The TMEM16A channel mediates the fast polyspermy block in <i>Xenopus laevis</i>

Abstract

In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm-a phenomenon known as the fast block to polyspermy. In the African clawed frog <i>Xenopus laevis</i>, this depolarization is driven by Ca²⁺-activated Cl^- efflux. Although the prominent Ca²⁺-activated Cl^- currents generated in immature <i>X. laevis</i> oocytes are mediated by <i>X. laevis</i> transmembrane protein 16a (xTMEM16A) channels, little is known about the channels that contribute to the fast block in mature eggs. Moreover, the gamete undergoes a gross transformation as it develops from an immature oocyte into a fertilization-competent egg. Here, we report the results of our approach to identify the Ca²⁺-activated Cl^- channel that triggers the fast block. By querying published proteomic and RNA-sequencing data, we identify two Ca²⁺-activated Cl^- channels expressed in fertilization-competent <i>X. laevis</i> eggs: xTMEM16A and <i>X. laevis</i> bestrophin 2A (xBEST2A). By exogenously expressing xTMEM16A and xBEST2A in axolotl cells lacking endogenous Ca²⁺-activated currents, we characterize the effect of inhibitors on currents mediated by these channels. None of the inhibitors tested block xBEST2A currents specifically. However, 2-(4-chloro-2-methylphenoxy)-<i>N</i>-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) and <i>N</i>-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) each reduce xTMEM16A currents by more than 70% while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we find that Ani9 and MONNA effectively diminish fertilization-evoked depolarizations. Additionally, these inhibitors lead to increased polyspermy in <i>X. laevis</i> embryos. These results indicate that fertilization activates TMEM16A channels in <i>X. laevis</i> eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.