Functional and fluorochrome analysis of an exocytotic mutant yields evidence of store-operated Ca2+ influx in Paramecium

Abstract

A non-discharge mutant of Paramecium tetraurelia (nd12-35°C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack 45Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25°C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35°C cells were stimulated, not only with AED (EC100 = 10-6 M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 mM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35°C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35°C as in 25°C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35°C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25°C cells). Furthermore, 35°C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35°C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation.