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J Biol Chem, Vol. 273, Issue 49, 32627-32635, December 4, 1998


Fatty Acid-mediated Calcium Sequestration within Intracellular Calcium Pools*

Krystyna E. Rys-SikoraDagger and Donald L. Gill§

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Intracellular Ca2+ pools play an essential role in generating Ca2+ signals. The heterogeneity of intracellular Ca2+ pools reflects the complex and dynamic character of the endoplasmic reticulum within which they reside. Translocation of Ca2+ between distinct subcompartments of the endoplasmic reticulum is mediated by a sensitive and specific GTP-activated process involving formation of reversible communicating junctions (Rys-Sikora, K. E., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 31607-31613). In the presence of palmitate at 10 µM or above, this GTP-activated mechanism mediates substantial Ca2+ accumulation within a specific Ca2+-pumping pool. The fatty acid- and GTP-dependent accumulation of Ca2+ was highly chain length-specific; pentadecanoate (C15) and palmitate (C16) were equally effective, whereas fatty acids of shorter or longer chain length were either marginally effective or devoid of effect. Fatty acids with one or more unsaturated carbons were without effect, regardless of chain length. Palmitate-induced Ca2+ accumulation was immediately terminated with 2 µM palmitoyl-CoA, a blocker of the GTP-activated Ca2+-translocating mechanism. The anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid completely prevented both palmitate- and oxalate-mediated GTP-dependent Ca2+ accumulation, with EC50 ~ 30 µM. Ca2+ sequestered in the presence of palmitate and GTP could be immediately and completely released by A23187, whereas the sequestered Ca2+ was remarkably resistant to release induced by inositol 1,4,5-trisphosphate (InsP3). In contrast, oxalate-sequestered Ca2+ within the same pool could be effectively released by either ionophore or InsP3. The results indicate that fatty acids are specifically transported into the lumen of a subset of Ca2+ pools, wherein they mediate substantial sequestration of Ca2+ in a distinct membrane-associated substate that is not readily releasable by opened InsP3-sensitive Ca2+ channels.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Cytosolic Ca2+ signals mediate control over a diverse array of cellular activities ranging from short-term responses such as contraction and secretion to longer term regulation of cell growth and proliferation (1-5). Intracellular pools of Ca2+ are fundamental elements in the generation of Ca2+ signals, yet little is known about the nature and distribution of the organelles that serve as Ca2+ pools (4). It is widely held that the majority of Ca2+ pools are contained within the endoplasmic reticulum (ER)1 or subcompartments thereof (1, 2, 4, 6, 7). Most of the ER appears to pump and accumulate Ca2+. However, the ER represents an extensive and heterogeneous network of cisternae undergoing continuous dynamic changes in morphology through active membrane trafficking events. It is therefore not surprising that Ca2+ pools are observed to be structurally and functionally heterogeneous in particular with respect to distribution of InsP3-sensitive Ca2+ release channels (8-11). In earlier studies, we identified and characterized distinct InsP3-sensitive and -insensitive intracellular Ca2+-pumping pools (8, 10). In addition, we and others have characterized a sensitive and specific guanine nucleotide-activated process that appears to mediate translocation of Ca2+ between organelles within cells (12-16) and that permits Ca2+ movement between InsP3-sensitive and -insensitive Ca2+ pools (8, 16-19). This Ca2+ translocation process is rapid, temperature-sensitive, and dependent on the hydrolysis of GTP and represents a movement of Ca2+ quite distinct from that activated by InsP3 (7, 8, 16). The process of Ca2+ transfer may involve a close interaction between ER subcompartments and the cytoskeleton and may be particularly evident after disruption or fragmentation of the ER (20). Although no specific GTP-binding protein has been identified as mediating the Ca2+ transfer, it is likely that transfer reflects the function of one or more of the class of small GTP-binding proteins that are known to control many of the membrane trafficking and translocation events that occur within the ER and other organelles of the secretory pathway (4, 16, 21).

The mechanism by which GTP activates Ca2+ transfer requires close contacts between membranes (7, 16), but the underlying process mediating Ca2+ transfer has not been elucidated. Work by Comerford and Dawson (22, 23) has suggested that GTP-induced Ca2+ movements may reflect the activation of fusion between membranes. Instead, we have interpreted our results to indicate that the rapid fluxes of Ca2+ observed in response to GTP reflect a process that may precede a subsequently activated membrane fusion event (16, 24). Indeed, studies have shown that acyl-CoA esters can not only block, but also reverse the action of GTP in inducing Ca2+ transfer (24). Thus, we revealed that fatty acyl-CoA esters allosterically modify a component of the GTP-activated process, resulting in a rapid termination and reversal of Ca2+ transfer. It is difficult to reconcile this rapid reversing action of acyl-CoA esters with a mechanism by which GTP induces Ca2+ movements via a membrane fusion event since, having taken place, membrane fusion is an essentially irreversible process. Instead, we concluded that GTP rapidly induces a communicating prefusion complex between membranes that allows Ca2+ transfer and that, later, may lead to full fusion between membranes (4, 24). To understand more about the progression of these events, we investigated the actions of agents that might modify the membrane and enhance the process of membrane fusion. Surprisingly, one class of agents used for such analysis, nonesterified fatty acids, induced a remarkable chain length-specific alteration in the accumulation of Ca2+ observed after activation of the GTP-induced Ca2+ transfer process. Our results indicate that specific fatty acids are transported into the lumen of a subset of Ca2+ pools, wherein they mediate substantial sequestration of Ca2+ in a distinct membrane-associated substate; although readily releasable by application of ionophore, the release of this fatty acid-complexed Ca2+ through opened InsP3-sensitive Ca2+ release channels is highly restricted.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Culture of Cells-- Cells of the hamster smooth muscle cell line DDT1MF-2 were cultured in Dulbecco's modified Eagle's medium with 2.5% serum (Calf-Plus, Inovar Chemicals, Inc., Gaithersburg MD) as described previously (25-27). Cells were passaged every 7 days, and media changes were performed on days 3 and 5 after passaging. Cells used in experiments had been grown for 4 days after passaging.

Cell Permeabilization-- The procedures for cell permeabilization were as described earlier (24). Briefly, suspensions of DDT1MF-2 cells (1 × 106 cells/ml) were incubated with 0.0025% saponin in an intracellular-like medium (ICM; comprising 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl2, and 10 mM Hepes-KOH, pH 7.0) at 37 °C until 95% permeabilization occurred (normally 2-3 min). After permeabilization, cells were washed twice in saponin-free ICM at 4 °C and kept cold before use in experiments. To avoid problems of lipid dilution of added hydrophobic compounds, the final cell concentration in all experiments was kept at exactly 5 × 105 cells/ml.

Calcium Flux Experiments-- Ca2+ flux measurements were conducted as described previously (24, 28, 29). The accumulation of Ca2+ in intracellular organelles was measured using permeabilized DDT1MF-2 cells (5 × 105 cells/ml) maintained with gentle stirring at 37 °C in ICM containing 50 µM CaCl2 (with 150 Ci/mol 45Ca2+), EGTA (to buffer free Ca2+ to exactly 0.1 µM), 3% polyethylene glycol, and 5 µM ruthenium red (to prevent mitochondrial Ca2+ accumulation) in a total volume of 2 ml. Effectors indicated in the figures (GTP, InsP3, and A23187) were added at the times shown. Fatty acids or oxalate, with or without GTP or DIDS when present, were added immediately prior to the start of uptake or as shown in the figures. At required times, 200-µl aliquots were removed from the stirred uptake medium, diluted immediately into 4 ml of ice-cold ICM containing 1 mM LaCl3, rapidly vacuum-filtered on glass-fiber filters (Schleicher and Schuell, type 31), washed, and counted. For the palmitate concentration curves, all additions of fatty acids with or without GTP were made prior to the start of the experiment. For the curves shown, Ca2+ loading was for 6 min, at which time three successive aliquots were taken, and accumulation was determined. In other experiments (data not shown), Ca2+ loading was monitored for 12 min as described. In fatty acid chain length specificity experiments, Ca2+ accumulation was assessed only after 12 min of Ca2+ loading. The figures show ATP-dependent Ca2+ accumulation with that component of Ca2+ retained by cells and filters in the absence of ATP subtracted (~0.1% of total Ca2+). All experiments shown are typical of at least three separate experiments (and in most cases, a considerably larger number).

Materials and Miscellaneous Methods-- InsP3 and A23187 were purchased from Calbiochem. ATP, GTP, EGTA, polyethylene glycol, saponin, ruthenium red, Hepes, DIDS, oxalate, and all fatty acids were purchased from Sigma. Fatty acids were each dissolved in methanol/Me2SO (1:1), sonicated, and stored at -20 °C in capped vials containing N2. Before addition to experiments, fatty acids were again sonicated and added to permeabilized cell suspensions in ICM in no more than 1% of the final 2-ml volume. All controls contained equivalent quantities of the solvent without fatty acid. Visible turbidity of solutions was only observed with palmitate at a final concentration of 300 µM or higher. The DDT1MF-2 cell line was originally obtained from Drs. James Norris and Lawrence Cornett (University of Arkansas). Free Ca2+ concentrations were controlled using EGTA, computing all complexes between EGTA, ATP, Ca2+, Mg2+, monovalent cations, and protons, as described previously (30).

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Palmitate Reverses GTP-activated Ca2+ Translocation, Resulting in Substantial Ca2+ Accumulation-- The role of distinct Ca2+ pools in accumulating, releasing, and transferring Ca2+ has been studied extensively in several permeabilized cell systems (16, 29), including cells from the DDT1MF-2 smooth muscle line. An important feature of the Ca2+ movements observed has been the role of a sensitive and specific guanine nucleotide-induced Ca2+ translocation process (4, 7, 8, 16). As shown in Fig. 1A, Ca2+ accumulated within internal pools of permeabilized DDT1MF-2 cells via SERCA pump activity in the presence of 1 mM ATP was rapidly released by addition of 20 µM GTP; more than half of the accumulated Ca2+ was released within 30 s. Thus, GTP caused release from only a proportion of the pools able to accumulate Ca2+ as compared with the action of the Ca2+ ionophore A23187, which released Ca2+ from all of the Ca2+-pumping pools. The action of GTP on Ca2+ pools has been studied extensively and is believed to result from a GTP-induced transfer of Ca2+ between distinct subcompartments (8, 13, 17-19, 31). This process requires close contact between membranes (17, 18) and the hydrolysis of GTP (7, 8, 13, 32) and can be fully reversed by fatty acyl-CoA esters (24). Ca2+ transfer is believed to result from the activation of junctional complexes between distinct Ca2+-pumping compartments of the endoplasmic reticulum (16, 24). Such junctions allow the passage of Ca2+ ions and perhaps other small molecules, but, since they can be reversed (24), appear not to represent fusion between membranes. Our studies indicate that the GTP-activated process may represent a rapid prefusion event (16, 24) and that, in a substantially longer period following the GTP-induced interaction, fusion between different vesicular components of the ER may take place (7, 16, 22, 23). Since, in permeabilized cells, a small component of the ER appears to exist as non-intact vesicular membranes, the rapid transfer process induced by GTP results in the fast release of a substantial proportion of the accumulated Ca2+ (7, 16), as observed in Fig. 1A.


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  		Fig. 1.   Influence of palmitate on GTP-induced Ca2+ movements in permeabilized DDT1MF-2 cells. ATP-dependent Ca2+ accumulation was measured as described for standard conditions under "Experimental Procedures." A, uptake was under control conditions (open circle ) or with 20 µM GTP (triangle ) or 5 µM A23187 (black-diamond ) added at 6.5 min. B, palmitic acid (100 µM) was added prior to the start of the experiment, and control uptake (bullet ) was monitored without further addition or with additions at 6.5 min of 20 µM GTP (black-triangle) or 5 µM A23187 (black-diamond ). The experiment is typical of at least 10 similar experiments.

To determine more about the mechanism of the GTP-induced Ca2+ transfer process, we investigated the actions of fatty acids. As stated earlier, the rationale for examining fatty acids was to determine whether such lipid molecules might modify the effectiveness of GTP by promoting membrane interactions and enhancing the rate of subsequent membrane fusion. The introduction of up to 100 µM palmitate into the Ca2+ uptake medium induced only a slight augmenting action on Ca2+ pump-mediated Ca2+ accumulation within the permeabilized DDT1MF-2 cells (Fig. 1B). However, in stark contrast to the results shown in Fig. 1A, addition of GTP in the presence of palmitate resulted in a rapid and dramatic increase in the accumulation of Ca2+ (Fig. 1B). Under these conditions, the action of GTP was biphasic, inducing initially a slight release of Ca2+ before this changed to a large and continuous increase in Ca2+ accumulation. The methyl ester of palmitate had no effect on Ca2+ accumulation (data not shown), indicating that the carboxyl group was important for the stimulation of Ca2+ uptake with GTP. Without GTP addition, the Ca2+ accumulated in the presence of palmitate could still be fully released with the Ca2+ ionophore A23187 (Fig. 1B), indicating no obvious difference in the state of accumulated Ca2+ induced by palmitate. Likewise, after loading pools in the presence of palmitate, passive Ca2+ release induced by Ca2+ pump inhibition with thapsigargin was unchanged (data not shown), suggesting no obvious alteration in Ca2+ release. As shown below (see Fig. 7), palmitate did not alter the function of Ca2+ release through InsP3 receptors. The large accumulation of Ca2+ induced by GTP in the presence of palmitate was dependent on Ca2+ pumping, with no change in Ca2+ accumulation being observed without ATP also being present. Under the standard conditions for Ca2+ accumulation, the low concentration of free Ca2+ (0.1 µM) and the presence of ruthenium red ensured that no mitochondrial Ca2+ uptake was occurring (7, 10). At a higher free Ca2+ concentration (10 µM), in the absence of ruthenium red, the accumulation of Ca2+ was predominantly mitochondrial (7), yet under this condition, palmitate and/or GTP again induced no alteration of the mitochondrial component of Ca2+ accumulation (data not shown).

The concentration dependence of the effect of palmitate on reversing GTP-induced Ca2+ fluxes in permeabilized cells is shown in Fig. 2. In this experiment, Ca2+ accumulation was measured after 6 min with the simultaneous addition of ATP and palmitate, with or without 20 µM GTP, all at the start of Ca2+ uptake. Under these conditions, concentrations of palmitate above 3 µM caused significant Ca2+ entry; the action of palmitate was half-maximal at ~25 µM and maximal at 100 µM. In the absence of GTP, little change in Ca2+ accumulation occurred with palmitate concentrations as high as 300 µM. If a longer time interval for Ca2+ accumulation was used, the total amount of Ca2+ that could be sequestered in the presence of GTP and palmitate continued to increase; after 12 min, total Ca2+ accumulation could reach as high as 8 nmol/106 cells (data not shown). Measured with a 12-min incubation, the sensitivity to palmitate was slightly increased, with half-maximal activation closer to 10 µM. Although 300 µM palmitate had no greater effect than 100 µM, introduction of concentrations beyond 300 µM induced a sharply concentration-sensitive decrease in Ca2+ accumulation in the presence or absence of GTP (data not shown). This reduction in Ca2+ accumulation at high palmitate levels likely reflects a generalized deleterious action of palmitate on the integrity of the membrane through detergent-like effects of the fatty acid. Indeed, at or above 300 µM, some turbidity following addition of palmitate indicated the formation of larger fatty acid complexes. The critical micelle concentration for fatty acids is highly dependent on ionic conditions and, although not known in the presence of the intracellular-like conditions used, was likely to have been exceeded at 300 µM palmitate. Physiological levels of free nonesterified fatty acids inside cells are difficult to assess. In serum, the level of total nonesterified fatty acid can be in the millimolar range, palmitate being one of the most abundant of fatty acids; however, a large proportion of this total fatty acid is bound to albumin and other serum proteins. Fatty acids inside cells are likely to be similarly bound to proteins and/or partitioned within membranes. Experiments with sarcoplasmic reticulum vesicles have revealed that palmitate in the mid-micromolar range is efficiently incorporated into the vesicle bilayer (33). These studies revealed that the monounsaturated C18 fatty acid oleate at these concentrations caused considerable disruption and fusion of SR vesicles, whereas, in contrast, palmitate induced no fusion of the vesicles and no morphological alteration in the membrane except for an increase in the lateral volume of the membranes (33).


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  		Fig. 2.   Concentration dependence of palmitate-induced reversal of GTP-mediated Ca2+ movements. Ca2+ accumulation was monitored with increasing concentrations of palmitate acid in the absence (open circle ) or presence (bullet ) of 20 µM GTP. Palmitate at the concentrations shown and GTP were added just prior to the start of Ca2+ uptake, which continued for 6 min, at which time three successive aliquots were rapidly taken, and radioactivity was determined as described under "Experimental Procedures." The measurements were carried out in duplicate, and results are the means ± S.D. of six determinations and are representative of four similar experiments.

Structural Specificity of Fatty Acid-mediated Stimulation of GTP-dependent Ca2+ Sequestration-- Important to assess was any structural requirement among fatty acids for stimulating GTP-dependent Ca2+ accumulation, particularly with respect to chain length and degree of saturation. Indeed, the results reveal a striking structural specificity for fatty acids. A comparison of the effects of saturated fatty acids of different chain length is shown in Fig. 3. In this experiment, each fatty acid was added at 100 µM at the beginning of Ca2+ uptake either with or without GTP, and uptake continued for a total of 12 min. The control condition (i.e. without fatty acid) revealed the ~50% inhibition of Ca2+ accumulation due to GTP, as a result of GTP-induced Ca2+ release. Both pentadecanoate (C15) and palmitate (C16) induced a profound reversal of this action, resulting in both cases in large GTP-dependent Ca2+ accumulation. Myristate (C14) and heptadecanoate (C17) in each case gave a slight effect, whereas laurate (C12), stearate (C18), and arachidate (C20) resulted in almost no change in the accumulation of Ca2+ observed in the presence of GTP. The results reveal that the action of fatty acids in promoting Ca2+ accumulation is narrowly restricted to those with a chain length of either 15 or 16 carbons. Fatty acids with chain lengths below 14 or above 17 induced little effect on Ca2+ fluxes in the presence or absence of GTP. Experiments also investigated the actions of unsaturated fatty acids. It was noted that the unsaturated fatty acids had significant inhibitory effects on Ca2+ accumulation at the higher levels (100 µM). Such effects may relate to the inhibitory effects of unsaturated fatty acids on Ca2+ pump activity as observed using SR vesicles (34-36) and, as described above, may be indicative of the greater effectiveness of the unsaturated fatty acids in disrupting the lipid bilayer, resulting in leak of Ca2+ (33). The experiment shown in Fig. 4 compared the actions of a range of different unsaturated fatty acids with that of palmitate, each added at a lower concentration (10 µM). With the longer incubation period of 12 min, the action of palmitate in inducing Ca2+ accumulation in the presence of GTP was clearly apparent even at 10 µM. In contrast, neither of the monounsaturated C16:1-Delta 9 fatty acids (either the cis form, palmitoleate, or the trans form, palmitoleidate) had any effect on Ca2+ accumulation. Moreover, heptadecenoate (C17:1-cis-Delta 10), oleate (C18:1-cis-Delta 9), linoleate (C18:2-cis-Delta 9,12), and arachidonate (C20:4-cis-Delta 5,8,11,14) also did not induce any reversal of the action of GTP. As discussed below, the specificity of action of fatty acids in inducing Ca2+ accumulation, particularly with respect to chain length of saturated fatty acids, may relate to differences in the ability of fatty acids to be transported into the ER and/or to different intrinsic abilities of fatty acids to bind to and sequester Ca2+.


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  		Fig. 3.   Chain length specificity of saturated fatty acid-stimulated reversal of GTP-mediated Ca2+ transfer. ATP-dependent Ca2+ accumulation was undertaken in the presence of a 100 µM concentration of each of the saturated fatty acids shown either under standard conditions (open bars) or in the presence of 20 µM GTP (closed bars). The experiment was undertaken as described for Fig. 2, except that aliquots were taken after 12 min of Ca2+ accumulation. The fatty acids tested were laurate (C12:0), myristate (C14:0), pentadecanoate (C15:0), palmitate (C16:0), heptadecanoate (C17:0), stearate (C18:0), and arachidate (C20:0). Incubations were carried out in duplicate. Results are the means ± S.D. of six determinations and are representative of three identical experiments.


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  		Fig. 4.   Inability of unsaturated fatty acids to reverse GTP-dependent Ca2+ transfer. Unsaturated fatty acids were each present at 10 µM and were added prior to monitoring Ca2+ accumulation either in the absence (open bars) or presence (closed bars) of 20 µM GTP. Three successive aliquots were rapidly taken after 12 min of Ca2+ accumulation as described for Fig. 3. The unsaturated fatty acids tested were palmitate (C16:0), palmitoleate (C16:1 cis), palmitoleidate (C16:1 trans), heptadecenoate (C17:1), oleate (C18:1), linoleate (C18:2), and arachidonate (C20:4). Incubations were conducted in duplicate, and results are the means ± S.D. of six determinations and are typical of three identical experiments.

What Is the Process by Which Fatty Acids Mediate GTP-dependent Ca2+ Accumulation?-- The actions of fatty acids in promoting the substantial GTP-dependent accumulation of Ca2+ could be explained by several possible mechanisms. First, palmitate could be augmenting the activity of the Ca2+ pump. Second, fatty acids could be promoting the action of GTP to induce fusion between membrane components of the ER, resulting in larger Ca2+ accumulation. Third, fatty acids might be entering the ER and mediating sequestration of Ca2+ within the lumen. Several lines of evidence support the latter conclusion. Direct effects of palmitate on the initial rate of Ca2+ pumping were not observed (Fig. 1, A and B). Others have reported that palmitate at 100 µM has no effect on intracellular Ca2+ pumping in permeabilized pancreatic cells (37), whereas saturated fatty acids including palmitate, stearate, and arachidate are reported to actually have an inhibitory effect on SERCA pumping in SR vesicles (38). The chain length specificity of the action of fatty acids did not appear consistent with their effect on promoting membrane fusion; indeed, experiments described below appear to more directly rule out membrane fusion as a mechanism mediating GTP- and palmitate-induced Ca2+ accumulation. Instead, the action of palmitate in promoting Ca2+ accumulation in the presence of GTP is strongly analogous with the action of the dicarboxylic acid oxalate (7, 16). We previously revealed that concentrations of oxalate in the millimolar range were also able to reverse the action of GTP, resulting in significant Ca2+ accumulation; the conclusions from a number of studies were that two distinct Ca2+-pumping pools exist, one of which is InsP3-releasable and oxalate-permeable, the other being InsP3-nonreleasable and impermeable to oxalate (reviewed in Ref. 16). As described above, Ca2+ transfer between these pools is mediated through interorganelle junctions activated by GTP hydrolysis, representing a rapid and reversible "prefusion" event (24). With oxalate present, Ca2+ can be sequestered within the oxalate-permeable pool as an oxalate precipitate; GTP then permits the additional Ca2+ pumped into the oxalate-impermeable pool to have access to oxalate, resulting in greatly increased Ca2+ accumulation. Even though the total pumping activity does not increase, oxalate acts as a sink for Ca2+ to provide a large increase in Ca2+ accumulation (19). As mentioned earlier, the GTP-induced release of Ca2+ observed without oxalate results from junctional activation between a small proportion of non-intact membrane vesicles and the larger ER continuum (7, 16, 32). Since oxalate-complexed Ca2+ cannot be transferred through the GTP-activated Ca2+ translocation process, this small leak does not prevent buildup of oxalate-complexed Ca2+ inside the ER (37).

Whereas the analogy between the actions of palmitate and oxalate was suggestive of a similar Ca2+-sequestering action of palmitate, there was also a major difference in the actions of the two agents, namely, the much greater sensitivity of the response to palmitate. Thus, measured under optimal conditions, the effect of palmitate was ~100-fold more potent than that of oxalate. In view of this, it was important to more definitively investigate a possible alternative effect of palmitate through enhancement of fusion between membranes of distinct pools. We recently revealed that fatty acyl-CoA esters can reverse the GTP-activated Ca2+ translocation process (24). As shown in Fig. 5, maximally activated uptake of Ca2+ promoted by GTP in the presence of palmitate could be immediately terminated by addition of 2 µM palmitoyl-CoA. If palmitate were stimulating Ca2+ accumulation by inducing GTP-dependent fusion between the Ca2+-pumping organelles, then such an effect would not be reversible; in other words, membrane fusion, once having occurred, is an irreversible event. We therefore conclude that the action of palmitate is to promote sequestration of Ca2+. There is ample precedent for believing this to occur. Thus, details of the binding of Ca2+ to palmitate have been measured (39). In the 10-80 µM palmitate range, Ca2+ binding was shown to be half-maximal at 30 µM Ca2+ and to occur with a stoichiometry of 0.4 mol of Ca2+/mol of palmitate. The rate of association was highly Ca2+-dependent and maximal at ~1 mM Ca2+. Considering that the luminal Ca2+ concentration is in the high micromolar to millimolar range (40-42), then entry of palmitate into the ER lumen could give rise to substantial Ca2+ sequestration within the lumen. The question of whether and how palmitate might cross the ER membrane was therefore addressed.


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  		Fig. 5.   Palmitoyl-CoA-induced reversal of GTP-mediated Ca2+ accumulation in the presence of palmitate. Ca2+ accumulation was monitored in the presence of 20 µM GTP together with 100 µM palmitate. At the arrow, incubations received either 2 µM palmitoyl-CoA (PCoA; bullet ) or no further addition (black-triangle). Results are representative of three identical experiments.

Does the Ability of Fatty Acids to Induce GTP-dependent Ca2+ Accumulation Represent Function of an ER Anion Transporter?-- In previous work (7, 16), we had speculated that the action of oxalate might be related to the function of anion channels present in the ER membrane. Thus, evidence has shown that relatively nonselective anion channels exist in the SR membrane of muscle cells and the ER membrane of nonmuscle cells, the function of which is considered to help equilibrate the charge buildup that would otherwise accompany rapid release of a large quantity of Ca2+ ions from the SR or ER (7, 16, 43-45). Plasma membrane anion channels have been characterized in detail, and the stilbene-disulfonic acid derivative DIDS has been widely used as an inhibitor of such channels (46). Anion channels in both the SR (45, 47-49) and ER (44, 50) have also been well described. In rabbit SR, there appear to be two different anion channels that are blocked by 8 and 80 µM DIDS, respectively (51), and in rat brain ER, anion channels are blocked by DIDS in the 15-100 µM range (52). We therefore investigated whether DIDS would modify the action of either palmitate or oxalate in promoting Ca2+ accumulation in the presence of GTP. As shown in Fig. 6, in both cases, 100 µM DIDS completely prevented the anion-mediated accumulation of Ca2+. The experiment reveals that the actions of 100 µM palmitate (Fig. 6A) and 2.65 mM oxalate (Fig. 6B) added at the start of Ca2+ uptake were very similar. In the presence of palmitate when 20 µM GTP was also present, Ca2+ accumulation was initially retarded (due to increased release, as described above) and then, within a few minutes, became greatly augmented. This biphasic effect is similar to that shown in Fig. 1A, but is significantly slower. The biphasic action most likely reflects the rate of palmitate entry. When added before GTP (Fig. 1A), there is a very rapid transition since palmitate has presumably equilibrated within the ER lumen; in Fig. 6A, entry of sufficient palmitate must take place before significant Ca2+ sequestration can occur, hence a lag of ~3 min. The effect of DIDS was to block any action of GTP and palmitate in promoting Ca2+ uptake. This action of DIDS was half-maximal between 10 and 30 µM in preventing both palmitate- and oxalate-dependent Ca2+ accumulation in the presence of GTP (data not shown), clearly within the sensitivity range described above for anion channels of the SR and ER (51, 52). DIDS had little inhibitory effect on the initial rate of ATP-dependent Ca2+ pumping in the absence of palmitate or oxalate; at longer time intervals, a slight reduction in Ca2+ accumulation may reflect the permissive role of anion channels in mediating charge equilibration and allowing an increase in the equilibrium level of Ca2+ to be attained within the ER.


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  		Fig. 6.   Inhibition of palmitate-induced (A) and oxalate-induced (B) GTP-dependent Ca2+ sequestration by the anion transport inhibitor DIDS. Ca2+ accumulation was measured in the presence and absence of 100 µM palmitate (A) or 2.65 mM oxalate (B). All additions were made prior to the start of Ca2+ accumulation. The conditions were as follows: A, control conditions without palmitate (+), 100 µM palmitate alone (bullet ), 100 µM palmitate with 20 µM GTP (black-triangle), 100 µM DIDS (black-diamond ), 100 µM palmitate with 100 µM DIDS (black-down-triangle ), and 100 µM palmitate with 100 µM DIDS and 20 µM GTP (black-square); B, control conditions without oxalate (+), 2.65 mM oxalate alone (open circle ), 2.65 mM oxalate with 20 µM GTP (triangle ), 100 µM DIDS (diamond ), 2.65 mM oxalate with 100 µM DIDS (down-triangle), and 2.65 mM oxalate with 100 µM DIDS and 20 µM GTP (). Details of the experiment are given under "Experimental Procedures." Results are typical of five identical experiments.

These results provide evidence to indicate that the actions of palmitate and oxalate are indeed reflections of a similar mechanism involving mediated anion entry. One question that arises is whether the movement of oxalate or palmitate to support the large accumulation of Ca2+ is directly mediated by a possible anion channel or whether the large sequestration of Ca2+ that occurs is facilitated as a result of anion channels permitting movement of other smaller ions (for example, Cl-), which themselves dissipate charge. Although we have not undertaken studies on the effects of anion replacement, it is interesting to note that there is considerable precedent for DIDS-sensitive long chain fatty acid transport. Thus, much evidence points to the movement of fatty acids across plasma membranes being mediated by specific transport proteins. Interestingly, the function of these transporters has been shown to be blocked by DIDS in the 40-200 µM range (53, 54); indeed, labeled DIDS has been revealed to specifically bind one such plasma membrane fatty acid transport protein, FAT, which has been isolated, sequenced, and expressed (55, 56). Controversy surrounds how important such fatty acid transport proteins really are for the movement of fatty acids across membranes. Whereas evidence suggests that such proteins exist (55, 57), some believe that the flip-flop of fatty acids across membranes is sufficiently fast that transport proteins are not necessary (58). Our data may throw light on this area. Thus, it is most unlikely that the charged hydrophilic dicarboxylic acid oxalate traverses membrane bilayers, yet both palmitate and oxalate mediate similar effects on GTP-induced Ca2+ movements, and the actions of both are prevented by DIDS. Thus, it may be reasonable to conclude that a DIDS-sensitive transporter for mono- or dicarboxylic acids at least facilitates the entry of the two Ca2+-complexing anions. A further very significant inference from these data is that fatty acid transport appears to be facilitated across intracellular membranes as well as the plasma membrane. Last, the question of how fatty acid-induced sequestration of Ca2+ within the ER has such narrow chain length specificity is interesting. It is unlikely that the Ca2+-binding properties of fatty acids are so stringently related to chain length; therefore, it appears more likely that the specificity of the effect we observed is derived from specificity of the transporter itself.

State of Intraluminal Ca2+ Sequestered in the Presence of Fatty Acids: Implications for the Location and Releasability of Fatty Acid-Ca2+ Complexes-- The effects of palmitate and oxalate on Ca2+ accumulation provide some important parallels. Their similarity of action and blockade by DIDS suggest that a similar anion transport mechanism facilitates both processes. And the promotion by GTP of the actions of both anions suggests that the same two differentially anion-permeable Ca2+ pools are functioning. Yet, despite these parallels, some significant differences in the state of the sequestered Ca2+ were observed for the two anions. The data in Fig. 7 compare the actions of InsP3 and A23187 on Ca2+ sequestered in the presence of GTP together with correspondingly effective concentrations of either palmitate or oxalate. With palmitate (Fig. 7A), after attainment of maximal Ca2+ sequestration, addition of a maximally effective InsP3 concentration (10 µM) induced the slow onset of a reduced rate of Ca2+ accumulation. With oxalate (Fig. 7B), introduction of InsP3 prevented any further Ca2+ accumulation and induced a modest release of Ca2+. A more striking difference in effect was observed upon addition of A23187. With palmitate (Fig. 7A), a remarkably rapid release of most of the accumulated Ca2+ was observed within a few seconds, whereas with oxalate (Fig. 7B), the action of A23187 was very much slower. In the latter case, there was almost no effect of A23187 after 30 s, and even after 4 min, scarcely more than half the Ca2+ had been released. These effects point to a significant difference in the state and/or location of Ca2+ sequestered with the two anions. Similar effects are revealed from the data shown in Fig. 8. In this case, InsP3 or ionophore was added from the beginning of Ca2+ accumulation. In the presence of palmitate (Fig. 8A), InsP3 without GTP prevented accumulation of Ca2+ within the InsP3-sensitive pool, indicating that the action of InsP3 was not inhibited by palmitate. With GTP present, substantial sequestration of Ca2+ was only reduced but not prevented by InsP3. In the presence of oxalate (Fig. 8B), InsP3 completely prevented accumulation of Ca2+ within the InsP3-sensitive pool whether or not GTP was present.


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  		Fig. 7.   Relative effectiveness of the Ca2+ ionophore A23187 and InsP3 in inducing Ca2+ release after Ca2+ sequestration in the presence of palmitate or oxalate. Ca2+ accumulation was examined in the presence of either 100 µM palmitate (A) or 2.65 mM oxalate (B). GTP-dependent Ca2+ uptake continued for 6.5 min, at which time either 10 µM InsP3 or 5 µM A23187 was added. The conditions were as follows: A, palmitate alone (bullet ), palmitate with 20 µM GTP (black-triangle), palmitate with 20 µM GTP followed by 10 µM InsP3 (IP3; black-square), and palmitate with 20 µM GTP followed by 5 µM A23187 (black-diamond ); B, oxalate alone (open circle ), oxalate with 20 µM GTP (triangle ), oxalate with 20 µM GTP followed by 10 µM InsP3 (), and oxalate with 20 µM GTP followed by 5 µM A23187 (diamond ). Results are representative of five identical experiments.


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  		Fig. 8.   Effects of InsP3 and A23187 on prevention of GTP-induced Ca2+ sequestration in the presence of palmitate or oxalate. Ca2+ uptake proceeded in the presence of 100 µM palmitate (A) or 2.65 mM oxalate (B) after the following additions were made at the start of Ca2+ accumulation. A, all conditions contained 100 µM palmitate with either no further addition (bullet ) or with addition of 20 µM GTP (black-triangle), 10 µM InsP3 (IP3; black-down-triangle ), 20 µM GTP and 10 µM InsP3 (black-square), and 5 µM A23187 (black-diamond ). B, all conditions contained 2.65 mM oxalate with either no further addition (open circle ) or with addition of 20 µM GTP (triangle ), 10 µM InsP3 (down-triangle), 20 µM GTP and 10 µM InsP3 (), and 5 µM A23187 (diamond ). This experiment is typical of three identical experiments.

These data indicate that, whereas the operational Ca2+ pools are likely the same, the state of Ca2+ sequestered with the two anions is different. Oxalate is known to form easily observable insoluble Ca2+ precipitates within the lumen of muscle SR vesicles (59, 60) or the ER of permeabilized liver cells (61). Such precipitates can redissolve, albeit slowly, if free luminal Ca2+ decreases, as reflected by the actions of InsP3 and A23187 (Fig. 7B). If the free luminal Ca2+ is prevented from reaching the threshold for precipitation as a result of the action of InsP3 or A23187, the Ca2+-oxalate complex does not form (Fig. 8B). With palmitate, the Ca2+ complex is relatively resistant to InsP3-induced release of Ca2+, but is remarkably sensitive to the action of A23187 (Fig. 7A). We conclude from this result that the Ca2+-palmitate complex is associated intimately with the membrane and as such is able to efficiently exchange Ca2+ with the ionophore, which itself is partitioned within the membrane. In contrast, the far slower release of oxalate-complexed Ca2+ by A23817 (Fig. 7B) reflects no particular advantage of the ionophore and the release of Ca2+ at a rate that reflects dissociation of Ca2+ from oxalate precipitated in the ER lumen. In this experiment, the more complete effect of A23187 as compared with InsP3 likely reflects ionophore-mediated release from both the InsP3-sensitive and -insensitive pools. Whereas the membrane-bound Ca2+-palmitate complex is highly accessible to ionophore, in contrast, this complex appears not to be easily releasable by InsP3. Thus, passage of Ca2+ through the InsP3 receptor channel is likely restricted to free uncomplexed luminal Ca2+. The slow rate of Ca2+ release by InsP3 may reflect the slower dissociation of Ca2+ from the Ca2+-palmitate complex due to a relatively higher affinity of palmitate for Ca2+ as compared with oxalate (the actual affinity of palmitate in the membrane may be enhanced by the presence of other negatively charged membrane lipids). Similarly, as shown in Fig. 8, InsP3 receptors are relatively less efficient in competing with palmitate for Ca2+ being pumped into the lumen of the pool in the presence of GTP (Fig. 8A) as opposed to oxalate (Fig. 8B). Interestingly, the rate of dissociation of Ca2+ from the Ca2+-palmitate complex may be even slower than that indicated in Fig. 7A. Thus, as shown in Fig. 9, in an extension of the experiment shown in Fig. 5, the action of InsP3 was compared on permeabilized cells either maximally accumulating Ca2+ in the presence of palmitate and GTP or after this maximal uptake had been blocked by palmitoyl-CoA. Whereas a small reduction in the rate of Ca2+ accumulation was observed in the former case (consistent with Fig. 7A), after fatty acyl-CoA addition, there was no release with InsP3. In this case, the accumulation of Ca2+ as a result of GTP-activated Ca2+ transfer has been terminated, and the palmitate-complexed Ca2+ is not released by InsP3. In other words, addition of palmitoyl-CoA has effectively isolated the InsP3-sensitive pool, which, although containing palmitate-complexed Ca2+, is not in a state releasable through InsP3 receptors. In contrast, the effect of InsP3 in the absence of fatty acyl-CoA likely reflects the ability of Ca2+ being pumped and transferred into the pool to be competed for and released by InsP3 receptors before it is sequestered with palmitate.


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  		Fig. 9.   Action of InsP3 applied after palmitoyl-CoA-induced reversal of GTP-induced Ca2+ accumulation in the presence of palmitate. Ca2+ accumulation was monitored in the presence of 20 µM GTP together with 100 µM palmitate. At the first arrow, incubations received either 2 µM palmitoyl-CoA (PCoA; bullet ) or no further addition (black-triangle). At the second arrow, incubations were continued either in the presence (IP3; black-square, black-diamond ) or absence (black-triangle, bullet ) of 10 µM InsP3. The experiment was the same as that shown in Fig. 5. Results are representative of three identical experiments.

Concluding Remarks-- A scheme depicting the carboxylic acid-induced sequestration of Ca2+ and the function of the two pools is presented in Fig. 10. The entry of either oxalate or palmitate is shown through a single transport system, the function of which is restricted to the InsP3-sensitive Ca2+ pool. GTP-induced transfer of Ca2+ occurs between this pool and a distinct Ca2+-pumping pool that does not contain either InsP3 receptors or the anion transporter. Palmitate is shown as being present only in the membrane, an inference highly likely considering the avid partitioning of fatty acids into membranes. The binding of two molecules of palmitate to one Ca2+ ion is consistent with the binding stoichiometry mentioned earlier (39). In contrast to palmitate, oxalate is shown as being within the lumen. An important message from this scheme is that, whereas separate pools of Ca2+ do appear to exist in cells, the Ca2+ within a single pool can exist in substates that are very different with respect to releasability by channels. Thus, pools are frequently defined on the relative effectiveness of a given release channel versus the action of an ionophore. On this basis, the palmitate-complexed Ca2+ would be defined as existing in a separate pool, yet every indication is that it is within the same pool as that complexed by oxalate and the same pool as that containing InsP3 receptors. Thus, fatty acids may provide an important sink of membrane-associated Ca2+ distinct from the bulk phase of Ca2+. Such a substate of Ca2+ can be compared with that bound to low affinity, high capacity, calcium-sequestering proteins within the SR or ER lumen (such as calsequestrin and calreticulin) that provide additional buffering capacity at high intraluminal Ca2+ levels; however, with these proteins, the bound Ca2+ is readily releasable through Ca2+ release channels (62). Whether fatty acids sequester significant Ca2+ within pools under physiological conditions is yet to be proven. However, considering that (a) significant quantities of nonesterified fatty acids are present inside cells, (b) fatty acids appear to be selectively transported into specific subcompartments of the ER, and (c) fatty acids are highly partitioned in the lipid bilayer, where they can bind substantial quantities of Ca2+, we conclude that fatty acid-mediated Ca2+ sequestration may be significant in the function of Ca2+ pools. Their role may be to provide a slowly exchangeable sink of Ca2+ in specific subcompartments of the ER. It is also intriguing that the binding of Ca2+ to fatty acids within such membranes may itself confer a signaling role, for example, by modifying the activity of ER membrane channel or pump proteins; thus, the transport of fatty acids into the ER described here may be a means for regulating such control.


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  		Fig. 10.   Scheme depicting the localization and actions of palmitate and oxalate in mediating Ca2+ sequestration within intracellular Ca2+ pools. A DIDS-sensitive anion channel is depicted as permitting entry of oxalate or palmitate into the InsP3-sensitive Ca2+ pool. Ca2+ is pumped into this pool and the InsP3-insensitive pool via the action of thapsigargin (TG)-sensitive SERCA Ca2+ pumps. Palmitate is shown exclusively attached to the inner membrane surface within the Ca2+ pool, where two molecules of palmitate are associated with one Ca2+ ion (39). In contrast, oxalate (shown as a dumbbell structure) reversibly complexes Ca2+ and remains within the lumen of the pool. Although the state and location of Ca2+ complexes are distinct for palmitate and oxalate, both can effectively create a Ca2+ sink, the filling of which is greatly augmented by the GTP-activated translocation process depicted as a junctional complex, allowing transfer of Ca2+ between the InsP3-sensitive and -insensitive pools (7, 16). Details of the scheme are provided under "Results and Discussion."


    ACKNOWLEDGEMENTS

We thank Drs. Tarun Ghosh, Richard Waldron, Carmen Ufret-Vincenty, Martin Schneider, Aristotle Kalivretenos, and Patricia Sokolove for discussions and technical advice.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL55426, National Science Foundation Grant MCB 9307746, and a grant-in-aid from the American Heart Association, Maryland Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Dermatology, University of Rochester Medical Center, Rochester, NY 14642.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Tel.: 410-706-2593/7247; Fax: 410-706-6676.

The abbreviations used are: ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; ICM, intracellular-like medium; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase.
    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

  1. Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve]
  2. Putney, J. W., Jr., and Bird, G. S. (1993) Endocr. Rev. 14, 610-631[CrossRef][Medline] [Order article via Infotrieve]
  3. Clapham, D. E. (1995) Cell 80, 259-268[CrossRef][Medline] [Order article via Infotrieve]
  4. Gill, D. L., Waldron, R. T., Rys-Sikora, K. E., Ufret-Vincenty, C. A., Graber, M. N., Favre, C. J., and Alfonso, A. (1996) Biosci. Rep. 16, 139-157[CrossRef][Medline] [Order article via Infotrieve]
  5. Thomas, A. P., Bird, G. S. J., Hajnóczky, G., Robb-Gaspers, L. D., and Putney, J. W., Jr. (1996) FASEB J. 10, 1505-1517[Abstract]
  6. Rossier, M. F., and Putney, J. W., Jr. (1991) Trends Neurosci. 14, 310-314[CrossRef][Medline] [Order article via Infotrieve]
  7. Gill, D. L., Ghosh, T. K., Bian, J., Short, A. D., Waldron, R. T., and Rybak, S. L. (1992) Adv. Second Messenger Phosphoprotein Res. 26, 265-308[Medline] [Order article via Infotrieve]
  8. Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239[CrossRef][Medline] [Order article via Infotrieve]
  9. Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653-1656[Abstract/Free Full Text]
  10. Bian, J., Ghosh, T. K., Wang, J. C., and Gill, D. L. (1991) J. Biol. Chem. 266, 8801-8806[Abstract/Free Full Text]
  11. Ghosh, T. K., Bian, J., Short, A. D., Rybak, S. L., and Gill, D. L. (1991) J. Biol. Chem. 266, 24690-24697[Abstract/Free Full Text]
  12. Dawson, A. P. (1985) FEBS Lett. 185, 147-150[CrossRef][Medline] [Order article via Infotrieve]
  13. Gill, D. L., Ueda, T., Cheuh, S. H., and Noel, M. W. (1986) Nature 320, 461-464[CrossRef][Medline] [Order article via Infotrieve]
  14. Cheuh, S.-H., and Gill, D. L. (1986) J. Biol. Chem. 261, 13883-13886[Abstract/Free Full Text]
  15. Thomas, A. P. (1988) J. Biol. Chem. 263, 2704-2711[Abstract/Free Full Text]
  16. Gill, D. L., Ghosh, T. K., Short, A. D., and Waldron, R. T. (1993) Handb. Exp. Pharmacol. 108, 625-649
  17. Chueh, S.-H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864[Abstract/Free Full Text]
  18. Mullaney, J. M., Chueh, S.-H., Ghosh, T. K., and Gill, D. L. (1987) J. Biol. Chem. 262, 13865-13872[Abstract/Free Full Text]
  19. Mullaney, J. M., Yu, M., Ghosh, T. K., and Gill, D. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2499-2503[Abstract/Free Full Text]
  20. Hajnoczky, G., Lin, C., and Thomas, A. P. (1994) J. Biol. Chem. 269, 10280-10287[Abstract/Free Full Text]
  21. Millman, J. S., and Andrews, D. W. (1997) Cell 89, 673-676[CrossRef][Medline] [Order article via Infotrieve]
  22. Comerford, J. G., and Dawson, A. P. (1988) Biochem. J. 249, 89-93[Medline] [Order article via Infotrieve]
  23. Comerford, J. G., and Dawson, A. P. (1993) Biochem. J. 289, 561-567
  24. Rys-Sikora, K. E., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 31607-31613[Abstract/Free Full Text]
  25. Ufret-Vincenty, C. A., Short, A. D., Alfonso, A., and Gill, D. L. (1995) J. Biol. Chem. 270, 26790-26793[Abstract/Free Full Text]
  26. Graber, M. N., Alfonso, A., and Gill, D. L. (1996) J. Biol. Chem. 271, 883-888[Abstract/Free Full Text]
  27. Graber, M. N., Alfonso, A., and Gill, D. L. (1997) J. Biol. Chem. 272, 29546-29553[Abstract/Free Full Text]
  28. Ghosh, T. K., Bian, J., and Gill, D. L. (1994) J. Biol. Chem. 269, 22628-22635[Abstract/Free Full Text]
  29. Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961[Abstract/Free Full Text]
  30. Gill, D. L., Chueh, S.-H., and Whitlow, C. L. (1984) J. Biol. Chem. 259, 10807-10813[Abstract/Free Full Text]
  31. Gill, D. L., Mullaney, J. M., and Ghosh, T. K. (1988) J. Exp. Biol. 139, 105-133[Abstract/Free Full Text]
  32. Gill, D. L., Ghosh, T. K., Mullaney, J. M., and Tarazi, F. I. (1989) Cell Calcium 10, 363-374[CrossRef][Medline]