Alveolar sacs are attached tightly at the cell membrane, thus allowing the formation of a subplasmalemmal space of ~15 nm width maintained by visible membrane-to-membrane links (Plattner et aI., 1991). These are un-identified proteins that superficially recall the feetlike connections at termi-nal SR cisternae in striated muscle. However, in ciliates, no tetrameric substructures have ever been shown to occur, in contrast to SR where tetrameric dihydropyridine receptors (acting as voltage sensors) in the cell membrane match tetrameric ryanodine receptor-type Ca2+-release channels in the SR membrane (Meissner, 1994). In Paramecium some drugs are inactive (Uingeet aI., 1995), and de- or hyperpolarization of the cell mem-brane potential does not cause Ca2+ release (Erxleben and Plattner, 1994).
The situation we postulate for Paramecium (Klaukeet al., 2000), is sum-marized in Figs. 4 and 5. A CaSR may be coupled to an unspecific cation-influx channel in the cell membrane. This may be associated with a Ca2 +-release channel in the nearby outer membrane of alveolar sacs. The precise entry pathway of Ca2+ is not yet known.
The first evidence for alveolar sacs serving as Ca stores came from EDX analyses inColeps(Faure-Fermietet aI., 1968), where Ca enrichment forms a conspicuously dense material in the lumen. particularly after precipitation in the insoluble form. Even the occurrence of a Ca pump has been precluded in these studies by enzyme cytochemistry. The isolation of alveolar sacs fromParameciumby Adoutte's group (Stellyet aI.,1991) allowed thorough analysis of 45Ca2+-uptake and -release kinetics (Uinge et al., 1995, 1996).
Structural analyses of Ca2+ dynamics during exocytosis stimulation have been performed on a subsecond time scale using ESI (Knollet al., 1993) and EDX methods (Hardtet al., 1998; Hardt and Plattner, 1999.2000). Still, Ca2+-release channels were identified only indirectly by their responsivity to SR activators, caffeine (Uingeet aI.,1995,1996; Klauke and Plattner, 1998) and 4-chloro-meta-cresol (4CmC; Klauke et aI., 1999). As with SR, to achieve maximal activation, caffeine has to be applied in concentrations up to 50 mM, whereas only :sI mM 4CmC is sufficient. 4CmC activates
,.
c:=
Cl
(2)Ca2+-release(J)Ca2 +/(polyvalentcation) ~annelsI-senSingreceptor(CaSR)
I
CELLMEMBRANE-STORECOUPLINGI
(activators:[Ca2+]o,[La3+J o' AED,polycations) ~~ •(j)SOC=store-operated Ca2 +influx CM SS OM-AS--'"ASJfT ~ O'J IM-ASI
CaSR0
unspecificcation(SaC)channelI
Caz+-releasechannels FIG.4HypotheticalcouplingofCa z ,releasefromalveolarsacsandCaz+influxbyasaC-typemechanism,followingCaSR activation,inthesequenceindicated(1-3).Coarsecrosshatch,unknownCaz+uptakemechanisminalveolarsacmembrane. Forabbreviations,seeFig.2. 4
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...
0,• I
Ca2 +-RELEASECHANNELSINSTORESI c:=
Cl
~
Ca2+-releasechannels (activators:caffeine, 4-CI-m-cresol) T IM-AS
CM SS OM-AS
~ .j:>. ... FIG.5AssumedlocalizationofCa2+-releasechannelsinalveolarsacsofParamecium.
ryanodine-receptor-type Ca2+-release channels even when mutated (Herrmann-Frank et al., 1996; Kabbara and Allen, 1999), and this drug is used in pathophysiology to test patients for malignant hyperthermia, which would entail fatality during anesthesia. In contrast to SR, ryanodine does not mobilize Ca2+ from alveolar sacs in situ or after isolation (Uinge et al., 1995).
We obtain considerable Ca2+ influx upon AED stimulation even at [Ca2+]o = 50 fLM. There would be sufficient driving force for entry into the cytosoL but what would be the situation with alveolar sacs (into which Ca2+may enter either directly or via a very fast indirect uptake mechanism operating at the subsecond level)? Actual [Ca2+] within alveolar sacs is unknown, yet decreases during store activation will further drive reuptake.
In ER-SR systems, estimates of [Ca2+]varied over the years by four orders of magnitude (Bygrave and Benedetti, 1996), yet values of ~50 fLM are now in consideration (Meldolesi and Pozzan, 1998a).
We expect Ca2+-release channels to be localized on the outer side of alveolar sacs, i.e., the side facing the cell membrane and/or facing trichocyst docking sites, whereas the inner region has been shown to contain the SERCA-type Ca2+pump heavily enriched (Plattner et al., 1999). This ar-rangement allows for site-directed Ca2+flux toward trichocyst docking sites and subsequent downregulation of [Ca2+]j increase. The plasmalemmal pump will "serve" the subplasmalemmal space, whereas Ca2+ sweeping into the cell interior will be handled by the organellar Ca2+ pump. This has been cloned (Hauseret al., 1998) and characterized pharmacologically and biochemically, particularly with regard to phospho intermediate forma-tion (Kissmehl et aI., 1998), as described in Section 1lI.EA.
In the alveolar sacs ofParamecium, calcium is bound to a high-capacity/
low-affinity CaBP ofthe calsequestrin type (Plattneret al.,1997b). We have established this by specific AB binding in immunofluorescence and by immunogold labeling, as well as by Western blot analysis of isolated frac-tions that also bound 45Ca2+ in overlays. Interaction with calreticulin has been excluded, and preadsorption with original calsequestrin from SR abol-ished AB binding.
This explains the high [Ca]= 43mMfound in alveolar sacs by EDX (Hardt and Plattner, 1999,2000), similar to values detected in the SR. Mobilization of Ca2+occurs during AED-stimulated exocytosis by a signal-transduction pathway to be established [because none of the known second messengers can release Ca2+from alveolar sacs (Uingeet al., 1995)]. We assume direct coupling to the cell membrane (Erxleben and Plattner, 1994; Erxlebenet al., 1997), which mediates store-operated Ca2+ influx (SaC-type mechanisms) via unspecific cation channels (Klauke et al., 2000). In fact, when [Sr2+]ois substituted for [Ca2+]o during synchronous (80 ms) AED-stimulated exo-cytosis in quenched-flow experiments, Sr is detected by EDX in alveolar sacs
,
CALCIUM IN CILIATED PROTOZOA 149
after only 80 ms, when -40% ofthe Ca has been released (Hardt and Plattner, 2000). Considering the somewhat variable pharmacological characteristics
• of SOC in different systems (Lewis, 1999), we therefore can reasonably as-sume it to exist as a functional component in ciliates. This mechanism is much faster than the lcRActype Ca2+-influx current measured in some other secre--. tory cells (Hoferet al., 1998); see Section ILB.
Alveolar sacs are also considered a Ca2T source during GTP- or Ba2
+-induced chemoresponses (Wassenberget al., 1997), because this is affected by previous exposure to SERCA inhibitors.
The biogenesis of alveolar sacs is unknown (Capdevilleet al., 1993). We presume each sac to be a closed compartment. We could not see any connecting holes, as reported by AlIen (1988), after fast freezing and freeze-fracturing (Flotenmeyer et aI., 1999). Such connections possibly are snap-shots of biogenetic formation by expansion, constriction, and cleavage.
Conclusions. Alveolar sacs are ample subplasmalemmal calcium stores containing a high-capacity/low-affinity CaBP of the calsequestrin type. They thus can store Ca in concentrations that occur in the SR of muscle cells.
During exocytosis stimulation. alveolar sacs release a large proportion of their calcium, but they do not participate in the regulation of ciliary function.