Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

Ca sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca²⁺ concentration is necessary and sufficient for this process. The predominant source of Ca²⁺ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca²⁺ to the cytosol. The ER store is (re)filled by the store-specific Ca²⁺-ATPase. Ultimately, the depleted ER is replenished by Ca²⁺ which enters from the extracellular space to the cytosol via store-operated Ca²⁺ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca²⁺ channels and plasma membrane Na⁺/Ca²⁺ exchangers are additional means for cytosolic Ca²⁺ entry. Cytosolic Ca²⁺ levels can be modulated by mitochondria, which can take up cytosolic Ca²⁺ via the Ca²⁺ uniporter and release Ca²⁺ into cytosol via the mitochondrial Na⁺/Ca²⁺ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca²⁺ sources generates cytosolic Ca²⁺ dynamics that can drive Ca²⁺-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Summary ATP-dependent Ca²⁺ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca²⁺-specific electrode and⁴⁵Ca²⁺. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na⁺,K⁺(Mg²⁺)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,⁴⁵Ca²⁺ uptake correlated with the RNA content and not with the Na⁺,K⁺(Mg²⁺)ATPase activity. The characteristics of nonmitochondrial Ca²⁺ uptake into leaky isolated cells and⁴⁵Ca²⁺ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg²⁺, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca²⁺ concentration of 2 and 2 mol/liter, respectively. Calcium uptake decreased at higher free Ca²⁺ concentration.⁴⁵Ca²⁺ uptake was dependent on monovalent cations (Rb⁺>K⁺>Na⁺>Li⁺>choline⁺) and different anions (Cl^–>Br^–>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–>SCN^–) in both preparations. Twenty mmol/liter oxalate enhanced⁴⁵Ca²⁺ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca²⁺ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca²⁺ concentration to 0.4 mol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca²⁺ concentration in pancreatic acinar cells.     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.     

Localization and regulation of Ca entry and exit pathways in exocrine gland cells

Studies of Ca²⁺ transport pathways in exocrine gland cells have been useful, chiefly because of the polarized nature of the secretory epithelial cells. In pancreatic acinar cells, for example, Ca²⁺ reloading of empty intracellular stores can occur solely via Ca²⁺ entry through the basal part of the plasma membrane. On the other hand, the principal site for intracellular Ca²⁺ release—with the highest concentration of inositol 1,4,5-trisphosphate (IP₃) receptors—is in the apical secretory pole close to the apical plasma membrane. This apical part of the plasma membrane contains the highest density of Ca²⁺ pumps and is therefore the principal site for Ca²⁺ extrusion. On the basis of the known properties of Ca²⁺ entry and exit pathways in exocrine gland cells, the mechanisms controlling Ca²⁺ exit and entry are discussed in relation to recent direct information about Ca²⁺ transport into and out of the endoplasmic reticulum (ER) and the mitochondria in these cells.     

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca²⁺ signalling and maintenance of Ca²⁺ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca²⁺-ATPase, Na⁺, K⁺-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca²⁺ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca²⁺ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca²⁺ entry, and their formation and rebuilding have an important regulatory role in cellular Ca²⁺ homeostasis.     

The endoplasmic reticulum (ER)-target protein Bik induces Hep3B cells apoptosis by the depletion of the ER Ca2+ stores

Bik, a BH3-only protein, was identified to induce cells apoptosis. In this study, we reported that Bik exclusively localized to endoplasmic reticulum rather than mitochondria. The apoptosis induced by Bik was inhibited in Hep3B cells, when TM domain of Bik was truncated. The ectopic overexpression of Bik protein caused the rapid and sustained elevation of the intracellular cytosolic Ca²⁺, which originated from the ER Ca²⁺ stores releasing. The Hep3B cells apoptosis induced by Bik was not prevented by establishing the clamped cytosolic Ca²⁺ condition, or by buffering of the extracellular Ca²⁺ with EGTA, suggesting that the depletion of ER Ca²⁺ stores rather than the elevation of cytosolic Ca²⁺ or the extracellular Ca²⁺ entry contributed to Bik-induced Hep3B cells apoptosis. The authors Xiaoping Zhao and Li Wang contributed equally to this work.     

STIM1 regulates store-operated Ca entry in oocytes

The single transmembrane-spanning Ca²⁺-binding protein, STIM1, has been proposed to function as a Ca²⁺ sensor that links the endoplasmic reticulum to the activation of store-operated Ca²⁺ channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca²⁺ entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca²⁺ stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca²⁺ stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca²⁺ influx after store depletion and subsequent Ca²⁺ add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca²⁺ channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca²⁺ levels in the store-depleted oocytes after Ca²⁺ add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca²⁺ store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca²⁺ entry.     

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.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Calcium uptake into acini from rat pancreas: Evidence for intracellular ATP-dependent calcium sequestration

Summary Intracellular ATP-dependent Ca²⁺ sequestration mechanisms were studied in isolated dispersed rat pancreatic acini following treatment with saponin or digitonin to disrupt their plasma membranes. In the presence of⁴⁵Ca²⁺ concentrations <10^–6 mol/liter, addition of 5 mmol/liter ATP caused a rapid increase in⁴⁵Ca²⁺ uptake exceeding the control by fivefold. ADP mimicked the ATP effect by 50 to 60%, whereas other nucleotides such as AMP-PNP, AMP-PCP, CTP, UTP, ITP, GTP, cAMP and cGMP did not. Maximal ATP-promoted Ca²⁺ uptake was obtained at 10^–5 mol/liter Ca²⁺ uptake by mitochondrial inhibitors was dependent on the Ca²⁺ concentration, indicating the presence of different Ca²⁺ storage systems. Whereas the apparent half-saturation constant found for mitochondrial Ca²⁺ uptake was 4.5×10^–7 mol/liter, in the presence of antimycin and oligomycin (nonmitochondrial uptake) it was 1.4×10^–8 mol/liter. In the absence of Mg²⁺ both ATP- and ADP-promoted Ca²⁺ uptake was nearly abolished. The Ca²⁺ ionophore and mersalyl blocked Ca²⁺ uptake. Electron microscopy showed electrondense precipitates in the rough endoplasmic reticulum of saponintreated cells in the presence of Ca²⁺, oxalate and ATP, which were absent in intact cells and in saponin-cells without ATP or pretreated with A23187. The data suggest the presence of mitochondrial and nonmitochondrial ATP-dependent Ca²⁺ storage systems in pancreatic acini. The latter is likely to be located in the rough endoplasmic reticulum.     

Formyl-peptide receptor like 1: a potent mediator of the Ca2+ release-activated Ca2+ current ICRAC

In electrically non-excitable cells, one major source of Ca²⁺ influx is through the store-operated (or Ca²⁺ release-activated Ca²⁺) channel by which the process of emptying the intracellular Ca²⁺ stores results in the activation of Ca²⁺ channels in the plasma membrane. Using both whole-cell patch-clamp and Ca²⁺ imaging technique, we describe the electrophysiology mechanism underlying formyl-peptide receptor like 1 (FPRL1) linked to intracellular Ca²⁺ mobilization. The FPRL1 agonists induced Ca²⁺ release from the endoplasmic reticulum and subsequently evoked I_CRAC-like currents displaying fast inactivation in K562 erythroleukemia cells which expresses FPRL1, but had almost no effect in K562 cells treated with FPRL1 RNA-interference and HEK293 cells which showed no FPRL1 expression. The currents were impaired after either complete store depletion by the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, or after inhibition of PLC by U73122. Our results present the first evidence that FPRL1 is a potent mediator in the activation of CRAC channels.     

Comparison of the Effects Exerted by Luminal Ca2+ on the Sensitivity of the Cardiac Ryanodine Receptor to Caffeine and Cytosolic Ca2+

Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca²⁺ entry through the plasma membrane, there is growing evidence that Ca²⁺ in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca²⁺ on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca²⁺ added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba²⁺ or Ca²⁺. The given ion served also as a charge carrier. Luminal Ca²⁺ effectively shifted the EC₅₀ for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca²⁺. Importantly, luminal Ca²⁺ exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca²⁺) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca²⁺ with the luminally located Ca²⁺ regulatory sites on the RYR2 channel complex.     

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca²⁺ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca²⁺ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca²⁺ level after activation; time to reach peak cytosolic free Ca²⁺ and the EC₅₀ of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca²⁺ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca²⁺ signaling.     

Role of STIM1 in the surface expression of SARAF

The store-operated Ca²⁺ entry-associated regulatory factor (SARAF), a protein expressed both in the endoplasmic reticulum and the plasma membrane, has been presented as a STIM1-interacting protein with the ability to modulate intracellular Ca²⁺ homeostasis. SARAF negatively modulates store-operated Ca²⁺ entry (SOCE) by preventing STIM1 spontaneous activation and regulating STIM1-Orai1 complex formation. In addition, SARAF is a negative regulator of Ca²⁺ entry through the arachidonate-regulated Ca²⁺ (ARC) channels. Here we explored the possible role of the surface expression of SARAF on the location of STIM1 in the plasma membrane. In NG115-401L cells, lacking a detectable expression of native STIM1, transfection with pHluorin-STIM1, which is able to translocate to the cell surface, enhances the plasma membrane location of SARAF as compared to cells transfected with YFP-STIM1, lacking the ability to translocate to the cell surface. These findings suggest that the surface location of SARAF is dependent on the expression of STIM1 in the plasma membrane.     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

Fatty acids, alcohol and fatty acid ethyl esters: Toxic Ca signal generation and pancreatitis

Pancreatitis, a potentially fatal disease in which the pancreas digests itself as well as its surroundings, is a well recognized complication of hyperlipidemia. Fatty acids have toxic effects on pancreatic acinar cells and these are mediated by large sustained elevations of the cytosolic Ca²⁺ concentration. An important component of the effect of fatty acids is due to inhibition of mitochondrial function and subsequent ATP depletion, which reduces the operation of Ca²⁺-activated ATPases in both the endoplasmic reticulum and the plasma membrane. One of the main causes of pancreatitis is alcohol abuse. Whereas the effects of even high alcohol concentrations on isolated pancreatic acinar cells are variable and often small, fatty acid ethyl esters – synthesized by combination of alcohol and fatty acids – consistently evoke major Ca²⁺ release from intracellular stores, subsequently opening Ca²⁺ entry channels in the plasma membrane. The crucial trigger for pancreatic autodigestion is intracellular trypsin activation. Although there is still uncertainty about the exact molecular mechanism by which this Ca²⁺-dependent process occurs, progress has been made in identifying a subcellular compartment – namely acid post-exocytotic endocytic vacuoles – in which this activation takes place.     

Ca2+ transport and chemoreception in Paramecium

Intracellular Ca²⁺ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca²⁺ pump and that a Ca²⁺-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca²⁺ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca²⁺ homeostasis and pump activity.Abbreviations EGTA ethyleneglycol tetra-acetate - ER endoplasmic reticulum - IBMX isobutyl methylxanthine - I _che index of chemokinesis - Mops 3-[N-morpholino] propanesulfonic acid - PEI phosphoenzyme intermediate - STEN sucrose, TRIS, EDTA, sodium chloride - TCA trichloroacetic acid - TRIS tris[hydroxymethyl] aminomethane