Microdomain Arrangement of the SERCA-type Ca²⁺ Pump (Ca²⁺-ATPase) in Subplasmalemmal Calcium Stores of Paramecium Cells

Abstract

SUMMARYWe localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented o ligomeric arrangements of ‫ف‬ 8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size ( ‫ف‬ 106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32 P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca 2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca 2+ pump may maintain or reestablish [Ca 2+ ] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca 2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.

Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx

“…Channels involved are only partially identified-they respond to the ryanodine-receptor activators, caffeine and 4-chloro-metacresol (4CI-m-C) [18], but not to inositol 1,4,5-trisphosphate (InsPJ) [28]. ASs possess a SERCA-type pump [30,31] located in that part of the ASs which faces the cell center, as we found by immuno- [32] and green fluorescent protein (GFP)-transfection labeling [33].…”

“…This is localized specifically in that part of ASs which faces the interior of the cell, i.e. the "inner AS membrane" [32]. For aspect (i), some authors envisage a Na+ /Ca2+ exchanger system, e.g.…”

“…In the SR. this is due to higher activity and to the much larger membrane area containing SERCA molecules (in the extensive longitudinal system [9]) as compared to the area containing Ca2+ -release channels (restricted to the relatively small terminal cisternae [53]). This microanatomical principle, small release versus large uptake area, is not applicable to Paramecium [32] and concomitantly refilling by SERCA activity is slow (see Table 2). Therefore, over longer periods after AED stimulation.…”

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells

“…Two types of Ca 2+ -pumps (Ca 2+ -ATPases) have been found in Paramecium, a plasmalemmal type activated by Ca 2+ /calmodulin [23] and a SERCA-type occurring in alveolar sacs [24,25], the established cortical Ca-stores of Paramecium [26][27][28][29][30]. The presence of a Ca 2+ -ATPase (pump) in ciliary membranes has been denied [23].…”

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

“…If so, this should be even more pronounced within a narrow subplasmalemmal space. In the Paramecium cell cortex, any Ca 2 +-release channels would have to be located on the outer part of alveolar sacs, because the part facing the cell center is densely studded with Ca 2 +-pump molecules [56]. In agreement with this postulate, the subplasmalemmal space, including that delineated by alveolar sacs and trichocysts, is the site where pp63/pf [36], as well as the relevant protein phosphatase PP2B/CaN [42] and protein kinases CK-2 and PKG [57,58] are heavily enriched according to immuno-EM studies (see Fig.…”