Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

Abstract

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.

Veratridine-Mediated Ca 2+ Dynamics and Exocytosis in Paramecium Cells

“…For both these agents we demonstrated, also by fluorochrome analysis, the occurrence of a cortical [Ca 2+ ] i transient orginating primarily from cortical pools ("alveolar sacs" [49]) and reinforced by superimposed Ca 2+ influx from the medium [14,15,26,27]. For trichocyst secretion induced by veratridine, the source of calcium is addressed in this paper.…”

“…Mn 2+ -quench of Fura-2 ( ex ‫ס‬ 360/380 nm/ em Ն 510 nm) was used to demonstrate veratridine-stimulated Mn 2+ entry by unspecific Me 2+ conducting channels [9,16]. Confocal analysis with Fluo-3 injected cells was carried out at video rate in an Odyssey XL, Noran Instruments (Middleton, WI, and Bruchsal, Germany), ex ‫ס‬ 488 nm/ em Ն 515 nm as previously described [14]. Calcein was analyzed by CLSM with the same filter set.…”

“…In 7S cells, veratridine-induced exocytotic response (membrane fusion and trichocyst secretory contents release by stretching ["decondensation"]) and [Ca 2+ ] i transients were correlated in time by confocal time series with alternating fluorescence and transmitted light pictures as previously described [14]. In nd9-28°C cells, veratridine-induced [Ca 2+ ] i transients were paralleled by internal trichocyst decondensation since these cells are incapable of membrane fusion, just as described for caffeine treatment [27].…”

“…This involves membrane fusion and contents discharge ("decondensation" ‫ס‬ severalfold stretching of pear-shaped contents to needles during expulsion through exocytotic openings), [37,38]. Membrane fusion requires a subplasmalemmal [Ca 2+ ] i increase [14,15,26,27], while decondensation of contents, with no Ca detectable by x-ray microanalysis [45], requires access of Ca 2+ to the secretory materials [5]. This normally occurs by influx of Ca o 2+ after formation of an exocytotic pore [37,38].…”

“…Finally, nonsecretory mutants, nd9 and tl, were used also for technical reasons, since these cells, in contrast to 7S cells, are not vigorously dislocated during quasiexplosive trichocyst expulsion. Therefore, these strains, though mobile, are much more easily and reliably amenable to 2 fluorochrome analysis of calibrated [Ca 2+ ] i transients, while 7S cells can be preferably used for semiquantitative, but fast, 1 analysis on a subsecond time scale [14,26,27].…”