In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) ␣1S subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation-contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681-L690 and residues L720 -Q765, respectively), claiming for each a key function in DHPR-RyR1 communication. To address whether residues 720 -765 of the II-III loop are sufficient to enable skeletal-type (Ca 2؉ entryindependent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to ␣1S) has no similarity to ␣1S in the regions R681-L690 and L720 -Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous ␣1S subunits) was unable to restore EC coupling and displayed strongly reduced Ca 2؉ current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit ␣1S residues L720 -L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724 -T755) completely restored bidirectional coupling, indicating its dependence on ␣1S loop residues 720 -764 but its independence from other regions of the loop. Thus, 45 ␣1S-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein-protein interaction required for bidirectional coupling.

The specific localization of L-type Ca2+ channels in skeletal muscle triads is critical for their normal function in excitation–contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca2+ channel α1S subunit restores Ca2+ currents, EC coupling, and the normal localization of α1S in the triads. In contrast, expression of the neuronal α1A subunit gives rise to robust Ca2+ currents but not to triad localization. To identify regions in the primary structure of α1S involved in the targeting of the Ca2+ channel into the triads, chimeras of α1S and α1A were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the α1S/α1A chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of α1A were not incorporated into triads, chimeras containing the COOH terminus of α1S were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607–1661) proximal to the putative clipping site of α1S. Transferring this triad targeting signal to α1A was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca2+-dependent EC coupling.

Ca(2+)-induced Ca(2+)-release (CICR)-the mechanism of cardiac excitation-contraction (EC) coupling-also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca(2+) release, by reconstituting dysgenic myotubes with the cardiac Ca(2+) channel alpha(1C) (Ca(V)1.2) subunit. Ca(2+) influx through alpha(1C) provides the trigger for opening the sarcoplasmic reticulum Ca(2+) release channels. Here we show that also the Ca(2+) channel alpha(1D) isoform (Ca(V)1.3) can restore cardiac-type EC-coupling. GFP-alpha(1D) expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca(2+) transients with the same efficiency as GFP-alpha(1C) despite threefold smaller Ca(2+) currents. In contrast, GFP-alpha(1A), which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca(2+) transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca(2+) channels and less on their current size. Combined patch-clamp/fluo-4 Ca(2+) recordings revealed that the induction of Ca(2+) transients and their maximal amplitudes are independent of the different current densities of GFP-alpha(1C) and GFP-alpha(1D). These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca(2+) gradients in the triad junctions.