Isolation And Identification Of Spider Toxin Receptors

Native toxins in nature are an important resource that can be utilized. At present, the animal toxins especially those from spiders have attracted many scientists'attention. There are a great variety of spider toxins that have diverse functions and exhibit a great application prospect in the neuronology and pharmacology. In recent years, the study of spider toxins has achieved significant progress, and many spider toxins that target voltage-gated ion channels in cell membrane such as K+, Na+-and Ca2+-channels have been characterized by electrophysiological techniques such as voltage clamp and patch clamp. These techniques have become the main means to investigate the action mechanism of the toxins. However, voltage clamp and patch clamp techniques have limitations in identification of the binding proteins of a toxin because they usually directionally use different types of ion channels or their subtypes to detect the activity of a toxin. In our present study, we used a biotin labeling-based strategy to investigate the function of huwentoxin I (HWTX-Ⅰ), a typical spider neurotoxin, via detecting and identifying its binding proteins (generally called receptors) in the neuron plasma membrane, so as to obtain more direct information on the action mechanism of the toxin.HWTX-Ⅰ is a neurotoxic peptide of3.75kDa and is isolated from the venom of spider S. huwena. It consists of33amino acid residues with three disulphide bonds. Electrophysiological experiments indicate that it can reversibly block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparation. For detecting the receptor of HWTX-Ⅰ in the junction of phrenic nerve-hemidiaphragm muscle, the purified HWTX-Ⅰ was labeled by Sulfo-NHS-Biotin and the biotinylated HWTX-Ⅰ (biotin-HWTX-Ⅰ) was used to interact with the plasma membrane proteins in rat diaphragm muscle rich in neuromuscular junctions. The binding proteins of HWTX-Ⅰ were isolated and identified in two ways. In the first way, the biotin-HWTX-Ⅰ was incubated with the plasma membrane debris first and then crosslinked by the crosslinker BS3. After that, the proteins were extracted with detergent-containing buffer and separated by SDS-PAGE. The gel-resolved proteins were transferred onto a PVDF membrane and probed as well as visualized with HRP-conjugated avidin (HRP-avidin). In the second way, the proteins in the plasma membrane were extracted and separated by SDS-PAGE. After proteins were transferred onto a PVDF membrane, the proteins on the membrane were renatured and then probed with biotin-HWTX-Ⅰ, followed by detection and visualization with HRP-avidin. The protein bands visualized in the two ways were analyzed by CapLC-MS/MS.Mass spectrometric and electrophysiological analyses indicated that the biotin-HWTX-Ⅰ prepared in the present study was coupled with a single biotin group and the biotinylation did not affect the bioactivity of HWTX-Ⅰ. In the first way to analyze the binding proteins, a band located around72kDa on the PVDF membrane was visualized using HRP-avidin, suggesting that this band most likely contained the binding proteins of HWTX-Ⅰ. For determining whether there was endogenous avidin binding site in the proteins, we made control experiments using unlabeled HWTX-Ⅰ or without using any HWTX-Ⅰ. The results showed that there was a band visualized at the same location in both cases, with the band visualized in the presence of unlabeled HWTX-Ⅰ being lighter than that in the absence of HWTX-Ⅰ. Theses data suggested that the plasma membrane proteins have a endogenous binding site for avidin, and, moreover, the binding site for avidin was most likely the same (or largely overlapped) as that for HWTX-I. In the other strategy to identify the receptor of HWTX-Ⅰ, the biotin-HWTX-Ⅰ was used to detect the potential binding proteins after the membrane proteins were gel-resolved and transferred onto a PVDF membrane. For determining the effect of protein renaturation on biotin-HWTX-Ⅰ binding to its binding proteins, the bindings of biotin-HWTX-Ⅰ to the proteins on PVDF membrane before and after renaturation were compared. The results indicated that the binding of HWTX-Ⅰ to its binding proteins was enhanced after the proteins on the PVDF were renaturated. At the same time, we used HRP-avidin to probe the proteins or biotin-HWTX-I on the PVDF membrane under different experimental conditions. The experimental results demonstrated that avidin at high concentrations could not completely prevent the HWTX-Ⅰ from binding to the potential binding site, and vice versa. These results further confirmed that the binding sites of avidin and HWTX-Ⅰ in the binding proteins are not completely overlapping.After the proteins contained in the band visualized by HRP-avidin were digested by trypsin and subjected to CapLC-MS/MS analysis, several candidate binding protein of HWTX-Ⅰ were identified, including voltage-gated calcium channel protein, voltage-dependent anion-selective channel protein, calcium ATPase, annexin, etc., suggesting that HWTX-Ⅰ may block neuromuscular transmission via interacting with multiple membrane proteins or ion channels.