Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca2+/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca2+-influx via unspecific cation channels

Abstract

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca2+] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca2+]i transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply increasing [Ca2+]o causes a similar rapid, short-lived [Ca2+]i transient. All these phenomena, (i¨Ciii), are compatible with activation of an extracellular ¡°Ca2+/(polyvalent cation)-sensing receptor¡± known from some higher eukaryotic systems, where this sensor (responding to Ca2+, La3+ and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (¡°alveolar sacs¡±) are physically linked to the cell membrane and they can also be activated by the Ca2+ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca2+ signal also in absence of Ca2+o we largely exclude a ¡°Ca2+-induced Ca2+ release¡± (CICR) mechanism. Our finding of increased cortical Ca2+ signals after store depletion and re-addition of extracellular Ca2+ can be explained by a ¡°store-operated Ca2+ influx¡± (SOC), i.e., a Ca2+ influx superimposing store activation. AED stimulation in presence of Mn2+o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOCtype Ca2+ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca2+/polyamine-sensing receptor, (ii) release of Ca2+ from subplasmalemmal stores, (iii) and Ca2+ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca2+] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca2+] microdomains (¡Ü0.5 mm, ¡Ü33 msec) upon stimulation.