Report Regulation of Store-Operated Calcium Channels by the Intermediary Metabolite Pyruvic Acid

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provided by Elsevier - Publisher Connector Current Biology 17, 1076–1081, June 19, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.05.041 Report Regulation of Store-Operated Calcium Channels by the Intermediary Metabolite Pyruvic Acid

Daniel Bakowski1 and Anant B. Parekh1,* CRAC channels exhibit Ca2+-dependent rapid inacti- 1 Department of Physiology, Anatomy, and Genetics vation, which develops within milliseconds and is trig- University of Oxford gered by the build-up of a microdomain of elevated Parks Road Ca2+ beneath each open channel [12–14]. Rapid inactiva- Oxford OX1 3PT tion is a general feature of CRAC channels expressed in United Kingdom different cell types [15]. Figure 1 compares the rate and extent of rapid inactivation in the presence of different Ca2+ chelators. After whole-cell dialysis of an RBL-1 Summary cell with a pipette solution containing a strong buffer (10 mM ethyleneglycol-bis(b-aminoethyl)-N,N,N0,N0- A rise in cytosolic Ca2+ concentration is used as a key tetraacetic acid [EGTA]) and the sarcoplasmic or endo- activation signal in virtually all animal cells, where it plasmic reticulum Ca2+-ATPase pump blocker thapsi- triggers a range of responses, including neurotrans- gargin (2 mM) to empty stores, ICRAC developed slowly mitter release, muscle contraction, and cell growth (Figure 1A). The characteristic current-voltage (I-V) rela- and proliferation [1, 2]. A major route for Ca2+ influx is tionship, taken at steady state, is shown in Figure 1B. through store-operated Ca2+ channels. One important Upon stepping to 2120mV (from a holding potential of 2+ intracellular target for Ca entry through store-oper- 0mV), ICRAC initially increased but then declined substan- ated channels is the mitochondrion, which increases tially during the pulse (Figure 1C), revealing rapid inacti- aerobic metabolism and ATP production after Ca2+ vation. Inactivation was a double-exponential process, uptake. Here, we reveal a novel feedback pathway with time constants of w10 and w100 milliseconds [13, whereby pyruvic acid, a critical rate-limiting substrate 14]. Inactivation was much less pronounced at 240mV for mitochondrial respiration, increases store-oper- (Figure 1C), where the driving force for Ca2+ influx through ated entry by reducing inactivation of the channels. the channels is reduced. Aggregate data over the voltage Importantly, the effects of pyruvic acid are manifest range of 240mV to 2120mV is shown in Figure 1J and the at physiologically relevant concentrations and mem- kinetics of inactivation at 2120mV is shown in Figure 1K. brane potentials. The reduction in the inactivation After dialysis with the faster Ca2+ chelator 1,2-bis(2-ami- of calcium release-activated calcium (CRAC) channels nophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA), by pyruvate is highly specific in that it is not mimicked ICRAC developed at a similar rate and extent to that of by other intermediary metabolic acids, does not re- EGTA (Figures 1D and 1E), but rapid inactivation was con- quire its metabolism, is independent of its Ca2+ buffer- siderably less (Figures 1F and 1J; p < 0.01 for each point ing action, and does not involve mitochondrial Ca2+ between 260mV and 2120mV). Inactivation kinetics uptake or ATP production. These results reveal a new were similar between BAPTA and EGTA (Figure 1K). and direct link between intermediary metabolism and Rapid inactivation in a mixture of EGTA and BAPTA ion-channel gating and identify pyruvate as a potential (5 mM each; Figures 1G–1I) lay between 10 mM EGTA signaling messenger linking energy demand to cal- and 10 mM BAPTA (Figure 1J). cium-channel function. To test whether metabolic signals regulated CRAC channels, we compared rapid inactivation in control cells (dialyzed cells with 5 mM EGTA and 2mM thapsigargin) Results and Discussion with cells dialyzed with 5 mM EGTA, 2mM thapsigargin, and 5 mM of different intermediary metabolites. Inclu- Ca2+ entry through store-operated calcium release- sion of pyruvic acid had quite significant effects on activated calcium (CRAC) channels is rapidly taken up CRAC-channel activity. Although the whole-cell current by mitochondria [3, 4], stimulating ATP production developed similarly under both conditions (Figure 2A) through activation of key Ca2+-dependent rate-limiting and with broadly similar I-V curves (Figure 2B), pyruvate enzymes of the Krebs cycle [5, 6]. In turn, by buffering dramatically reduced the extent of rapid inactivation cytoplasmic Ca2+, mitochondria reduce Ca2+-depen- (Figure 2C), over a range of voltages (Figure 2D; p < dent inactivation of CRAC channels and thus maintain 0.01 for each voltage). The time constants of rapid inac- channel activity [7, 8]. Not all of the effects of mitochon- tivation were unaffected by pyruvate (Table S1 in the dria on CRAC channels can be explained by Ca2+ buffer- Supplemental Data available online). ing, however [9], and it is possible that mitochondrially- The concentration of pyruvic acid in nonstimulated derived signals might help control this ubiquitous cells is w0.5 mM [16], but it can double upon stimulation Ca2+-entry pathway [4], as has been suggested for insu- [16]. Hence pyruvate concentrations in the low millimolar lin secretion and Na+-channel activity [10, 11]. Here, we range are likely to occur physiologically. To see whether have tested this idea by examining the effects of numer- these concentrations impacted rapid inactivation, we in- ous intermediary metabolites on CRAC-channel activity. cluded different concentrations of pyruvate (0.1–5 mM) in the pipette solution and measured the extent of rapid inactivation at 2120mV (Figure 2E). The relation- *Correspondence: [email protected] ship could be fitted with a Hill-type equation, yielding Control of Calcium Channels by Pyruvic Acid 1077

Figure 1. Rapid Ca2+-Dependent Inactivation

of ICRAC Panels depict whole-cell recording with (A–C) 10 mM EGTA in the patch pipette, (D–F) 10 mM BAPTA, and (G–I) a mixture of 5 mM EGTA and 5 mM BAPTA. (A), (D), and (G)

plot the development of ICRAC versus whole- cell recording time; (B), (E), and (H) show the current-voltage relationship taken when each current had reached steady state (measured at 280mV from voltage ramps span- ning from 2100mV to +100mV in 50 ms). (C), (F), and (I) show the time course of rapid inactivation at 2120mV and 240mV. To evoke rapid inactivation, cells were clamped at 0mV, and then 250 msec hyperpolarizing steps were applied to the voltages indicated. (J) plots aggregate data for the conditions shown (each point represents 9 cells for EGTA, 9 cells for BAPTA, and 5 cells for EGTA plus BAPTA). The y axis indicates the percent of fast inactivation, determined by measurement of the current remaining at the end of each pulse divided by the peak amplitude (1 ms after the onset of the pulse). Error bars are contained within each symbol. For all voltages except 240mV, p < 0.01 between EGTA and BAPTA. In (K), inactivation time constants are plotted at 2120mV for each chelator (9 cells for EGTA, 9 cells for BAPTA, and 5 cells for EGTA plus BAPTA). There were no signiﬁcant differences between the fast or slow time constants for each condition. Error bars indicate the standard error of the mean.

a half-maximal pipette concentration (EC50)ofw1.6 mM. concentration would be considerably lower than that in The effects of pyruvic acid were discernible as soon as the pipette. We estimated the rate of diffusion of pyruvic ICRAC started to develop, which was generally within acid into the cell [17] (Figure 2F; calculated for a pipette 50–100 s of the onset of whole-cell recording. At these concentration of 5 mM). After 50–100 s, cytoplasmic times, pyruvic acid would not have equilibrated between pyruvic acid would be w1 mM, well within the physiolog- the patch pipette and the cytosol, and the latter ical range. This estimation assumes that pyruvic acid is

Figure 2. Pyruvate Dramatically Reduces Ca2+-Dependent Rapid Inactivation of CRAC Channels

(A) shows ICRAC development after dialysis with EGTA and pyruvate, (B) shows the steady-state current-voltage relationship, and (C) shows the rate and extent of rapid inactivation, for both 5 mM EGTA and 5 mM EGTA plus pyruvate (with thapsigargin in both cases). Aggregate data is summarized in (D) (7 cells each for each condition; p < 0.01 between points over the entire range; error bars are contained within the symbols). (E) shows the dependence of inactivation on pyruvate concentration. The data could be ﬁt- ted with a Hill-type equation, yielding a Hill

coefﬁcient of w1 and KD of 1.6 mM (4 cells for each point). (F) plots the predicted rise in cytosolic pyruvate concentration after dialysis with 5 mM pyruvate. An average capaci- tance of 15 pF and an average series re- sistance of 10 Megaohms were used. Error bars indicate the standard error of the mean. Current Biology 1078

Figure 3. Neither Mitochondrial Ca2+ Buffer- ing nor ATP Production Mediate the Effects of Pyruvic Acid (A) Inclusion of ruthenium red (100 mM) in the pipette failed to reverse the effects of pyruvate on rapid inactivation (4 cells for each condition). (B) The effects of pyruvate were not mimicked by a variety of pyruvate-derived intermediary metabolites (4–7 cells per bar). (C) Inhibition of mitochondrial ATP synthesis with oligomycin (1 mg/ml; 15 min pretreat- ment) failed to reverse the effects of pyruvate (6 cells for each condition). (D) Raising Mg-ATP (to 10 mM) was less effective than pyruvate (5 cells for each condition). (E) Plot of the time course of inward (at 280mV) and outward (at +80mV) currents af-

ter dialysis with 5 mM Na ATP/1 mM MgCl2 (w3.8 mM free ATP; w30 mM free Mg2+). After w220 s, a large outward current developed, accompanied by a smaller inward one. (F) Plot of the current-voltage relationship taken at 546 s in (E). (G) Comparison of the time course of rapid inactivation at points ‘‘a’’ and ‘‘b’’ in (E). The traces were scaled to have the same peak amplitude. Note that less inactivation is seen after the outward current (TRPM7) develops. Error bars indicate the standard error of the mean. neither metabolized nor removed from the cell but simply that seen in the presence of pyruvic acid (Figure 3D; equilibrates between the cytosolic compartment and the p < 0.01). Free ATP is normally kept at very low levels in patch pipette. Because pyruvic acid is extensively me- healthy cells, with the vast majority of ATP being bound tabolized, the simulation overestimates the rise of its in- to Mg2+. However, free ATP in the millimolar range is tracellular concentration, and hence the effects probably sometimes used experimentally as a mobile Ca2+ buffer, occur at even lower concentrations. so we designed experiments to see whether high levels We tested whether the effects of pyruvic acid could of free ATP could affect rapid inactivation of CRAC be explained by mitochondrial Ca2+ buffering. However, channels. Such experiments are complicated because dialysis with ruthenium red, an inhibitor of the mitochon- of the generation of a nonselective cation current that drial Ca2+-uptake channel and which suppresses mito- is activated by low levels of Mg2+/Mg-ATP and is likely chondrial buffering in RBl-1 cells [8], failed to prevent mediated by Ca2+-, Mg2+-, and Cs+-permeable TRPM7 the reduction in rapid inactivation evoked by pyruvate channels [18, 19]. To reduce TRPM7 activation, we (Figure 3A). used a pipette solution containing 2 mM Mg-ATP along To see whether other intermediary metabolites could with 5 mM Na ATP (free ATP was calculated to be substitute for pyruvate, we tested a range of dicarbox- 3.8 mM and free Mg2+ was 30 mM). Under these condi- ylic and tricarboxylic acids generated from the Krebs tions, development of the TRPM7 current was slightly cycle (Figure 3B). Dialysis with 5 mM succinic acid, citric delayed, so ICRAC initially activated in relative isolation. acid, oxaloacetic acid, or a-ketoglutaric acid were all Figure 3E plots the development of inward current (black much less effective (Figure 3B). Two main intracellular circles, measured at 280mV) and outward current (white pathways for pyruvate metabolism are its conversion circles, measured at +80mV) after whole-cell dialysis to acetyl coenzyme A, which enters the Krebs cycle, with 5 mM EGTA, 5 mM Na-ATP, and 2 mM Mg-ATP. and to phosphoenolpyruvate, which is metabolized to By 200 s, a time when ICRAC had fully developed, no de- oxaloacetic acid. Because a variety of Krebs-cycle inter- tectable outward current was present, indicating little mediates and oxaloacetate failed to affect rapid inacti- activation of TRPM7. Within 200 s, the extent of rapid vation, pyruvate metabolism by these major pathways inactivation was considerably more with 5 mM Na-ATP is not necessary for the effects reported here. and 2 mM Mg-ATP than with pyruvate (57.0 6 1.55 for Because free ATP can buffer cytoplasmic Ca2+,we ATP versus 45 6 2.8% for pyruvate ; p < 0.01), demon- considered that the effects of pyruvate were mediated strating that the effects of pyruvate were not mediated through mitochondrial ATP generation. However, inhibi- by an increase in free ATP. After w220 s, a clear outward tion of the mitochondrial F1-F0ATPsynthase with oligo- current developed, and this was accompanied by a small mycin failed to alter the effects of pyruvate on rapid rise in inward current (Figure 3E). The I-V relationship inactivation (Figure 3C), and the inactivation in pyruvate (Figure 3F) revealed the prominent outward current at and oligomycin was similar to that seen in pyruvate. potentials positive to +50mV, characteristic of TRPM7 Moreover, because the vast majority of intracellular channels [18]. Importantly, hyperpolarizing steps to ATP is in the form of Mg-ATP, we increased Mg-ATP in 2120mV at these later time points revealed less rapid the standard pipette solution to 10 mM (free Mg2+, inactivation of the whole-cell current (Figure 3G, which w0.95 mM; free ATP, w270 mM). However, rapid inacti- compares rapid inactivation before and after the devel- vation was still prominent and was signiﬁcantly less than opment of TRPM7, labeled ‘‘a’’ and ‘‘b’’ in Figure 3E). Control of Calcium Channels by Pyruvic Acid 1079

Figure 4. Effect of Pyruvate on Na+-Conducting CRAC Channels (A) The graph compares the additional Ca2+-binding ratio provided by various intermediary metabolites against the extent of rapid inactivation. Points from left to right show 5 mM EGTA (standard) and then 5 mM EGTA plus the following: 5 mM succinate, 5 mM pyruvate, 10 mM Mg-ATP, 5mMa-ketoglutaric acid, 5 mM Na-ATP, and 5 mM citric acid.

(B and C) Ten millimolars of pyruvate alone failed to activate any resolvable ICRAC, whereas 5 mM EGTA and 5 mM pyruvate evoked robust current. Aggregate data from experiments as in (B) are summarized in (C) (7 cells for each condition). (D) In divalent-free external solution, hyperpolarizing pulses from 0mV to 2120mV evoke noninactivating currents in 5 mM EGTA versus 5 mM EGTA plus 5 mM pyruvate (5 cells each).

(E) Current-voltage relationships for ICRAC in divalent-free solution are shown for a cell dialyzed with 5 mM EGTA alone and for one dialyzed with 5 mM EGTA and pyruvate. A larger outward current was consistently observed in pyruvate-dialyzed cells. The inset shows the outward currents on an expanded scale. (F) Mean inward current at 290mV (left-hand panel) and mean outward current at +90mV (right-hand panel) normalized to the size of the corre- sponding inward current are measured for cells dialyzed with either 5 mM EGTA (6 cells) or 5 mM EGTA plus pyruvate (7 cells). Error bars indicate the standard error of the mean.

We attribute this difference to the contaminating pres- not through a Ca2+-buffering action. First, the affinity of 2+ ence of the noninactivating TRPM7 current, which would pyruvate for Ca is low (KD of w12 mM), orders of mag- 2+ mask the true inactivation of ICRAC. In a previous study, nitude below the range of cytosolic Ca rises in most mitochondrially-derived ATP was claimed to act as a lo- cells. Second, we calculated the additional exogenous 2+ 2+ cal Ca buffer [20], preventing the slow inactivation of Ca -binding ratio (kB), which reflects the buffering ca- CRAC channels that develops over tens of seconds. In pacity of each ligand [21], for each metabolic acid. The those experiments, Jurkat T cells were dialyzed with kB of the standard pipette solution is dominated by the up to 10 mM Na-ATP in the presence of 10 mM EGTA, 5 mM EGTA present, with small contributions from glu- and no precautions were taken to eliminate TRPM7. tamic acid and free ATP (from Mg-ATP). The kB from Hence those recordings were likely severely contami- each metabolic acid on top of this background is plotted nated by the development of TRPM7, masking the inac- against the extent of rapid inactivation in Figure 4A. Cit- tivation of CRAC channels [20]. Furthermore, interpreta- rate has a 50-fold greater Ca2+-binding ratio than does tion of those authors’ findings is complicated by the fact pyruvate, yet it is much less effective in reducing rapid that the extent of inactivation they reported was very dif- inactivation. Similarly, increasing free ATP adds more ferent between 10 mM Mg-ATP and 2 mM Na-ATP [20], to the total kB than does 5 mM pyruvate, yet it is less even though free ATP was similar in both cases (w1 and effective in reducing inactivation. Hence the effect of 1.3 mM). Hence it is difficult to see how these effects pyruvate on rapid inactivation does not correlate with could actually be mediated by free ATP. Indeed, we see cytoplasmic Ca2+ buffering. no difference in the extent of rapid inactivation between We considered the possibility that pyruvate evoked 5mMNa+-ATP and 2 mM Mg-ATP (56.3 6 1.6%) and a noninactivating current, developing in parallel with 10 mM Mg-ATP (57.2% 6 2.1), where free ATP differs ICRAC. However, dialysis with up to 10 mM pyruvate by more than 10-fold (w3.8 and 0.27 mM, respectively). failed to activate a detectable current (Figure 4B; aggre- Some intermediary metabolic acids like citric acid gate data in Figure 4C). Moreover, the amplitude of ICRAC bind Ca2+ and thus function as intracellular Ca2+ buffers. in 5 mM EGTA (22.21 6 0.17 pA/pF at 280mV) was sim- Two arguments suggest that the effects of pyruvate are ilar to that seen in cells dialyzed with 5 mM EGTA and Current Biology 1080

pyruvate (22.30 6 0.24 pA/pF; p > 0.2). Hence the ef- Acknowledgments fects of pyruvate cannot be explained by the generation of a non-store-operated current that develops in parallel This work was supported by a (Medical Research Council) MRC program grant to A.B.P. with ICRAC. Collectively, we have found that pyruvate reduces the Received: April 13, 2007 rapid inactivation of CRAC channels and it does so with- Revised: May 11, 2007 2+ out buffering cytosolic Ca . Because rapid inactivation Accepted: May 15, 2007 is mediated at the channel itself [13, 22], it is likely that Published online: June 14, 2007 pyruvate interferes with Ca2+ by either competing at a binding site on the channel or by modifying the efficacy 2+ of Ca to mediate inactivation. We tested the specificity References of the pyruvate effect on rapid inactivation by using divalent-free external solution, in which Na+ replaces 1. Carafoli, E. (2002). Calcium signalling: A tale for all seasons. Proc. Natl. Acad. Sci. 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