©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ATP-dependent Regulation of Sodium-Calcium Exchange in Chinese Hamster Ovary Cells Transfected with the Bovine Cardiac Sodium-Calcium Exchanger (*)

Madalina Condrescu , Jeffrey P. Gardner , Galina Chernaya , Joseph F. Aceto , Chris Kroupis , John P. Reeves (§)

From the (1) Department of Physiology and Hypertension Research Center, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey 07103 and The Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na]and stimulate Cainflux by Na/Ca exchange. Depletion of cellular ATP inhibited Ca uptake by 40% or more and reduced the half-maximal Naconcentration for inhibition of Ca uptake from 90 to 55 m M. ATP depletion also reduced the rate of rise in [Ca]when [Na]was reduced and inhibited the decline in [Ca]when high [Na]was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na] inhibition profile for Ca uptake. Thus, ATP depletion inhibits both the Cainflux and Caefflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Naand Cawith the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.


INTRODUCTION

The Na/Ca exchanger is a major Caefflux mechanism in cardiac myocytes and several other cell types. Studies conducted with cardiac sarcolemmal membrane patches (1, 2, 3, 4) and squid giant axons (5) have defined two different regulatory processes for the exchanger. These appear to involve two distinct inactive states of the carrier, the first promoted by the presence of cytosolic Na(Na-dependent inactivation) and the second promoted by the absence of cytosolic Ca(secondary Caactivation). The presence of cytosolic ATP attenuates Na-dependent inactivation and increases the affinity of the secondary activation site for cytosolic Ca. ATP also alters the kinetics of the exchange process in a variety of cell types. In dialyzed squid giant axons, the presence of ATP markedly reduces the Kfor cytosolic Caand for extracellular Na(6, 7, 8) . In adult heart myocytes (9, 10) and cultured vascular smooth muscle cells (11) , ATP depletion using metabolic inhibitors reduces Caentry via Na/Ca exchange by more than 80%.

The mechanism by which ATP regulates exchange activity is unknown. DiPolo and Beaugé (8, 12, 13) have suggested that in squid giant axons, the exchanger is regulated by a phosphorylation process, although the kinases/phosphatases involved have not been identified. Studies with cardiac sarcolemmal patches are not consistent with the phosphorylation hypothesis, however, and suggest that exchange activity is indirectly regulated by aminophospholipid translocase activity (2) . This ATP-dependent enzyme maintains a high density of phosphatidylserine at the cytosolic surface of the bilayer and it is thought that the asymmetric distribution of this lipid is essential for maximal exchange activity (2) .

The cardiac Na/Ca exchanger has been cloned by Philipson and his colleagues (14) . The exchanger is a protein of 938 amino acids with 11 putative transmembrane regions and a large hydrophilic domain between the fifth and sixth transmembrane segments. Deletion of 440 out of the 520 amino acids of the hydrophilic domain does not alter the kinetics of the exchange process, but eliminates secondary activation by cytosolic Ca(15) . In this report, we use transfected Chinese hamster ovary cells permanently expressing either the native bovine cardiac exchanger (CK1.4 cells; Ref. 16) or the deletion mutant described above to examine the effects of cellular ATP depletion on Na/Ca exchange activity. ATP depletion strongly inhibits the Caefflux mode of Na/Ca exchange and potentiates the inhibitory effect of extracellular Naon Cainflux. These effects are absent or reduced in cells expressing the deletion mutant. This suggests that ATP-dependent regulation, like secondary activation by cytosolic Ca, is mediated by regions within the hydrophilic domain of the exchanger. Portions of these results were presented previously in abstract form (17, 18) .


EXPERIMENTAL PROCEDURES

Cells

CK1.4 cells were prepared by transfection of dhfrChinese hamster ovary cells with a mammalian expression vector (pcDNA-NEO; Invitrogen) containing a cDNA insert coding for the bovine cardiac Na/Ca exchanger (16) . CK138 cells were prepared similarly except that the nucleotides coding for amino acids 241-681 were deleted from the insert by cleavage with BclI at positions 1079 and 2399 and religation; the BclI site at position 1079 was introduced in clone p17 (19) by site-directed mutagenesis using the method of Kunkel (20) , provided commercially as the Bio-Rad Muta Gene 2 kit. Expression of the expected mutation in CK138 cells was verified by RNA extraction and reverse transcriptase-polymerase chain reaction using a commercial kit (Invitrogen cDNA Cycle and TA Cloning kits). The properties of the canine exchanger with an identical deletion were previously described (15) . The cells were grown in Iscove's modified Dulbecco's medium containing 10% fetal calf serum and G418 (16) . In some of the immunoprecipitation experiments, COS cells transiently transfected with the cardiac Na/Ca exchanger were used as described (19) .

Ca Uptake Assay

The cells were grown to confluence in 24-well plastic dishes coated with fibronectin; the plates were preincubated overnight with culture medium containing 17 µg/ml fibronectin prior to adding the cells. The fibronectin coating was required to prevent ATP-depleted cells from detaching from the plate during the transport assay. The medium was removed and the cells were preincubated for 30 min at 37 °C in nominally Ca-free PSS() containing (in m M) 140 NaCl, 5 KCl, 1 MgCl, and 20 m M Mops, buffered to pH 7.4 (37 °C) with Tris (Mops/Tris); 0.4 m M ouabain was also added to inhibit the Na,K-ATPase and load the cells internally with Na( cf. Ref. 16) and 10 m M glucose was either included or omitted as specified in individual experiments. To assay Ca uptake, the preincubation medium was removed by aspiration and replaced with 200 µl of assay medium (see below) containing 10 µCi/ml CaCl. After the desired interval, Ca uptake was terminated by adding 1 ml of quenching medium (room temperature) containing (in m M) 100 MgCl, 10 LaCl, and 10 Mops/Tris, pH 7.4. The quenching medium was immediately aspirated and the wells were washed 3 additional times by the repeated addition and aspiration of quenching medium (1 ml). The quenching and washing procedure was completed within 7-8 s. The normal assay medium contained (in m M) 5 KCl, 1 CaCl, 20 Mops/Tris (pH 7.4) and the amounts of NaCl and NMDG (adjusted to pH 7.4 with HCl) described in individual experiments; 10 m M glucose was included where specified. For assays lasting longer than 1 min, 0.4 m M ouabain was also included in the assay medium. Occasional variations in this protocol are described in the text. Data are presented as the mean values (± S.E.) for the number of individual experiments given in the figure legends.

ATP Depletion

CK1.4 cells were preincubated in Ca-free PSS containing 0.4 m M ouabain for 30 min in either the presence (control) or absence of 10 m M glucose. The medium was replaced with fresh medium containing oligomycin (2.5 µg/ml) or oligomycin plus 2 µ M rotenone and the incubation continued for an additional 10-30 min. Cellular ATP levels were assayed by the firefly luciferase method (Calbiochem kit).

Fura-2 Measurements

Cells were grown to confluence on coverslips and then preincubated for 30 min in PSS containing 1 m M CaCl, 5 µ M fura-2-AM, 0.125 m M sulfinpyrazone (to reduce transport of fura-2 out of the cell; Ref. 16), with or without 0.4 m M ouabain as indicated. For ATP-depleted cells, glucose was omitted from the medium and 2.5 µg/ml oligomycin was included; for control cells, oligomycin was omitted and 10 m M glucose was included. The coverslips were mounted in a quartz cuvette and superfused with PSS containing 1 m M CaClwith or without ouabain, glucose, and/or oligomycin as indicated. To initiate Cainflux by Na/Ca exchange, the perfusion medium was changed to one with the same composition except that [Na] was reduced to 40 m M (K substitution). Fluorescence was monitored at 505 nm on a SPEX Fluorolog CM3 spectrofluorimeter at excitation wavelengths of 340 and 380 nm. All fluorescence values were corrected for autofluorescence. Data are presented as the ratio of fluorescence at 340/380 nm excitation.

BCECF Measurements

Cells were grown to confluence on coverslips and then preincubated for 30 min in PSS containing 1 m M CaCland 5 µ M BCECF-AM. Control cells and ATP-depleted cells were treated as described under ``Fura-2 Measurements.'' Coverslips were washed three times with PSS, mounted in the quartz cuvette, and superfused with PSS containing 1 m M CaCland glucose or oligomycin as indicated. Changes in pHwere produced by including 20 m M NHCl in both the low Na-PSS and the 140 m M Na-PSS added subsequently. Fluorescence was monitored at 530 nm with excitation wavelengths of 440 and 503 nm. Cytosolic pH was calculated from the 503/440 ratio and a calibration curve (21) established in separate coverslips subjected to 5 µg/ml nigericin in potassium-substituted (145 m M) PSS (pH6.4-7.8).

Immunoprecipitation

CK1.4 cells or transfected COS cells in a 60-mm plate were incubated 1 h (37 °C) in 1.5 ml of phosphate-free Dulbecco's modified Eagle's medium; the medium was replaced with 1.5 ml of fresh medium and [P]orthophosphate (1 mCi/ml, 8-9 Ci/µmol) and incubated for 4 h at 37 °C. The cells were then washed in cold buffer containing (in m M) 150 NaCl, 20 sodium phosphate (pH 7.0), 50 NaF, 10 sodium pyrophosphate, 5 EDTA, 5 EGTA, 1 each ortho- and meta-vanadate, 0.2 phenylmethylsulfonyl fluoride, 1 benzamidine, 0.1% bovine serum albumin, and 5 µg/ml each aprotinin, leupeptin, and pepstatin. The cells were lysed with 2% Triton X-100 and 0.5% sodium deoxycholate in the above buffer and sonicated on ice (3 15 s). After centrifugation for 15 min in an Eppendorf centrifuge, the supernatant was precleared with 1 µl of preimmune serum + 50 µl of 50% protein A-agarose previously equilibrated in lysis buffer. After 1 h at 4 °C, the mixture was centrifuged; 3 µl of immune serum was added to the supernatant and the mixture was incubated 4 h or overnight at 4 °C. After centrifugation, 50 µl of 50% protein A-agarose was added to the supernatant and the mixture was incubated 1 h (4 °C) and centrifuged. The protein A-agarose was washed extensively with ice-cold lysis buffer, resuspended in 30 µl of sample buffer, and applied to one lane of a gel for SDS-polyacrylamide gel electrophoresis. Transfection of COS cells and S labeling/immunoprecipitation were carried out as described previously (19) . The antibody used for immunoprecipitation was prepared against a fusion protein containing the NH-terminal portion of the exchanger (19) .

Cytochalasin D Treatment

Cells were incubated in Ca-free PSS plus 0.4 m M ouabain with or without 1 µ M cytochalasin D and then assayed for Ca uptake as described above. In some experiments, the cells were preincubated for an additional 30 min with 1 µ M cytochalasin D added directly to the culture media prior to the 30 min preincubation period; no difference was observed between the effects of the different protocols.

Fluorescein Isothiocyanate-Phalloidin Staining

Cells were grown to 20-40% confluence on Aclar plastic coverslips (Proplastics) and were subjected to cytochalasin D treatment or ATP depletion. The cells were then washed 3 times with phosphate-buffered saline (PBS) followed by fixation in 4% paraformaldehyde solution in PBS for 10 min. Coverslips were washed 3 more times with PBS and extracted with acetone at -20 °C for 4 min. Samples were air-dried and 1.4 units of fluorescein isothiocyanate-labeled phalloidin (Molecular Probes) previously dissolved in 180 µl of PBS were placed on each coverslip for 30 min at room temperature. Cells were then washed 3 times with PBS and the coverslips were mounted on slides using the SlowFade antifade reagent in glycerol/PBS (Molecular Probes).

Materials

Rotenone and cytochalasin D were obtained from Sigma. The oligomycin was a mixture of oligomycins A, B, and C (65% A) and was obtained from Sigma (catalog No. O4876). Fura-2, BCECF, and SlowFade were obtained from Molecular Probes.


RESULTS

ATP Depletion and Ca Uptake in CK1.4 Cells

To determine the effects of ATP depletion on Na/Ca exchange activity, CK1.4 cells were preincubated for 30 min in the absence of glucose and then treated with a combination of mitochondrial inhibitors: rotenone (2 µ M) and oligomycin (2.5 µg/ml). This treatment reduced cellular ATP levels by 95% within 10 min of adding the inhibitors (data not shown). The inhibitors were much less effective in the presence of 10 m M glucose; under these conditions, ATP levels declined by 30% within the first 2 min of incubation and remained at this level for the duration of the incubation period. The effects of these inhibitors (with and without glucose present) on Ca uptake are shown in Fig. 1. In these experiments, the cells were loaded internally with Naby including 0.4 m M ouabain in the preincubation and assay media; Ca uptake was assayed in either 150 m M NMDG ( circles) or 150 m M NaCl ( squares). Under these conditions, Ca uptake reflects the activity of the Na/Ca exchanger operating in the ``reverse,'' or Cainflux, mode (16) . As shown, 10 min of ATP depletion ( open symbols, Fig. 1 ) reduced the rate of Ca uptake in the NMDG medium by approximately 30% ( left panel); a more pronounced inhibition was observed after 30 min of ATP depletion ( right panel). Despite the reduced initial Ca uptake, the total Ca accumulation during the later portions of the time course was higher in ATP-depleted than in control cells. In the presence of 150 m M extracellular Na, total Ca accumulation was reduced compared to the NMDG medium and ATP depletion reduced uptake still further. The inhibition of Ca uptake by extracellular Nais characteristic of Na/Ca exchange activity and reflects the competition between Naand Cafor transport sites on the exchange carrier (22, 23) .


Figure 1: Effect of ATP depletion on Ca uptake by CK1.4 cells. Left panel, ouabain-treated CK1.4 cells were depleted of ATP for 10 min using oligomycin and rotenone as described under ``Experimental Procedures.'' For the control cells, 10 m M glucose was present during the exposure to oligomycin and rotenone. Ca uptake ( n = 3) was assayed in either 150 m M NMDG ( circles) or 150 m M NaCl ( squares) containing 1 m MCaCl, 5 m M KCl, and 20 m M Mops buffered to pH 7.4 (37 °C) with Tris. The mitochondrial inhibitors (±10 m M glucose) were included in the assay media. Right panel, CK1.4 cells were depleted of ATP for 30 min as described under ``Experimental Procedures.'' Ca was assayed as described above ( n = 3).



To examine the effects of extracellular Nain more detail, we measured the rates of Ca uptake (15 s) at various Naconcentrations after 10 min of ATP depletion. The results, shown in Fig. 2 A, indicate that Nainhibited Cauptake more effectively in ATP-depleted cells than in control cells. The data in panel A are plotted as the percentage of the rate seen in the absence of Naand represent the mean (± S.E.) of 10-11 independent experiments. The ICfor inhibition by Nawas 90 m M in ATP-replete cells versus 57 m M for ATP-depleted cells; the Hill coefficients for the Naconcentration profiles were similar for the ATP-replete (2.4) and ATP-depleted (2.0) cells. The shift in the Nainhibition curve was not simply a consequence of the lower rate of Ca uptake in the ATP-depleted cells. Ca uptake could be inhibited to a similar extent by treating the cells with 1 m M N-ethylmaleimide, or by including 1 m M CaClin the preincubation media (24) , without producing a shift in the Nainhibition curve (data not shown). The effects of ATP depletion were fully reversed within 10 min of adding 10 m M glucose to the ATP-depleted cells; cellular ATP levels were also restored under these conditions, although only to 40% of initial values (data not shown).

We also examined the effects of ATP depletion on the Caconcentration profile for Ca uptake. As shown in Fig. 2 B, control cells exhibited a typical Michaelis-Menten type of behavior, with a Vof 7.6 nmol/mg protein/15 s and an apparent Kof 0.19 m M, as determined from a Lineweaver-Burk plot (not shown). For the ATP-depleted cells, the rates of Ca uptake appeared to saturate quite sharply at concentrations above 0.4 m M Cawith a half-maximal [Ca] of approximately 0.1 m M. It is very unlikely that these rates of Ca uptake represent true initial rates of Na/Ca exchange (see ``Discussion'') and so it is difficult to interpret these data in strict kinetic terms. It is important to note, however, that ATP depletion was not associated with a shift in the Caconcentration profile to higher concentrations, and so the increased effectiveness of Naas an inhibitor of Ca uptake does not appear to be due to a reduced affinity of the exchanger for Ca.

ATP Depletion and Ca Uptake in CK138 Cells

A large (520 amino acid) hydrophilic domain lies between the fifth and sixth putative transmembrane regions of the Na/Ca exchange protein and appears to reside on the cytoplasmic side of the membrane (14) . The results of Matsuoka et al. (15) indicated that 440 of the 520 amino acids in the hydrophilic domain could be deleted without obvious changes in the kinetic characteristics of exchange activity. The deletion altered the regulatory behavior of the exchanger, however, since secondary activation of Na-dependent Cainflux by [Ca]was not observed (15) . To determine whether ATP-dependent regulation of exchange activity was also altered in this mutant, we prepared CHO cells that express the deleted form of the exchanger (see ``Experimental Procedures''), which we designate CK138 cells.

In contrast to the behavior of wild-type cells, 10 min of ATP depletion did not affect the concentration profile for inhibition of Ca uptake by Na(Fig. 3 A). The characteristics of the mutant cells were similar to those of ATP-depleted wild-type cells (IC= 60 m M). The Caconcentration profile (Fig. 3 B) for the CK138 cells was also similar in ATP-replete versus ATP-depleted CK138 cells; half-maximal Cauptake occurred at 0.1 m M Caunder both conditions, again similar to the Caconcentration dependence of ATP-depleted wild-type cells (compare Fig. 2B). Note that the rates of Ca uptake by the CK138 cells were uniformly lower than for the CK1.4 cells and tended to be less affected by ATP depletion (compare Fig. 2 B and Fig. 3 B; see legends for Figs. 2 and 3 for additional data). [Ca]Measurements-With Ca uptake measurements, it is difficult to distinguish an effect on the exchanger itself from secondary effects due to loss of Casequestration by intracellular organelles. Therefore, we examined the effects of ATP-depletion on exchanger-mediated changes of [Ca]in fura-2-loaded cells (Fig. 4); in these experiments, an increase in the ratio of fluorescence with excitation at 340 versus 380 nm reflects an increase in [Ca]. ATP depletion was carried out using CK1.4 cells ( upper panel) or CK138 cells ( lower panel) grown on glass coverslips; the cells were preincubated in glucose-free PSS for 30 min and then loaded with fura-2 for an additional 30 min in glucose-free PSS containing 2.5 µg/ml oligomycin with or without 0.4 m M ouabain as indicated. For the control cells, 10 m M glucose was present throughout and oligomycin was omitted. In the presence of glucose, a reduction in extracellular [Na] to 40 m M (potassium substitution) produced a rise in in the 340/380 ratio which was faster and more extensive in ouabain-treated ( trace 3) than in untreated ( trace 4) cells. Restoration of 140 m M Naproduced a rapid decline in the 340/380 ratio in both batches of cells. The results with the CK1.4 cells ( upper panel) are similar to those reported previously (16) ; the CK138 cells ( lower panel) behaved nearly identically to the CK1.4 cells.


Figure 3: Effect of ATP depletion on Na-dependent inhibition ( A) and [Ca] dependence ( B) of Ca uptake in CK138 cells. Ouabain-treated CK138 cells were incubated with oligomycin and rotenone for 10 min, with ( closed circles) or without ( open circles) 10 m M glucose, as described under ``Experimental Procedures.'' For the data in panel A, the rate of Ca uptake in the NMDG media were 2.5 ± 0.2 and 2.1 ± 0.2 nmol/mg protein/s with and without ATP, respectively. For panel A, n = 5, and for panel B, n = 3.




Figure 2: [Na] and [Ca] dependence of Ca uptake in ATP-depleted versus control CK1.4 cells. Left panel, inhibition by Naof Ca uptake. Ouabain-treated CK1.4 cells were incubated with oligomycin and rotenone for 10 min in the presence ( closed circles) or absence ( open circles) of glucose as described under ``Experimental Procedures.'' Ca uptake (15 s) was assayed as described under ``Experimental Procedures'' in mixtures of 150 m M NMDG and 150 m M NaCl to generate the Naconcentrations shown ( n = 10-11). The data are presented as the % of Cauptake observed in the Na-free medium. The portion of Cauptake occurring independently of exchange activity under these conditions cannot be reliably determined and so no correction for this has been applied. The rates of Ca uptake in the NMDG media were 7.2 ± 0.5 and 4.2 ± 0.4 nmol/mg protein/s with and without ATP, respectively. Right panel, [Ca] dependence of Na/Ca exchange in ATP-depleted versus control cells. Ouabain-treated CK1.4 cells were treated with oligomycin and rotenone for 10 min, with ( closed circles) or without ( open circles) 10 m M glucose present as described under ``Experimental Procedures.'' The cells were assayed for Ca uptake (15 s) in 150 m M NMDG, 5 m M KCl, 20 m M Mops/Tris (pH 7.4) at the indicated concentrations of CaCl( n = 6).



For both the wild-type and mutant cells, ATP depletion ( traces 1 and 2) slowed the rise in the 340/380 ratio when [Na]was reduced to 40 m M. However, the 340/380 ratio did not reach a plateau, as observed in control cells, but continued to rise throughout the duration of the period in low [Na]. This probably reflects the loss of Capump activity of both intracellular organelles and the plasma membrane under the ATP-depleted conditions. Another difference between ATP-depleted and control cells was that ouabain treatment had no effect on the response of the ATP-depleted cells. Presumably, ATP depletion itself inhibits Na,K-ATPase activity sufficiently that any additional effects of ouabain are negligible.

When [Na]was restored from 40 to 140 m M in the ATP-depleted CK1.4 cells, only a slight decline in the 340/380 ratio was evident. With ATP-depleted CK138 cells, a more pronounced reduction in the 340/380 ratio was observed, but the decline was still considerably slower than observed in the presence of glucose. A large part of this effect probably reflects the loss of intracellular Casequestration and/or plasma membrane Ca-ATPase activity due to lack of ATP. However, since the decline in [Ca]does not appear to be as severely compromised in the CK138 cells, the results suggest that the exchanger continues to catalyze Caefflux in the CK138 cells but is inhibited by ATP depletion in the CK1.4 cells.

ATP Depletion and pH

Since acidic conditions inhibit Na/Ca exchange (25) , we examined the effects of ATP depletion on pHusing BCECF fluorescence as an intracellular pH indicator. After 30 min of ATP depletion with 2.5 µg/ml oligomycin under glucose-free conditions, pHdeclined from 7.46 ± 0.05 to 6.85 ± 0.1 (mean ± S.E.; n = 4). To determine whether the effects of ATP depletion could be explained by the inhibitory effects of cytosolic acidification, we assayed Ca uptake at various [Na]during cytosolic acidification induced by the NHCl prepulse technique (26) . Cells that were preloaded with 20 m M NHCl and assayed in NH-free media exhibited a reduction in pHof 0.3 pH units; this treatment reduced the rate of Ca uptake by 20% but had no effect on the [Na]inhibition profile (data not shown).

In a second series of experiments, we alkalinized ATP-depleted cells with 20 m M NHCl during Na/Ca exchange; the results are shown in Fig. 5. These experiments are similar to those depicted in Fig. 4, except that all the traces are for ouabain-treated cells and for some of the traces (depicted in bold), 20 m M NHCl was included in the 40 m M Namedium and in the 140 m M Namedium added subsequently. In ATP-replete cells, the NHCl treatment increased pHto a peak value of 7.82 ± 0.02 ( n = 3), followed by a gradual decline in pHby less than 0.1 unit over the subsequent 8 min (data not shown). In ATP-depleted cells, the NHCl treatment raised pHto a stable value of 7.32 ± 0.09 ( n = 4), close to that observed in ATP-replete cells in the absence of NHCl. The changes in [Na]during these experiments did not evoke any detectable changes in pH.


Figure 4: Na/Ca exchange activity and [Ca] in ATP-depleted and control CK1.4 and CK138 cells. Upper panel, CK1.4 cells were grown on coverslips, and loaded with fura-2 in the presence ( traces 1 and 2) or absence ( traces 3 and 4) of 2.5 µg/ml oligomycin, with ( traces 1 and 3) or without ( traces 2 and 4) 0.4 m M ouabain, as described under ``Experimental Procedures.'' The data depict the ratio of the fluorescence obtained by excitation at 340 and 380 nm as described under ``Experimental Procedures.'' At the times indicated by the lower bar, the perfusion medium was changed from 140 m M Na-PSS to PSS containing 40 m M Na/100 m M KCl and back to 140 m M Na-PSS. Each trace is the mean of eight to nine individual experiments. Lower panel, the procedure was identical to that described for the upper panel except that CK138 cells were used. Traces are the mean values for three to five experiments.



As shown in Fig. 5( upper panel), cytosolic alkalinization ( traces 1 and 4) accelerated the increase in the 340/380 ratio upon [Na]reduction in both ATP-depleted and ATP-replete cells, but had little effect on the rate of decline in [Ca]when [Na]was restored to 140 m M; after an initial small decline, [Ca]remained elevated in ATP-depleted cells after restoration of 140 m M Na, whether or not NHCl was present. A similar pattern of results was observed in the case of CK138 cells expressing the deleted form of the exchanger ( lower panel, Fig. 5); again, a less pronounced effect of ATP depletion on Na-dependent Caefflux was observed in the CK138 cells compared to the CK1.4 cells. Thus, cytosolic alkalinization accelerates the increase in [Ca]resulting from exchange-mediated Caentry in ATP-depleted cells but does not overcome the inhibitory effects of ATP depletion on Caefflux.


Figure 5: Effect of cytosolic alkalinization on Na/Ca exchange and [Ca] in ATP-depleted and control CK1.4 cells ( upper panel) and CK138 cells ( lower panel). The procedure was identical to that described in Fig. 4 except that all cells were ouabain-treated and that, for traces 1 and 4 (indicated in bold), 20 m M NHCl was included in the 40 m M Naperfusion medium and in the 140 m M Namedium added subsequently.



Although the results in Fig. 5 seem to imply that the Cainflux mode of the exchanger recovered fully from the effects of ATP depletion upon cytosolic alkalinization, this is not necessarily the case. The rise in [Ca]during exchange-mediated Caentry is likely to be buffered by the Casequestering activities of intracellular organelles under ATP-replete conditions, but not, or much less so, in ATP-depleted cells. Thus, the loss of intracellular Casequestration probably understates the actual inhibition of exchange activity in the fura-2 experiments, and may exaggerate the loss of exchange activity in the Ca experiments. In this regard, it should be noted that the presence of NHCl during Ca uptake assays after 30 min of ATP depletion did not restore the rate of Ca influx to the levels seen with ATP-replete cells (data not shown).

Phosphorylation of the Exchanger?

The hydrophilic cytoplasmic domain of the exchanger contains several possible sites for phosphorylation by different protein kinases. Indeed, a bacterially expressed protein containing the entire hydrophilic domain fused to the Escherichia coli maltose binding protein was a substrate for in vitro phosphorylation by either protein kinase A or casein kinase II; the maltose binding protein itself was not phosphorylated under these conditions (data not shown). To determine whether the exchanger expressed in CK1.4 cells is phosphorylated, we incubated the cells with [P]phosphate to label the intracellular nucleotide pool. As shown in Fig. 6, we were unable to detect labeled exchanger upon immunoprecipitation of the exchanger from P-labeled CK1.4 cells ( left panel) although several P-labeled contaminating proteins could be detected and S-labeled exchanger was easily observed ( right panel). As described previously (16) , the expressed exchanger appeared as 2 bands at 150 and 120 kDa when immunoprecipitated from CK1.4 cells. Additional experiments (data not shown) were carried out with transfected COS cells, which express higher levels of S-labeled exchanger than the CHO cells; nevertheless, we were still unable to detect P labeling of the exchanger in the transfected COS cells (data not shown). The results suggest that the exchanger is not phosphorylated in either CK1.4 cells or COS cells. However, we cannot completely rule out the possibility that the exchanger is dephosphorylated during our sample preparation procedures (despite the presence of fluoride, pyrophosphate, and vanadate to inhibit phosphatases), and so this conclusion must remain tentative.

We also examined the effects of various agents which alter protein phosphorylation. No effects on exchange activity were observed after 30 min exposure of CK1.4 cells to a nonspecific protein kinase inhibitor (staurosporine, 1 µ M), or to calphostin C (1 µ M), a relatively specific inhibitor of protein kinase C (data not shown). Moreover, the protein phosphatase inhibitors okadaic acid (1 µ M) and calyculin (50 n M) had no affect on the decline in exchange activity, or the alterations in Na-dependent inhibition, when CK1.4 cells were depleted of ATP (data not shown). Finally, the recovery of exchange activity when 10 m M glucose was restored to CK1.4 cells after 30 min of ATP-depletion was not affected by the presence of staurosporine (1 µ M) (data not shown).

Effects of ATP Depletion and Cytochalasin D on the Actin Cytoskeleton

ATP depletion produces extensive alterations in the cellular cytoskeleton in a number of cells (27, 28, 29) . To assess the effects of ATP depletion on the actin cytoskeleton in the CK1.4 cells, they were stained with fluorescein isothiocyanate-labeled phalloidin, a fluorescent agent that interacts with polymerized actin (30) . As shown in Fig. 7, panel A, the cells exhibited a network of actin filaments extending throughout the cytoplasm; stress fibers and local concentrations of fibrous actin in cortical regions of the cell were clearly apparent. After 10 min of ATP depletion (see ``Experimental Procedures''), the filamentous network seemed less sharply defined and punctate aggregates of actin appeared throughout the cell (Fig. 7, panel B). After 30 min of ATP depletion (Fig. 7, panel C), much of the actin had aggregated to a diffuse region in the center of the cell. The effects of 30 min of ATP depletion were fully reversible within 15 min of replacing the medium with fresh PSS containing 10 m M glucose (data not shown). The results indicate that ATP depletion produced a profound reorganization of actin microfilaments in these cells.


Figure 7: Effect of ATP depletion on actin cytoskeleton. CK1.4 cells were grown on plastic coverslips and stained with fluorescein isothiocyanate-palloidin as described under ``Experimental Procedures.'' The treatments shown are: A, control cells treated with oligomycin + rotenone in the presence of 10 m M glucose; B, cells treated for 10 min with oligomycin + rotenone in the absence of glucose (10 min ATP depletion); C, cells after 30 min of ATP depletion; and D, cells after 1 h exposure to 1 µ M cytochalasin D. Magnification is approximately 500. We thank Dr. John Connor, Roche Institute of Molecular Biology, for use of the microscope.



CK1.4 cells were treated for 1 h with 1 µ M cytochalasin D, an agent that binds to the barbed end of actin microfilaments and alters their state of polymerization in intact cells (30) . As shown in Fig. 7, panel D, the microfilament network was no longer visible in cytochalasin D-treated cells and most of the actin appeared to reside in intensely stained aggregates that were dispersed throughout the cell. Thus, cytochalasin D also promoted redistribution of cellular actin, although the effects were not precisely identical to those produced by ATP depletion.

Effect of Cytochalasin D Treatment on Na/Ca Exchange Activity

The data in Fig. 8 A indicate that the effects of ATP depletion on the Nainhibition profile could be mimicked by treating cells with 1 µ M cytochalasin D. As shown, the cytochalasin D treatment reduced the ICfor extracellular Nafrom 85 m M to approximately 50 m M, similar to the effects of ATP depletion in the CK1.4 cells (compare with Fig. 2A). As shown in panel B of Fig. 8, cytochalasin D treatment reduced the rate of Ca uptake at all Caconcentrations tested. This experiment was carried out with a different clone of transfected CHO cells (C16-3 cells) instead of CK1.4 cells; comparable effects of cytochalasin D were observed with the CK1.4 cells.


Figure 8: Effect of cytochalasin D on Na/Ca exchange in C16-3 cells. A, cells were preincubated with ( closed circles) or without ( open circles) 1 µ M cytochalasin D in Ca-free PSS + 0.4 m M ouabain as described under ``Experimental Procedures.'' The cells were then assayed for the rate (15 s) of Ca uptake at various Naconcentrations ( n = 5). B, cells were preincubated with 1 µ M cytochalasin D (see ``Experimental Procedures'') and assayed for the initial rate of Ca uptake at the indicated Caconcentrations ( n = 5).



The effects of cytochalasin D required a minimum of 30 min to develop, and were manifest at concentrations as low as 50 n M. Cytochalasin D did not alter cellular ATP levels or pH(data not shown). Moreover, agents that affect microtubule organization (10 µ M nocodazole or colchicine) had no effect on Na/Ca exchange activity in these cells although staining with anti--tubulin antibodies revealed clear evidence of microtubular disruption. The results suggest that the change in the concentration profile for inhibition of Cauptake by Nais related to the breakdown of the actin cytoskeleton. As in the case of ATP depletion, the effects of cytochalasin D on the Nainhibition profile were not observed in cells expressing the deletion mutant of the Na/Ca exchanger (CK138 cells; data not shown).

Additional experiments were carried out with cytochalasin D at a Caconcentration of 0.2 m M instead of 1.0 m M. As shown in Fig. 9, left panel, the Na-inhibition curve for CK1.4 cells was again displaced to lower concentrations in cytochalasin D-treated cells. At the lower Caconcentration, half-maximal inhibition occurred at approximately 40 m M Nain control cells versus 27 m M Nain the cytochalasin D-treated cells. These concentrations are about half of those observed at 1 m M Ca, reflecting the increased effectiveness of Naas a competitor of Cainflux at the lower Caconcentrations. As shown in the right panel of Fig. 9, cytochalasin D had no significant effect on the Na-inhibition curve for the CK138 cells, which exhibited half-maximal inhibition at approximately 28 m M Na, i.e. the same as for the cytochalasin D-treated CK1.4 cells. The results are consistent with those obtained at higher Caconcentrations (Fig. 8), and illustrate the competitive nature of the inhibitory effect of Naon Cauptake.


Figure 9: Effect of cytochalasin D on Ca uptake in CK1.4 and CK138 cells at 0.2 m M Ca. The experiment was conducted as described in the legend to Fig. 8, except that that Caconcentration in the assay media was 0.2 m M rather than 1.0 m M. Results are the means of five experiments for the CK1.4 cells and six experiments for the CK138 cells. The rate of Ca uptake (15 s) in the absence of Na, with and without cytochalasin D treatment, were 3.1 ± 0.1 and 4.2 ± 0.2 nmol/mg protein for the CK1.4 cells, and 0.55 ± 0.04 and 0.75 ± 0.06 nmol/mg protein for the CK138 cells.



Surprisingly, cytochalasin D treatment had no discernible effect on the changes in [Ca]induced by Naremoval and restoration in ouabain-treated CK1.4 cells (Fig. 10). Thus, despite the inhibition of Ca uptake by cytochalasin D, the rate of rise in the 340/380 ratio upon [Na]reduction was essentially identical with and without cytochalasin D. More importantly, disruption of the actin cytoskeleton did not inhibit the decline in [Ca]upon restoration of Na, as was observed in ATP-depleted cells. We speculate that the Ca uptake experiments are more sensitive to changes in Casequestration than the [Ca]experiments, and that changes in the actin cytoskeleton may alter the intracellular distribution of Casequestering organelles. This might partially explain why exchange-mediated Ca uptake is inhibited by cytochalasin D but the increase in [Ca]is not. The decline in [Ca]when high Nais restored in the fura-2 experiments (Fig. 10) reflects the combined effects of several different processes, including decreased Caentry by Na/Ca exchange, Caefflux by the plasma membrane Ca-ATPase as well as the Na/Ca exchanger, and Casequestration by intracellular organelles. Thus, the absence of an effect does not necessarily imply that Na-dependent Caefflux via the exchanger was unaffected by cytochalasin D, since intracellular Casequestration and Caefflux via the plasma membrane CaATPase could make any effects on Na-dependent Caefflux difficult to detect.


Figure 10: Na/Ca exchange activity and [Ca] in cytochalasin D-treated cells. CK1.4 cells were grown on coverslips and incubated for 30 min in PSS containing 0.2 m M Ca, 0.4 m M ouabain, 5 µ M fura-2-AM, and 0.25 m M sulfinpyrazone in the presence ( trace 2) or absence ( trace 1) of 1 µ M cytochalasin D. Coverslips were placed in the cuvette and perfused with 140 Na-PSS + 0.2 m M Ca; at the times indicated by the lower bar, the perfusion medium was changed to PSS containing 40 m M Na+ 100 m M KCl and then back to 140 m M Na-PSS. The traces shown are the average of four individual traces. Exchange activity had increased markedly in the CK1.4 cells used for these experiments in comparison to those used in Fig. 4, probably due to the use of a different batch of fetal calf serum in culturing the cells. In order to obtain results that were comparable to those in Fig. 4, [Ca] was reduced from 1 m M to 0.2 m M for these experiments.




DISCUSSION

Previous reports have established that Na/Ca exchange activity is regulated by ATP in various cell types (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . The results presented here indicate that ATP depletion reduces the rate of [Na]-dependent Cainflux mediated by the exchanger and shifts the concentration profile for inhibition of Cauptake to lower Naconcentrations. The increased sensitivity of the ``reverse mode'' of the exchanger to inhibition by Nawas an unexpected finding since studies with dialyzed squid axons have shown that the concentration of Narequired to activate Caefflux by Na/Ca exchange increases in the absence of ATP (7, 8) . The increased Nainhibition, combined with the reduced turnover of the exchanger, may be a protective mechanism to prevent excessive Caentry via the exchanger during periods of ATP depletion ( e.g. during cardiac ischemia). The effectiveness of high [Na]in this regard can be inferred from the fura-2 results in Figs. 4 and 5. Despite the absence of cellular ATP and the loss of Caefflux mechanisms, [Ca]does not substantially increase in ATP-depleted cells until [Na]is reduced from 140 to 40 m M.

The rates of Ca uptake in these experiments reflect Na/Ca exchange activity but should not be taken as true initial rate measurements. The accumulated Carepresents a large increase in cellular Cacontent and the uptake process therefore involves intracellular Casequestration and other Cabuffering processes as well as Na/Ca exchange. Thus, with an intracellular water volume of 6 µl/mg protein (16) , the amount of Caaccumulated in 15 s by ATP-replete cells (7.5 nmol/mg protein; Fig. 2) corresponds to >1 mmol/liter cell water. The increase in [Ca]under these conditions is in the micromolar or submicromolar range (16) , indicating that nearly all of the Caentering the cell is bound by intracellular Cabuffers or sequestered by internal organelles. Therefore, it is important to determine whether the effects of ATP depletion reflect changes in Na/Ca exchange activity or changes in intracellular Cahandling.

We attempted to minimize the effects of organellar Casequestration in the Ca experiments by blocking mitochondrial Cauptake with oligomycin + rotenone for both control (glucose present) and ATP-depleted cells. In other experiments, which will be described in a separate publication, we found that thapsigargin, an agent which inhibits SERCA-type ATPases and blocks Casequestration by the endoplasmic reticulum, did not inhibit exchange-mediated Ca uptake in the CK1.4 cells (31) and did not displace the Na-inhibition curve. Thus, the effects of ATP depletion on Ca uptake cannot be explained solely by the loss of Casequestration by mitochondria and/or the endoplasmic reticulum. Furthermore, cells expressing the deletion mutant of the exchanger (CK138 cells) exhibited an altered response to ATP depletion compared to the CK1.4 cells; this indicates that some of the effects of ATP depletion on Ca uptake involve the exchanger itself, since effects on organellar Casequestration ought to be the same in both cell types ( cf. below). Despite these considerations, we do not claim that organellar Casequestration has been eliminated in our studies or that it plays no role in the observed responses to ATP depletion. Indeed, the loss of intracellular Casequestration with ATP depletion probably exaggerates the apparent decline in exchange activity in the Ca experiments, while understating the actual loss of exchange activity in the fura-2 experiments. Thus, after 30 min of ATP depletion, the rate of Ca uptake was profoundly inhibited (Fig. 1 B), while the fura-2 results (Fig. 4) suggested that the rise in [Ca]due to exchange activity was inhibited by only 50%.

A second factor that must be considered is an indirect modulation of exchange activity through effects of ATP depletion on pH. Thus, pHdecreased by 0.6 units after 30 min of ATP depletion and this could significantly inhibit Na/Ca exchange activity (25) , alter intracellular Cabuffering, and/or inhibit organellar Casequestration. The experiments with NH-induced acidification and alkalinization (Fig. 5) support the idea that exchange activity is inhibited by cellular acidification. However, the decline in pHseems less likely to have contributed to the shift in the Nainhibition curve since no tendency in this direction was noticed in cells acidified by the NH-prepulse technique.

The results with cytochalasin D suggest that the displacement of the Na-inhibition curve during ATP depletion is related to the breakdown of the actin cytoskeleton. The precise mechanism(s) involved are uncertain. One possibility is that the competition between external Naand Cafor transport sites on the exchanger is modified by ATP depletion and/or cytoskeletal breakdown. This modulation of transport kinetics could occur directly, through altered contacts between the exchanger itself and elements of the cytoskeleton, or indirectly through structural or ionic changes in the exchanger's cytosolic environment. The former alternative is compatible with a recent report that the exchanger binds to the cytoskeletal protein ankyrin (32) . The second alternative is comprised of a large number of possibilities, including changes in the local Naor Caconcentrations, and alterations in secondary Caactivation; these changes, acting singly or in concert, could alter the kinetics of the Na/Ca exchanger at the extracellular transport sites. Whatever the precise mechanisms, the interactions which modify the Nainhibition profile are absent in cells expressing the exchanger deletion mutant. This suggests that these interactions are mediated by the exchanger's central hydrophilic domain.

An alternate interpretation is that the cytoskeleton maintains the wild-type exchanger in close proximity to a Casequestering organelle, thereby promoting an efficient coupling between Cainflux and Casequestration. This spatial relationship would presumably be lost during cytoskeletal breakdown, or in the exchanger deletion mutant, thereby reducing Ca influx and perhaps altering the Nainhibition profile as well. For example, a reduced coupling between Cainflux and local Casequestration could lead to a buildup of [Ca] beneath the cytoplasmic surface which would reduce net Ca entry by promoting Ca/Ca exchange or Na-dependent Ca efflux. While we cannot eliminate this possibility, we believe that an alteration in Na/Ca competition at the exchanger's external surface provides a more straightforward explanation for the shift in the Na-inhibition profile.

Another effect of ATP depletion on exchange activity is apparent in the fura-2 studies, in which ATP-depleted cells show only a slight decline in [Ca]when 140 m M Nais restored to the cells after a period of [Na]reduction (Figs. 4 and 5). In part, this probably reflects the loss of organellar Casequestration and plasma membrane Capump activity due to the absence of ATP. However, it seems likely that the rate of Na-dependent Caefflux is inhibited by ATP depletion as well. Previous studies have shown that ATP depletion increases the Kfor Catransport at the cytosolic surface and decreases the affinity of the secondary Caactivation site for Ca(1, 2, 3, 4, 5, 6, 7, 8) . It has recently been shown, through the use of mutants defective in secondary Caactivation, that filling of the activation site with Caregulates Caefflux as well as Cainflux (33) . Thus, the apparent absence of Na-dependent Caefflux in ATP-depleted CK1.4 cells could reflect the reduced affinity of the exchanger for Caions at either the transport sites, the secondary Caactivation sites, or both. The absence of secondary Caactivation in the exchanger deletion mutant (15) may partly explain the reduced sensitivity of Caefflux to ATP depletion in the CK138 cells.

There are two general hypotheses as to the mechanism by which ATP regulates exchange activity. Studies with internally dialyzed squid axons suggest that exchange activity is regulated by a phosphorylation mechanism (8, 12, 13) . Our results with the CK1.4 cells do not support the phosphorylation hypothesis. A phosphorylated form of the exchanger was not detected by immunoprecipitation of the exchanger from either transfected CHO cells or COS cells. Moreover, inhibitors of protein kinases and phosphatases did not modify exchange activity or alter the response of cells to ATP depletion. Nevertheless, we cannot rule out this possibility because it is conceivable that a phosphorylated form of the exchanger escaped detection by our procedures and/or that the inhibitors used did not act against the kinases/phosphatases involved. The other mechanism that has been proposed for ATP-dependent regulation invokes an ATP-dependent aminophospholipid translocase to maintain an asymmetric distribution of negatively charged phospholipids between the cytoplasmic and extracellular membrane surfaces (2) . We did not test the phospholipid translocase hypothesis in this study. However, we note that the asymmetric distribution of acidic phospholipids has been suggested to involve interactions with the cytoskeleton as well as the aminophospholipid translocase (reviewed in Ref. 34).

In summary, our results describe several new characteristics of ATP-dependent regulation of Na/Ca exchange activity. First, in agreement with previous reports (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) , ATP depletion inhibits both Cainflux and Caefflux mediated by the exchanger. Second, the potency of extracellular Naas an inhibitor of exchange-mediated Cainflux is increased in ATP-depleted cells. Third, the effects of ATP on exchange activity probably do not involve direct phosphorylation of the exchanger. Fourth, exchange activity is modulated by changes in the actin cytoskeleton. And finally, the effects of the actin cytoskeleton on the Na-inhibition profile require the presence of the exchanger's central hydrophilic domain. While the precise mechanism(s) of ATP-dependent regulation of exchange activity remain to be determined, the actin cytoskeleton is likely to emerge as a physiologically important regulator of this transport activity, as it is for several other carrier-mediated transport processes (35) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL49932. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 201-982-3890; Fax: 201-982-7950; E-mail: reeves@umdnj.edu.

The abbreviations used are: PSS, physiological salt solution; BCECF, 2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; Mops, 3-( N-morpholino)propanesulfonic acid; NMDG, N-methyl- D-glucamine; CHO, Chinese hamster ovary.


REFERENCES
  1. Collins, A., Somlyo, A. V., and Hilgemann, D. W. (1992) J. Physiol. 454, 27-57 [Abstract]
  2. Hilgemann, D. W., and Collins, A. (1992) J. Physiol. 454, 59-82 [Abstract]
  3. Hilgemann, D. W., Matsuoka, S., Nagel, G. A., and Collins, A. (1992) J. Gen. Physiol. 100, 905-932 [Abstract]
  4. Hilgemann, D. W., Collins, A., and Matsuoka, S. (1992) J. Gen. Physiol. 100, 933-961 [Abstract]
  5. DiPolo, R., and Beaugé, L. (1987) J. Gen. Physiol. 90, 505-525 [Abstract]
  6. DiPolo, R. (1974) J. Gen. Physiol. 64, 503-517 [Abstract/Free Full Text]
  7. Blaustein, M. P. (1977) Biophys. J. 20, 79-111 [Abstract]
  8. DiPolo, R., and Beaugé L. (1991) Ann. N. Y. Acad. Sci. 639, 100-111 [Medline] [Order article via Infotrieve]
  9. Haworth, R. A., Goknur, A. B., Hunter, D. R., Hegge, J. O., and Berkoff, H. A. (1987) Circ. Res. 60, 586-594 [Abstract]
  10. Haworth, R. A., and Goknur, A. B. (1992) Circ. Res. 71, 210-217 [Abstract]
  11. Smith, J. B., and Smith, L. (1990) Am. J. Physiol. 252, C302-C309
  12. DiPolo, R., and Beaugé, L. (1993) J. Physiol. 462, 71-86 [Abstract]
  13. DiPolo, R., and Beaugé, L. (1994) Am. J. Physiol. 266, C1382-C1391
  14. Nicoll, D. A., Longoni, S., and Philipson, K. D. (1990) Science 250, 562-565 [Medline] [Order article via Infotrieve]
  15. Matsuoka, S., Nicoll, D. A., Reilly, R. F, Hilgemann, D. W., and Philipson, K. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3870-3874 [Abstract]
  16. Pijuan, V., Zhuang, Y., Smith, L., Kroupis, C., Condrescu, M., Aceto, J. F., Reeves, J. P., and Smith, J. B. (1993) Am. J. Physiol. 264, C1066-C1074
  17. Condrescu, M., Aceto, J. F., Kroupis, C., and Reeves, J. P. (1993) Biophys. J. 64, A399 [Abstract]
  18. Condrescu, M., Aceto, J. F., Kroupis, C., Gardner, J. P., and Reeves, J. P. (1994) Biophys. J. 66, A332
  19. Aceto, J. F., Condrescu, M., Kroupis, C., Nelson, H., Nelson, N., Nicoll, D., Philipson, K. D., and Reeves, J. P. (1992) Arch. Biochem. Biophys. 298, 553-560 [Medline] [Order article via Infotrieve]
  20. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  21. Thomas, J. A., Buchsbaum, R. N., Zimniak, A., and Racker, E. (1979) Biochemistry 18, 2210-2218 [Medline] [Order article via Infotrieve]
  22. Baker, P. F., Blaustein, M. P., Hodgkin, A. L., and Steinhardt, R. A. (1969) J. Physiol. 200, 431-458 [Medline] [Order article via Infotrieve]
  23. Reeves, J. P., and Sutko, J. L. (1983) J. Biol. Chem. 258, 3178-3182 [Abstract/Free Full Text]
  24. Condrescu, M., Chernaya, G., Aceto, J. F., Gardner, J. P., and Reeves, J. P. (1994) Biophys. J. 66, A332
  25. Philipson, K. D., Bersohn, M. M., and Nishimoto, A. Y. (1982) Circ. Res. 50, 287-293 [Abstract]
  26. Boron, W. F., and DeWeer, P. (1976) J. Gen. Physiol. 67, 91-112 [Abstract]
  27. Nelson, W. J., and Veshnock, P. J. (1987) Nature 328, 533-536 [CrossRef][Medline] [Order article via Infotrieve]
  28. Bershadsky, A. D., Gelfand, V. I., Svitkina, T. M., and Tint, I. S. (1980) Exp. Cell. Res. 127, 421-429 [Medline] [Order article via Infotrieve]
  29. Sanger, J. W., Sanger, J. M., and Jockusch, B. M. (1983) Eur. J. Cell Biol. 31, 197-204 [Medline] [Order article via Infotrieve]
  30. Cooper, J. A. (1987) J. Cell Biol. 105, 1473-1478 [Medline] [Order article via Infotrieve]
  31. Reeves, J. P., Chernaya, G., Aceto, J. F., Gardner, J. P., and Condrescu, M. (1994) Biophys. J. 66, A148
  32. Li, Z., Burke, E. P., Frank, J. S., Bennett, V., and Philipson, K. D. (1993) J. Biol. Chem. 268, 11489-11491 [Abstract/Free Full Text]
  33. Matsuoka, S., Nicoll, D. A., Hryshko, LV., Levitsky, D. O., Weiss, J. N., and Philipson, K. D. (1995) J. Gen. Physiol., in press
  34. Devaux, P. F. (1991) Biochemistry 30, 1163-1173 [Medline] [Order article via Infotrieve]
  35. Mills, J. W., and Mandel, L. J. (1994) FASEB J. 8, 1161-1165 [Abstract/Free Full Text]

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