Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors.

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.

Degradation of inositol 1,4,5-trisphosphate receptors during cell stimulation is a specific process mediated by cysteine protease activity.

Inositol 1,4,5-trisphosphate (InsP3) receptors are down-regulated in response to chronic activation of certain cell surface receptors because their degradation is accelerated. Studies on the nature of the down-regulatory process and the protease(s) responsible for receptor degradation are described here. InsP3 receptor down-regulation was not accompanied by parallel changes in the concentrations of several other relevant proteins (endoplasmic reticulum Ca2+-ATPase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and protein kinases alpha and epsilon). Thus, the down-regulatory process selectively targets InsP3 receptors for degradation. Furthermore, down-regulation was unaffected by brefeldin A and NH4Cl, indicating that InsP3 receptor degradation occurs without removal of receptors from the endoplasmic reticulum and independently of functional lysosomes. Analysis of InsP3 receptor immunofluorescence confirmed that the receptors are not redistributed prior to or during down-regulation. Finally, of a range of protease inhibitors tested, only N-acetyl-Leu-Leu-norleucinal blocked down-regulation. Thus, cysteine protease activity accounts for InsP3 receptor degradation and analysis of proteolysis in permeabilized cells indicates that this activity is calpain. Thus, InsP3 receptor down-regulation appears to result from the highly selective calpain-mediated degradation of InsP3 receptors. Calpain activity may be stimulated by the high concentrations of Ca2+ that are thought to be found in the vicinity of activated InsP3 receptors.

pH regulation in mouse sperm: identification of Na(+)-, Cl(-)-, and HCO3(-)-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation.

Intracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHi in these cells are not understood. The pHi of mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na(+)-dependent Cl(-)-HCO3- exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates in N-methyl-D-glucamine+ Cl- or in 0.25 M sucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na(+)-H+ antiport pathway and the Na(+)-independent Cl(-)-HCO3- exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHi increases (from 6.54 +/- 0.08 to 6.73 +/- 0.09) that require a functional Na(+)-, Cl(-)-, and HCO3(-)-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHi during capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHi regulator.

Identification of a calsequestrin-like protein from sea urchin eggs.

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.

Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis.

Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate.