A RYANODINE-SENSITIVE CALCIUM STORE IN ASCIDIAN EGGS MONITORED BY WHOLE-CELL PATCH-CLAMP RECORDINGS

Using whole cell patch clamp recordings on unfertilized eggs of the as cidian Ciona intestinalis, we are able to detect ryanodine receptors w ithin the oocytes. Our approach is based on measurements of the voltag e-activated inward calcium currents. Two types of Ca2+ currents have b een described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, wh en applied either externally or internally from the mouth of a patch p ipette. Caffeine application mimics fertilization which transiently de creases the high threshold Ca2+ current density during the first meiot ic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This p artial decrease in Ca2+ current density elicited by caffeine or ryanod ine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarizati on-induced Ca2+ current intensity allows monitoring of an intracellula r calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was o btained by immunological staining with antibodies against mammalian sk eletal muscle ryanodine receptor. Ryanodine receptors were asymmetrica lly localized in the cortex of Ciona eggs. We discuss the methodologic al relevance of our patch-clamp approach, in connection with the possi ble biological role of such a ryanodine receptor in the early stages o f development.