Intracellular coupling via limiting calmodulin

Summary - Measurements of cellular Ca 2+ -calmodulin concentrations have suggested that competition for limiting calmodulin may couple calmodulin-dependent activities. Here we have directly tested this hypothesis. We have found that in endothelial cells the amount of calmodulin bound to nitric oxide synthase and the catalytic activity of the enzyme both are increased ~ 3-fold upon changes in the phosphorylation status of the enzyme. Quantitative immunoblotting indicates that the synthase can bind up to 25% of the total cellular calmodulin. Consistent with this, simultaneous determinations of the free Ca 2+ and Ca 2+ -calmodulin concentrations in these cells performed using indo-1 and a fluorescent calmodulin biosensor (K d = 2 nM) indicate that increased binding of calmodulin to the synthase is associated with substantial reductions in the Ca 2+ -calmodulin concentrations produced, and an increase in the [Ca 2+ ] 50 for formation of the calmodulin-biosensor complex. The physiological significance of these effects is confirmed by a corresponding 40% reduction in calmodulin-dependent plasma membrane Ca 2+ pump activity. An identical reduction in pump activity is produced by expression of a high-affinity (K d = 0.3 nM) calmodulin biosensor, and treatment to increase calmodulin binding to the synthase then has no further effect. This suggests that the observed reduction in pump activity is due specifically to reduced calmodulin availability. Increases in synthase activity thus appear to be coupled to decreases in the activities of other calmodulin targets through reductions in the size of a limiting pool of available calmodulin. This exemplifies what is likely to be a ubiquitous mechanism for coupling among diverse calmodulin-dependent activities.

and 10 glucose) and incubated at room temperature for 30 minutes in the presence or absence of 50 M forskolin and 0.5 mM IBMX with either 100 M L-NAME or 100 M L-arginine. This medium was then removed and cells were stimulated by the addition of 0.5 mL of a buffer containing 1 M ionomycin, in addition to the above concentrations of L-NAME or L-arginine, and forskolin/IBMX. After 5 min, the supernatant was collected and treated to reduce nitrate to nitrite using a procedure involving catalysis with cadmium metal (Nitralyzer II, World Precision Instruments). Nitrite was then converted to NO and measured using an NO-specific electrode as described by the manufacturer (ISO-NOP MARK II, World Precision Instruments).
Simultaneous measurement of free Ca 2+ and Ca 2+ -CaM concentrations -Biosensor ECFP and EYFP fluorescence was collected using 480/40M and 535/30M emission filters, while indo-1 fluorescence was collected using 405/30M and 485/25M filters (Chroma Technology, Brattleboro, VT). Alternating 340 (indo-1) and 435 (biosensor) excitation light was provided by a DeltaRAM rapid-switching monochromator (PTI International) coupled with a custom-made 410/30M-460LP microscope polychroic (Chroma Technology, Brattleboro, VT). There is no detectable spillover between indo-1 and biosensor channels in this system. Alternating indo-1 (405/485) and biosensor (480/535) emission ratios were determined at 1-second intervals. Subconfluent BAECs transiently expressing CaM biosensors were incubated with indo-1/AM (6 µM) and the specified pharmacological agents or equal volumes of vehicle control (DMSO) for 30 minutes. Free Ca 2+ concentration was estimated from indo-1 emission ratios using a 460 nM K d value determined in BAECs by comparison with the response of a CaM-based Ca 2+ biosensor (9). Free Ca 2+ -CaM concentrations were determined from biosensor emission ratios as described previously and are "effective" values for free (Ca 2+ ) 4 -CaM, as they are calculated based on biosensor K d values for this fully liganded CaM species (9). Thermodynamic coupling effects and differences in the mechanisms controlling CaM-binding to different targets make it difficult to precisely define the CaM species in equilibrium with a CaM-biosensor complex under all conditions in the cell, hence we prefer the generic "Ca 2+ -CaM" designation. Nevertheless, calculated effective values are useful for comparative purposes, and as a means of evaluating, based on its affinity for (Ca 2+ ) 4 -CaM, whether a given target is likely to compete successfully for CaM in the cell.

RESULTS AND DISCUSSION
For several reasons, we chose to use endothelial cells for our initial investigations of coupling among calmodulin target activities. First, endothelial nitric oxide synthase (eNOS) is a CaM-binding protein of undisputed physiological importance (13), whose catalytic activity is increased up to 20-fold by Ca 2+ -CaM (4). Second, CaM binding to eNOS can be manipulated experimentally, as it is known to be influenced by in vivo phosphorylation at one or more residues (14). Thr-497 (Thr-495 in the human sequence) in the putative CaM-binding domain appears to be a particularly important determinant of CaM-binding affinity. Dephosphorylation at this site is associated with a significantly increased CaM-binding ability of eNOS, whereas phosphorylation occurs constitutively, and is correlated with decreased binding (12,14).
Conditions that cause Thr-497 dephosphorylation have generally been found to also produce phosphorylation at Ser-1179, so this site may also play a role in controlling CaM binding to the synthase (15). The focus of this study is not on eNOS phosphorylation per se, but is instead on how physiologically relevant changes in the phosphorylation status of eNOS affect CaM availability in the endothelial cell.
To increase CaM binding to eNOS in BAECs, we used combined treatment with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX), which has previously been shown to mimic, albeit in a more sustained manner, agonist-evoked Thr-497 dephosphorylation and Ser-1179 phosphorylation (15). In our hands, FSK/IBMX treatment produces a ~3-fold increase in the amount of CaM bound to eNOS in cell homogenates ( activity, nitric oxide (NO) production was measured. This activity is also increased ~3-fold, and is completely inhibited by the NOS inhibitor L-NAME (Fig. 1B).
Changes in CaM availability are seen as changes in the apparent free Ca 2+ -CaM concentrations produced at comparable free Ca 2+ concentrations. Therefore, we have simultaneously measured both free Ca 2+ and free Ca 2+ -CaM concentrations produced in BAECs in single cells using indo-1 and CaM biosensors. We have previously developed fluorescence biosensors to monitor dynamic changes in Ca 2+ -CaM concentrations in living cells (8,9). In this study, the ability to simultaneously monitor free Ca 2+ and free Ca 2+ -CaM has allowed us for the first time to precisely assess their relationship under different experimental conditions. Figures Pretreatment with FSK/IBMX causes a ~ 3-fold reduction in the peak free Ca 2+ -CaM concentrations produced in response to ionomycin, with a slight increase in the peak free Ca 2+ concentrations (Fig. 2D). Hence, the observed reduction in free Ca 2+ -CaM is due to reduced CaM availability, not a decrease in free Ca 2+ concentration. The mean Ca 2+ concentration producing 50% of the peak BSCaM 2 fractional response ([Ca 2+ ] 50 value) is increased from 295 ± 18 nM to 502 ± 84 nM (p < 0.05, n = 6) by FSK/IBMX treatment (Fig. 2E). Blocking eNOS activity with 100 M L-NAME does not alter the effects of FSK/IBMX, indicating that increased NO production is not a contributing factor. To verify that changes in CaM availability could account for the observed changes in both maximal biosensor response and Ca 2+ sensitivity, we have determined in vitro how formation of the CaM-BSCaM 2 complex at a fixed CaM concentration is affected by increasing amounts of a CaM-binding peptide (nPEP) (16) (Fig. 2F).
At a low peptide concentration, the Ca 2+ sensitivity of the biosensor response is reduced, followed at higher concentrations by decreases in both the Ca 2+ sensitivity and magnitude of the response. Thus, changes in CaM availability are sufficient to explain the observed changes in both the magnitude and Ca 2+ sensitivity of BSCaM 2 response in BAECs (Fig. 2E). A potential difficulty with the results presented so far is that the biosensor may itself reduce CaM availability enough to exaggerate the effects of FSK/IBMX treatment. To address this potential problem, and to confirm the physiological significance of the observed changes in CaM availability, we have investigated the effect of FSK/IBMX and biosensor expression on plasma membrane Ca 2+ pump (PMCA) activity. Among the major routes for cytosolic Ca 2+ removal, only the PMCA has been shown to depend directly on Ca 2+ -CaM, which is bound with an apparent dissociation constant of 4-10 nM and increases PMCA activity up to 10-fold (17). To determine PMCA activity we first inhibited the SERCA pump with thapsigargin and the Na + -Ca 2+ exchanger by replacing Na + in cell buffers with an equimolar amount of N-methyl-Dglucamine. L-NAME was also applied as a precaution against NO-dependent effects. Under these conditions PMCA activity is directly proportional to the Ca 2+ extrusion rate (18,19). Since PMCA activity is itself Ca 2+ -dependent, extrusion rates were determined for cells grouped on the free Ca 2+ concentration (300-600 nM) at the start of each extrusion time course (Fig. 3A).
Relaxation times ( ) for Ca 2+ extrusion from individual cells were then estimated by fitting the first 50 seconds of extrusion time courses to a mono-exponential (Fig. 3A). A similar approach has been used to assess PMCA activity in endothelial cells (18,19).
As seen in Fig. 3B and 3D, BSCaM 2 expression does not alter PMCA activity, but FSK/IBMX treatment reduces it by ~ 40% in both wild-type and BSCaM 2 -expressing BAECs.
This effect may also partly explain the slight increase in peak free Ca 2+ concentrations observed in cells treated with FSK/IBMX. We reasoned that if the effect of FSK/IBMX on PMCA activity is due specifically to reduced CaM availability it should be reproduced by a CaM antagonist. We therefore expressed a CaM biosensor with a 0.3 nM K d for Ca 2+ -CaM (BSCaM 0. 3

) in BAECs.
This high-affinity biosensor reduces PMCA activity to essentially the same extent as does FSK/IBMX treatment of wild-type BAECs (Fig. 3C and 3D). Peak free Ca 2+ -CaM concentrations are reduced to ~ 2 nM in cells expressing BSCaM 0.3 (Fig. 4A and 4B), compared with ~ 8 nM in cells expressing BSCaM 2 . Most important, subsequent FSK/IBMX treatment has no further effect on PMCA activity (Fig. 3C and 3D) or CaM availability (Fig. 4). Interestingly, FSK/IBMX treatment of cells expressing BSCaM 2 also reduces free Ca 2+ -CaM to ~2 nM (