Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca²⁺ release in the cell cortex. Here, we show that this Ca²⁺ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca²⁺ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca²⁺ release during meiotic maturation. The Ca²⁺ wave was inhibited by the classical antagonists of the InsP₃-linked Ca²⁺ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca²⁺ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP₃, despite the massive release of Ca²⁺, implying that exocytosis of the cortical granules requires not only a Ca²⁺ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca²⁺ signals and in regulating cortical granule exocytosis.     

Actin,more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca²⁺ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca²⁺ increase in the fertilized egg can be recapitulated by several Ca²⁺-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca²⁺ waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca²⁺ signaling. As it is well known that Ca²⁺ regulates the structure of the actin cytoskeleton, our finding that Ca²⁺ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca²⁺ signaling may form a ‘positive feedback loop’ that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the ‘acrosome process,’ a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.     

The role of the actin cytoskeleton in calcium signaling in starfish oocytes

Ca(2+) is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca(2+) to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca(2+) signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca(2+)-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca(2+) signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca(2+)-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.     

Alteration of the cortical actin cytoskeleton deregulates Ca2+ signaling, monospermic fertilization, and sperm entry

Background

When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca²⁺) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.

Methodology/Principal Findings

We have measured changes in intracellular Ca²⁺ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP₃ receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.

Conclusions/Significance

Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca²⁺ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.     

Latrunculin A depolarizes starfish oocytes

Depolymerization of the actin cytoskeleton may liberate Ca²⁺ from InsP₃-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca²⁺ influx in others. However, no information is available on the modulation of membrane potential (V_m) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V_m in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V_m which reached the threshold of activation of voltage-gated Ca²⁺ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca²⁺-free sea water, while it was abolished upon removal of external Na⁺. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca²⁺ from InsP₃Rs. The Ca²⁺ signal in turn activates a Ca²⁺-dependent Na⁺ entry, which causes the positive shift in V_m and stimulates the VGCC.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

Background

Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca²⁺ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca²⁺ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.

Methodology/Principal Findings

By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca²⁺ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca²⁺; iii) GDPβS has little effect on the amplitude of the Ca²⁺ peak, but significantly expedites the initial Ca²⁺ waves induced by InsP₃ photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca²⁺ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca²⁺ signaling.

Conclusions/Significance

Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca²⁺ release.     

Protein Kinase Activities Associated with Actin Cytoskeleton in Xenopus laevis Oocytes and Eggs

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [-³²P]ATP has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [-³²P]ATP in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca²⁺-dependent kinases, as well as MAP, p34^cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin

Physico-chemical properties and interaction with actin of an actin- depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.     

Mitochondrial Calcium uniporters are essential for meiotic progression in mouse oocytes by controlling Ca2+ entry

ObjectivesThe alteration of bioenergetics by oocytes in response to the demands of various biological processes plays a critical role in maintaining normal cellular physiology. However, little is known about the association between energy sensing and energy production with energy‐dependent cellular processes like meiosis.Materials and methodsWe demonstrated that cell cycle‐dependent mitochondrial Ca²⁺ connects energy sensing to mitochondrial activity in meiosis progression within mouse oocytes. Further, we established a model in mouse oocytes using siRNA knockdowns that target mitochondrial calcium uniporters (MCUs) in order to inhibit mitochondrial Ca²⁺ concentrations.ResultsDecreased numbers of oocytes successfully progressed to the germinal vesicle stage and extruded the first polar body during in vitro culture after inhibition, while spindle checkpoint‐dependent meiosis was also delayed. Mitochondrial Ca²⁺ levels changed, and this was followed by altered mitochondrial masses and ATP levels within oocytes during the entirety of meiosis progression. Abnormal mitochondrial Ca²⁺ concentrations in oocytes then hindered meiotic progress and activated AMP‐activated protein kinase (AMPK) signalling that is associated with gene expression.ConclusionsThese data provide new insight into the protective role that MCU‐dependent mitochondrial Ca²⁺ signalling plays in meiotic progress, in addition to demonstrating a new mechanism of mitochondrial energy regulation by AMPK signalling that influences meiotic maturation.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

Aster formation in sand dollar eggs by micro-injection of calcium buffers and centriolar complexes isolated from starfish sperm

Centriolar complexes isolated from sperm of the starfish, Asterina pectinifera, could initiate irregular cleavage by injecting them into the fertilized eggs of the sand dollar, Scaphechinus mirabilis. Unfertilized sand dollar eggs could be activated when Ca²⁺ concentration in the egg cytoplasm increased by means of microinjecting calcium buffers in which the concentration of free Ca²⁺ was controlled by mixing calcium salt and calcium chelator at various proportions. When the centriolar complexes were injected into Ca²⁺-activated eggs, aster formation was induced in 25 out of the 55 eggs injected with calcium buffers at an intracellular Ca²⁺ level above 2.9 μM. The number of asters detected in one egg was quite variable, ranging from 2 to 20. The pronucleus in injected eggs was seen to migrate towards the induced asters. Eight of those 25 eggs cleaved.     

A Highlights from MBoC Selection: Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT–dependent neurite outgrowth in Aplysia bag cell neurons

Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca²⁺; however, mechanistic roles for Ca²⁺ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca²⁺ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca²⁺ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca²⁺) often termed a ''Ca²⁺ wave''. While the speed and number of oscillations of the Ca²⁺ wave varies between species, the change in intracellular Ca²⁺ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca²⁺-dependent events. Here we present a protocol for imaging the Ca²⁺ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca²⁺ wave and the downstream changes associated with it.     

The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling

Background

Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca²⁺. Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP₃) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.

Methodology/Principal Findings

In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca²⁺ and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca²⁺ wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca²⁺ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.

Conclusions/Significance

Our results suggest that PIP2 plays comprehensive roles in shaping Ca²⁺ waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.     

Physiological inducers of the acrosome reaction

This article reviews recent studies on physiological inducers of the acrosome reaction in starfish. Upon encountering the jelly coat of eggs, starfish sperm undergo the acrosome reaction in response to a cooperation of three jelly components: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of steroidal saponins named Co-ARIS, and an oligopeptide presumably having an activity to increase the intracellular pH of sperm. ARIS induces the acrosome reaction in high Ca²⁺ or high pH sea water. In normal sea water, both ARIS and Co-ARIS are required for the induction. In addition to ARIS and Co-ARIS, a third jelly component, the oligopeptide, is necessary to mimic the full capacity of the jelly coat to induce the acrosome reaction. ARIS and Co-ARIS cooperatively increase the intracellular Ca²⁺ by stimulating Ca²⁺ channels, while the oligopeptide increases the intracellular pH by stimulating Na⁺/H⁺ exchange systems. When sperm meet the eggs, both changes are simultaneously achieved in them and thus they undergo the acrosome reaction.     

Regulation of the Cardiac Na+-Ca2+ Exchanger by the Endogenous XIP Region

The cardiac sarcolemmal Na⁺-Ca²⁺ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na⁺-dependent inactivation. This inactivation is manifested as a partial decay in outward Na⁺-Ca²⁺ exchange current after application of Na⁺ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na⁺ affinities. Regulation of the exchanger by nontransported, intracellular Ca²⁺ was also modified by the XIP-region mutations. Binding of Ca²⁺ to the intracellular loop activates exchange activity and also decreases Na⁺-dependent inactivation. XIP-region mutants were all still regulated by Ca²⁺. However, the apparent affinity of the group 1 mutants for regulatory Ca²⁺ was decreased. The responses of all mutant exchangers to Ca²⁺ application or removal were markedly accelerated. Na⁺-dependent inactivation and regulation by Ca²⁺ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na⁺-induced inactivated state, but that the XIP region is also involved in regulation by Ca²⁺.