Purification of a New Dimeric Protein from Cliona vastifica Sponge, which Specifically Blocks a Non-L-Type Calcium Channel in Mouse Duodenal Myocytes

Abstract

Marine sponges are synthesizing a wide variety of peptidic and organic molecules with biological activities. Multiple-step purification ofCliona vastifica extract led to a new dimeric peptide (mapacalcine; M_r = 19,064) that is composed of two homologous chains, each containing nine cysteins. This protein has been found to selectively block a new calcium conductance characterized in mouse duodenal myocytes with an IC₅₀ value of ∼0.2 μm. The mapacalcine-sensitive current was a non-L-type calcium current activated from a holding potential of −80 mV that persisted during stimulation of the cell at high frequencies (0.1–0.2 Hz) within 5–10 min. Time constants of inactivation were similar for both L-type and non-L-type calcium currents. The non-L-type calcium current of duodenal myocytes was not blocked by the pharmacological agents specific for N-, L-, P-, or Q-type calcium channels. Mapacalcine was unable to block T-type calcium current in portal vein myocytes as well as voltage-dependent potassium currents and calcium-activated chloride currents in duodenal and portal vein cells. Mapacalcine did not affect caffeine-induced calcium responses, indicating that it did not interfere with intracellular calcium stores. Competition experiments on mouse intestinal membranes showed that mapacalcine did not interact with dihydropyridines receptors. These data suggest that mapacalcine may be a specific inhibitor of a new type of calcium current, first identified in duodenal myocytes.