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 u-agatoxin IIA (u-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 a 1I channel stably expressed in HEK cells, u-Aga-IIA partially inhibited T-type current with an EC 50 of 1.05 5 0.62 nM. u-Aga-IIA also partially blocked a 1B calcium channels with a higher efficacy than its effect on a 1I channel, with a comparable EC 50 of 0.1750.056 nM. u-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 u-Aga-IIA isoform, u-Agatoxin IIC (u-Aga-IIC). Recombinant u-Aga-IIC exhibited similar RP-HPLC elution profiles as u-Aga-IIA and blocked a 1I/ a 1B channels with high potency (EC 50 of 1.01 5 0.38 and 0.1650.049, respectively). The high affinities of u-Aga-IIA and u-Aga-IIC for a 1I and a 1B 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 u-Aga-IIC, a valuable tool has become available for further studies of calcium channel pharmacology and function. 2873-PlatCaveolin-3 Inhibits Ca v 3.2 (a1H) Currents and Regulates Hypertrophic Signaling in Ventricular Myocytes
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