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bioRxiv · 10.1101/270629

The TMEM16A Channel Mediates the fast polyspermy block in Xenopus Laevis

Wozniak, K. L.·Phelps, W. A.·Tembo, M.·Lee, M. T.·Carlson, A. E.
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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, Xenopus laevis, this depolarization is driven by a Ca2+-activated Cl- efflux. Although the prominent Ca2+-activated Cl- currents generated by immature X. laevis oocytes are conducted by xTMEM16A channels, little is known about which channels contribute to fertilization-competency in mature eggs. Moreover, the gamete undergoes a gross transformation as it matures 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. Querying published proteomics and RNA-seq data, we identified two Ca2+-activated Cl- channels expressed in fertilization-competent X. laevis eggs: xTMEM16A and xBEST2A. Furthermore, transcripts for these channels increase in abundance during gamete maturation. To determine if either of these mediates the fast block, we characterized exogenously expressed xTMEM16A and xBEST2A using pharmacologic inhibitors. None of the inhibitors tested blocked xBEST2A currents specifically. However, Ani9 and MONNA each reduced xTMEM16A currents by more than 70%, while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we found that Ani9 and MONNA effectively diminished fertilization-evoked depolarizations. These results indicate that fertilization activates TMEM16A channels in X. laevis eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.\n\nHIGHLIGHTSO_LIProtein for the channels xBEST2A and xTMEM16A is present in X. laevis eggs.\nC_LI\n\nO_LIThe inhibitors MONNA and Ani9 effectively block xTMEM16A compared to xBEST2A.\nC_LI\n\nO_LIXenopus laevis fertilization opens TMEM16A to trigger egg depolarization.\nC_LI\n\nO_LIThe TMEM16A-mediated depolarization is critical for the fast block to polyspermy.\nC_LI

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Wozniak, K. L., Phelps, W. A., Tembo, M., Lee, M. T., Carlson, A. E.. 2018-02-23. The TMEM16A Channel Mediates the fast polyspermy block in Xenopus Laevis. https://doi.org/10.1101/270629

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