Penetratin Peptide Potentiates Endogenous Calcium-Activated Chloride Currents in Xenopus oocytes

Abstract

Calcium-activated chloride currents (CaCCs) are required for epithelial electrolyte and fluid secretion, fertilization, sensory transduction and excitability of neurons and smooth muscle. Defolliculated Xenopus oocytes express a robust CaCC formed by a heterologous group of proteins including transmembrane protein 16A (TMEM16A) and bestrophins. Penetratin, a 17-amino acid peptide, potentiated endogenous oocyte CaCCs by ~50-fold at 10 μM, recorded using a two-electrode voltage clamp. CaCC potentiation was rapid and dose-dependent (EC₅₀ = 3.2 μM). Penetratin-potentiated currents reversed at −18 mV and were dependent on the extracellular divalent cations present, showing positive regulation by Ca²⁺ and Mg²⁺ but effective block by Zn²⁺ (IC₅₀ = 5.9 μM). Extracellular Cd²⁺, Cu²⁺ and Ba²⁺ resulted in bimodal responses: CaCC inhibition at low but potentiation at high concentrations. Intracellular BAPTA injection, which prevents activation of CaCCs, and the Cl⁻ channel blockers niflumic acid and DIDS significantly reduced potentiation. In contrast, the K⁺ channel blockers Cs⁺, TEA, tertiapin-Q and halothane had no significant effect. This pharmacological profile is consistent with penetratin potentiation of zinc-sensitive CaCCs that are activated by influx of extracellular Ca²⁺. These findings may stimulate basic research on CaCCs in native cells and may lead to development of novel therapeutics targeting disorders caused by insufficient chloride secretion.