Presentation Abstract

Session Title: PLATFORM BE: Voltage-gated Ca Channels
Location: Room 307
Presentation Number: 2872-Plat
Presentation Time: 3/9/2011 1:00:00 PM
Abstract Title: A SPIDER TOXIN AND ITS RECOMBINANT ISOFORM BLOCK T-TYPE AND N-TYPE CALCIUM CHANNELS WITH HIGH AFFINITY
Author Block: Xiao Zhang, Li Dai, Michael E. Adams.
University of California, Riverside, Riverside, CA, USA.
Abstract Body: T-type calcium channels are widely distributed in diverse tissues and dysfunctions of these channels contribute to a variety of disorders and diseases. Notably, few specific ligands are available for physiological identification of T-type calcium channels. Here we identify ω-agatoxin IIA (ω-Aga-IIA), a polypeptide toxin purified from venom of American Funnel Web spider, Agelenopsis aperta, as a high affinity low voltage-activated (T-type) calcium channel antagonist. In whole cell recordings of the human α1I channel stably expressed in HEK cells, ω-Aga-IIA partially inhibited T-type current with an EC50 of 1.05 ± 0.62 nM. ω-Aga-IIA also partially blocked α1B calcium channels with a higher efficacy than its effect on α1I channel, with a comparable EC50 of 0.17±0.056 nM. ω-Aga-IIA partially inhibited T-type and N-type calcium current at saturating concentrations without shifting the I-V curve. We also developed a heterologous expression system (E. coli) and a modified on-column protein refolding method for production of a ω-Aga-IIA isoform, ω-Agatoxin IIC (ω-Aga-IIC). Recombinant ω-Aga-IIC exhibited similar RP-HPLC elution profiles as ω-Aga-IIA and blocked α1I/α1B channels with high potency (EC50 of 1.01 ± 0.38 and 0.16±0.049, respectively). The high affinities of ω-Aga-IIA and ω-Aga-IIC for α1I and α1B calcium channels indicates the presence of an evolutionarily conserved binding site on high- and low voltage-activated calcium channels. With the successfully production and refolding of recombinant ω-Aga-IIC, a valuable tool has become available for further studies of calcium channel pharmacology and function.
Commercial Relationship:  X. Zhang: None. L. Dai: None. M.E. Adams: None.



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