Control of IP(3)-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin.
1. Elementary events of Ca2+ release (Ca2+ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP(3)Rs) at low concentrations of IP(3). Ca(2+) puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP(3) into the cell. However, cells appear to have sufficient concentrations of IP(3) (0.1-3.0 microM) to induce Ca2+ release under resting conditions. 2. Here, we investigated Ca2+ puff activity in non-stimulated Xenopus oocytes using confocal microscopy. The fluorescent Ca2+ dye indicators Calcium Green 1 and Oregon Green 488 BAPTA-2 were injected into oocytes to monitor basal Ca2+ activity. 3. In this preparation, injection or overexpression of parvalbumin, an EF-hand Ca(2+)-binding protein (CaBP), induced Ca2+ puffs in resting Xenopus oocytes. This activity was inhibited by heparin, an IP(3)R channel blocker, and by mutation of the Ca(2+)-binding sites in parvalbumin. 4. Ca2+ puff activity was also evoked by injection of low concentrations of the Ca2+ chelator EGTA, but not by calbindin D(28k), another member of the EF-hand CaBP superfamily. 5. BAPTA and the Ca2+ indicator dye Oregon Green 488 BAPTA-1 evoked Ca2+ puff activity, while the dextran conjugate of Oregon Green 488 BAPTA-1 did not. These data indicate that a Ca(2+) buffer must be mobile in order to increase Ca2+ puff activity. 6. Together, the data indicate that some IP(3)Rs spontaneously release Ca2+ under resting concentrations of IP(3). These elementary Ca2+ events appear to be below the level of detection of current imaging techniques. We suggest that parvalbumin evokes Ca2+ puffs by coordinating the activity of elementary IP(3)R channel openings. 7. We conclude that Ca2+ release can be evoked not only by hormone-induced increases in IP(3), but also by expression of mobile cytosolic CaBPs under resting concentrations of IP(3).
John
L M
LM
Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Mosquera-Caro
M
M
Camacho
P
P
Lechleiter
J D
JD
D000818:Animals; D002021:Buffers; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002264:Carboxylic Acids / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D010320:Parvalbumins / Q000494:pharmacology / Q000502:physiology*; D011930:Reaction Time / Q000187:drug effects; D064030:S100 Calcium Binding Protein G / Q000494:pharmacology; D014982:Xenopus laevis
D002021:Buffers; D064026:Calbindins; D002264:Carboxylic Acids; D002614:Chelating Agents; D004396:Coloring Agents; C436864:Oregon Green 488 carboxylic acid; D010320:Parvalbumins; D064030:S100 Calcium Binding Protein G; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium