The Tarantula Toxin omega-Avsp1a Specifically Inhibits Human Ca(V)3.1 and Ca(V)3.3 via the Extracellular S3-S4 Loop of the Domain 1 Voltage-Sensor

Inhibition of T-type calcium channels (Ca(V)3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of Ca(V)3. However, subtype-specific Ca(V)3 inhibitors for therapeutic purposes or for studying the physiological roles of Ca(V)3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. ("Amazonas Purple", Peru) tarantula venom inhibited specific T-type Ca-V channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin omega-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 mu M) and synthetic omega-Avsp1a (10 mu M) inhibited 90% of Ca(V)3.1 and Ca(V)3.3, but only 25% of Ca(V)3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive Ca(V)3.2 and more sensitive Ca(V)3.3. Our results suggest that domain-1 of Ca(V)3.3 is important for the inhibitory effect of omega-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of omega-Avsp1a.