PLATFORM BE: Voltage-gated Ca Channels
3/9/2011 1:00:00 PM
A SPIDER TOXIN AND ITS RECOMBINANT ISOFORM BLOCK T-TYPE AND N-TYPE CALCIUM CHANNELS WITH HIGH AFFINITY
, Li Dai, Michael E. Adams.
University of California, Riverside, Riverside, CA, USA.
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,
as a high affinity low voltage-activated (T-type) calcium channel antagonist. In whole cell recordings of the human α
channel stably expressed in HEK cells, ω-Aga-IIA partially inhibited T-type current with an EC
of 1.05 ± 0.62 nM. ω-Aga-IIA also partially blocked α
calcium channels with a higher efficacy than its effect on α
channel, with a comparable EC
of 0.17±0.056 nM. ω-Aga-IIA partially inhibited T-type and N-type calcium current at saturating concentrations without shifting the
curve. We also developed a heterologous expression system (
) 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 α
channels with high potency (EC
of 1.01 ± 0.38 and 0.16±0.049, respectively). The high affinities of ω-Aga-IIA and ω-Aga-IIC for α
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.
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