and enhanced the persistent sodium current (I(NaP)) at suprathreshold potentials in concentration-dependence, whereas it hardly affected the fast inactivation. BmK I was also able to augment the subthreshold I(NaP) at high concentrations (>100nM) with disruption of the open-state deactivation. The increased I(NaP) accelerated the firing frequency in the oocytes that fired repetitively after electrode punctures, as well as raised the baseline potential and induced bursting of spikes in the quiescent oocytes. These results demonstrated that BmK I could target rNav1.2a and induce the I(NaP) by preventing the development of slow inactivation and deactivation from the open-state, leading to the enhancement of membrane excitability, which may be involved in the BmK I-induced epilepsy.
Subject(s)
Scorpion Venoms/toxicity , Sodium Channels/drug effects , Animals , Female , Membrane Potentials/drug effects , NAV1.2 Voltage-Gated Sodium Channel , Nerve Tissue Proteins , Oocytes/metabolism , Rats , Sodium Channels/genetics , Xenopus laevis/geneticsABSTRACT
In the present study, the pharmacological effects of BmK AS, a beta-like scorpion toxin on rNav1.2 alpha-subunit expressed in Xenopus laevis oocytes were investigated using a two-electrode voltage-clamp recording. It was found that the voltage dependence of rNav1.2 inactivation was significantly shifted towards positive membrane potential by 500 nM BmK AS, whereas the activation curves of rNav1.2 were unruffled at the same dosage. The inactivation curves of both slow and fast inactivation currents were positively moved about 12.8 and 9.7 mV, respectively. In addition, the persistent currents of rNav1.2 were invariable. The effects of BmK AS on the rNav1.2 inactivation were opposite to the previous results found in the peripheral sensory neurons. The results suggested that Nav1.2 might be the target of BmK AS in the central nervous system, and BmK AS might have an excitatory effect on the central neuron through enhancing Nav1.2.
Subject(s)
Nerve Tissue Proteins/metabolism , Oocytes/drug effects , Oocytes/metabolism , Peptides/pharmacology , Scorpion Venoms/pharmacology , Sodium Channels/metabolism , Xenopus laevis , Animals , Ion Channel Gating/drug effects , NAV1.2 Voltage-Gated Sodium Channel , RatsABSTRACT
In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.