COMMUNICATION
The Relationship between the Free Concentrations of Ca2+ and Ca2+-calmodulin in Intact Cells*

Anthony PersechiniDagger and Benjamin Cronk

From the Department of Pharmacology & Physiology, University of Rochester Medical Center, Rochester, New York 14642

    ABSTRACT
Top
Abstract
Introduction
References

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 µM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of ~ 45 nM is produced when the free Ca2+ concentration exceeds 3 µM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1 µM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is ~ 2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (Kd <=  10 nM) are efficiently activated throughout the cell, but efficient activation of low-affinity targets (Kd >=  100 nM) occurs only where free Ca2+-calmodulin concentrations can be locally enhanced.

    INTRODUCTION
Top
Abstract
Introduction
References

Calmodulin (CaM)1 is responsible for Ca2+-dependent regulation of the activities of a vast array of different putative target proteins, including enzymes, ion pumps and channels, and cytoskeletal proteins. CaM has four Ca2+-binding sites, and its fully liganded form, (Ca2+)4-CaM, plays a prominent role in target binding and regulation, whereas its Ca2+-free form is generally not bound to targets (1, 2). Putative targets include those binding (Ca2+)4-CaM with high (Kd <=  10 nM), intermediate (10 nM < Kd < 100 nM), and low (Kd >=  100 nM) affinities. Among those with high affinities are myosin light chain kinase, calcineurin, neuronal nitric oxide synthase, phosphodiesterase, and cGMP-gated ion channels (2-5). Targets with intermediate affinities include CaM kinase IV, G protein-coupled receptor kinase 5, A-kinase anchoring protein, and synapsin (6-9). Those with low affinities include the CaM-dependent adenylate cyclases, G protein-coupled receptor kinase 2, spectrin, Gbeta gamma , and caldesmon (7, 10-16). Knowledge of the physiological free concentrations of (Ca2+)4-CaM is crucial if we are to evaluate whether a putative target activity may be regulated by CaM in the cell. In this study we have investigated the global relationship between the concentrations of free Ca2+ and (Ca2+)4-CaM in intact cells using stably expressed indicator proteins.

    EXPERIMENTAL PROCEDURES

Indicators were expressed under control of a cytomegalovirus promoter/enhancer using the pcDNA3.1 or pcDNA6 vectors supplied by InvitroGen (Carslbad, CA). HEK-293 cells were transfected using LipofectAMINE according to the instructions of the manufacturer (Life Technologies, Gaithersburg. MD). Mixed-clonal populations of stably transfected cells used in experiments were grown under drug selection (G418 or Zeocin) for at least 6 weeks. The CaM-binding sequences in the indicators are altered versions of the avian smooth muscle myosin light chain kinase sequence: R1RKWQKTGHA10VRAIGRL (17). According to this numbering scheme, the sequences in the indicators have the following changes: FIP-CBSM-38 and FIP-CA37, R1Q, R2Q; FIP-CBSM-39, R12Q, R16Q; and FIP-CBSM-41, R2Q. In vitro cooperativity coefficients and Kd values for the indicators were determined in buffered saline as described previously (18, 19). Values for the Ca2+ indicator were also determined in cells; the lack of a reliable buffering system for CaM precluded doing this with the CaM indicators.

Indicator responses were monitored in individual cells using a standard microscope photometry system with dual photomultiplier tubes (Photon Technology Int., Monmouth Junction, NJ). The emitted light from single cells was isolated using an adjustable diaphragm, and emission intensities were determined by photon counting using 200 msec integration times. Excitation light at 430 nm was supplied by a monochromator. A 455DCLP microscope dichroic was used, and emitted light was distributed to the two photomultipliers using a 510DCLP dichroic cube fitted with D535/25 and D480/40 bandpass filters. Dichroics and filters were obtained from Chroma Technologies (Brattleboro, VT). All measurements were made in cells expressing n5 µM indicator as assessed by comparing the fluorescence intensities of cells with the intensities of indicator standards. Cells were incubated in a buffered saline solution containing 141 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM HEPES, 10 mM glucose, pH 7.4, and added CaCl2 and/or Ca2+-chelators as indicated. Free (Ca2+)4-CaM and Ca2+ concentrations were calculated from indicator emission ratios using an equation of the form,
[L]<SUB>free</SUB>=K′<SUB>d</SUB><FENCE><FR><NU>R−R<SUB><UP>min</UP></SUB></NU><DE>R<SUB><UP>max</UP></SUB>−R</DE></FR></FENCE><SUP>1/n</SUP> (Eq. 1)
where L is Ca2+ or (Ca2+)4-CaM, Kd' is the ligand concentration when the emission ratio (R) is midway between its maximum (Rmax) and minimum (Rmin) possible values and n is the cooperativity coefficient for the response. Kd' is related to the true titration midpoint (Kd) according to the following equation,
K′<SUB>d</SUB>=K<SUB>d</SUB><FENCE><FR><NU>s<SUB>f,2</SUB></NU><DE>s<SUB>b,2</SUB></DE></FR></FENCE><SUP>1/n</SUP> (Eq. 2)
where sf,2 and sb,2 are the indicator emission intensities at 535 nm when ligand-free or ligand-saturated, respectively. The sf,2/sb,2 value is 1.8 for FIP-CBs and 1.5 for FIP-CAs.

    RESULTS AND DISCUSSION

The indicator proteins used in this study are similar to those we have described previously (18, 19). The main difference is in the green fluorescent protein variants used to generate ligand-dependent fluorescence resonance energy transfer (FRET), which have been replaced by the ECFP and EYFP variants described by Miyawaki et al. (20). The advantages of these are that FRET can be monitored as the 480/535 emission ratio, and the ECFP donor fluorophore can be excited at a longer wavelength of 430 nm. To estimate physiological (Ca2+)4-CaM values, we have made measurements in cells expressing indicators with different affinities, which were generated by varying the CaM-binding linker sequence between the GFP variants. The Ca2+-indicator protein used in some experiments is similar to the CaM indicators, but incorporation of the complete amino acid sequence of CaM renders it directly responsive to changes in the free Ca2+ concentration (18). Stably transfected cells were produced that express one of three different CaM indicators, FIP-CBSM-41 (Kd = 2 nM), FIP-CBSM-38 (Kd = 45 nM), and FIP-CBSM-39 (Kd = 400 nM), or the Ca2+ indicator, FIP-CA37 (Kd = 0.6 µM). The CaM indicators are freely distributed but FIP-CA37 is confined to the cytoplasm, probably because of its higher molecular weight (data not shown). The morphology and growth kinetics are similar for control and indicator-expressing HEK-293 cells.

To determine Rmax, the 480/535 emission ratio for fully liganded indicator, in cells expressing the CaM indicators, the cells were permeabilized with 50 µM beta -escin, followed by addition of 10 µM CaM, which diffuses into the cells producing the maximum possible indicator response. The indicator response is specific to Ca2+-liganded CaM and is reversed by a high-affinity CaM-binding peptide (Fig. 1A). To determine Rperm, the emission ratio corresponding to the free (Ca2+)4-CaM concentration produced at a saturating free Ca2+ concentration, cells were permeabilized with 15 µg/ml alpha -toxin (alpha -hemolysin) in a buffered saline solution containing 1.3 mM added CaCl2 (Fig. 1B). There is no loss of indicator fluorescence from alpha -toxin permeabilized cells, and addition of 20 µM CaM or 50 µM CaM-binding peptide has no effect on the indicator response, demonstrating that the cells are not permeable to proteins or even small peptides. Indicator emission ratios identical to Rperm are produced in cells incubated with 5 µM ionomycin and 5 mM CaCl2. Because the FIP-CA37 response is CaM-independent, Rperm is equivalent to Rmax, and we refer only to the latter value (Fig. 1B). To determine Rmin, the emission ratio for ligand-free indicator, cells were permeabilized with alpha -toxin in 3 mM BAPTA (Fig. 1B). Indicator emission ratios identical to Rmin are observed in intact resting cells. Rmax/Rmin values are consistently 2 for the CaM indicators and 1.6 for the Ca2+ indicator. This difference is because of a higher Rmin value for the Ca2+ indicator (Fig. 1B), which is expected based on a comparison of the in vitro emission spectra for the two types of indicator (data not shown).


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Fig. 1.   Representative time courses for the 480/535 emission ratios in cells expressing FIP-CBSM-41. A, a cell preincubated in buffered saline containing 3 mM BAPTA was permeabilized with beta -escin, followed by addition of 10 µM CaM and 5 mM CaCl2, which produces the full indicator response. Subsequent addition of excess CaM-binding peptide completely reverses this response. The peptide used has the sequence: KRRWKKNFIAVSAANRFKK-amide, and is based on the CaM-binding domain in skeletal muscle myosin light chain kinase (41). Its Kd for (Ca2+)4-CaM is ~1 nM. Inset, the effect of changing the order of addition of CaM and CaCl2 on the indicator response. Only the portion of the curve showing the response to added CaM is presented. B, histogram plots of the mean values for Rmin, Rperm, and Rmax determined in indicator-expressing cells as described in the text. The standard error is given for each value (n = 15). Inset, a plot of the free (Ca2+)4-CaM value calculated for each Rperm value versus the apparent Kd value of the expressed indicator.

The free (Ca2+)4-CaM concentrations produced in cells expressing each CaM indicator were calculated from Rperm values as described under "Experimental Procedures." A value of 2.3 ± 0.5 nM was determined for cells expressing FIPCBSM-41, and values of 23 ± 2 nM and 45 ± 4 nM were determined for cells expressing FIP-CBSM-38 and FIP-CBSM-39. A plot of these values versus the apparent Kd values for the corresponding indicators suggests that the physiological global maximum free (Ca2+)4-CaM concentration is ~45 nM (Fig. 1B, inset). We have found that Rperm corresponds to only 60% of the full indicator response in cells expressing FIP-CBSM-41, which has a 2 nM Kd value, and is significantly less than this in cells expressing the lower-affinity indicators (Fig. 1B). If we assume that the native CaM-binding proteins in the cell have Kd values for (Ca2+)4-CaM that are >= 2 nM, this observation suggests that on a molar basis CaM-binding proteins outnumber CaM by a factor of ~2.

To define the relationship between the intracellular concentrations of free Ca2+ and (Ca2+)4-CaM in greater detail, we used dibromo-BAPTA to control the free Ca2+ concentration in alpha -toxin permeabilized cells (21). Effective control of intracellular free Ca2+ is demonstrated by the FIP-CA37 responses in cells incubated at different calculated free Ca2+ concentrations (Fig. 2A). A cooperativity coefficient of 1.7 and an apparent Kd value of 0.9 µM were derived from these data; respective in vitro values obtained with the same Ca2+ buffering system are 1.8 and 0.6 µM. The FIP-CBSM-41 or FIP-CBSM-38 responses and corresponding free (Ca2+)4-CaM concentrations are presented in Fig. 2, B and C, respectively. Data for the two indictors are fit by binding curves with cooperativity coefficients of 2.6 and apparent Kd values of 1 and 1.1 µM, respectively. Because these indicators bind (Ca2+)4-CaM, not Ca2+, the apparent Kd values are actually the free concentrations of Ca2+ producing half-maximal free (Ca2+)4-CaM concentrations. Despite the 20-fold difference in the affinities of the two indicators, the apparent Kd values determined in cells expressing them are similar, which points to a physiological value of ~1 µM for this parameter (Fig. 2C).


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Fig. 2.   The responses of expressed indicators in alpha -toxin permeabilized cells at different free Ca2+ concentrations. Each point represents the mean ± S.E. of 15 determinations. A, the binding curve for the FIP-CA37 data has a cooperativity coefficient of 1.7 and apparent Kd value of 0.9 µM. The emission ratios (B) and corresponding free (Ca2+)4-CaM concentrations (C) in cells expressing FIP-CBSM-38 (black-square) and FIP-CBSM-41 (). The binding curves for these data both have a cooperativity coefficient of 2.6 and have apparent Kd values of 1 and 1.1 µM, respectively.

Based on the observation that Ca2+ is bound more tightly to the C-terminal EF hand pair in CaM than to the N-terminal pair, it has been proposed that CaM targets might be associated with (Ca2+)2-CaM at resting free Ca2+ concentrations (22). Binding of a tryptic fragment of CaM containing only the C-terminal EF hand pair produces about half the maximal indicator response in vitro seen with intact CaM (data not shown). Hence, if there were significant levels of indicator complexes involving just the C-terminal EF hand pair of CaM in resting cells, they would have been detected.

To investigate the relationship between the free concentrations of Ca2+ and (Ca2+)4-CaM under more physiological conditions, we stably expressed FIP-CBSM-38 or FIP-CA37 in HEK-293 cell line expressing the receptor for thyrotropin releasing hormone (TRH), which exhibits reproducible transients in intracellular free Ca2+ in response to this agonist (19, 23). We determined time courses for the concentrations of free Ca2+ and free (Ca2+)4-CaM produced after addition of TRH (Fig. 3B), and combined the time-aligned transients to produce the relationship in Fig. 3A. The cooperativity coefficient and apparent Kd values derived from these data are 2.7 and 0.9 µM, essentially identical to the values derived from data measured in alpha -toxin-permeabilized cells expressing the CaM indicator. The maximum free (Ca2+)4-CaM concentration of 16 nM is slightly less than the value determined in permeabilized cells. This may reflect a lack of spatial alignment in the indicator signals, because FIP-CA37 is cytoplasmic and FIP-CBSM-38 is freely distributed in the cell.


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Fig. 3.   Changes in the free concentrations of Ca2+ and (Ca2+)4-CaM produced in response to agonist in cells expressing the receptor for thyrotropin releasing hormone. A, the relationship between the free Ca2+ and (Ca2+)4-CaM concentrations measured in parallel experiments in cells expressing FIP-CA37 or FIP-CBSM-38. The binding curve fit to these data has a cooperativity coefficient of 2.7 and an apparent Kd value of 1 µM. B, time courses for the free Ca2+ or (Ca2+)4-CaM concentrations produced after addition of TRH and BAPTA to cells expressing FIP-CA37 or FIP-CBSM-38. The two traces are each the average of responses from 10 individual cells. BAPTA was added along with TRH to inhibit store-operated Ca2+ entry, which otherwise could produce a variable sustained elevation in the free Ca2+ concentration. Data from these traces were used to generate the relationship seen in panel A.

These studies represent the first quantitative evaluations of the relationship between the concentrations of free Ca2+ and (Ca2+)4-CaM in cells. We find that no detectable Ca2+-liganded CaM is produced in the cell below a free Ca2+ concentration of 0.2 µM. A maximum free (Ca2+)4-CaM concentration of ~45 nM is produced at a free Ca2+ concentration of 3 µM, and a half-maximal concentration is produced when the free Ca2+ concentration is 1 µM. The total concentration of CaM-binding sites appears to exceed the total concentration of CaM by a factor of ~2, consistent with investigations of the mobility of labeled CaM that suggest its concentration in the cell is matched or exceeded by the concentration of CaM-binding sites (24). Given a total CaM concentration of ~10 µM (25-27), only a minute fraction of the (Ca2+)4-CaM produced is free in the cell. This underscores the importance of directly determining the free (Ca2+)4-CaM concentration, as it clearly cannot be deduced from the total CaM concentration. The free Ca2+ concentration producing a half-maximal free (Ca2+)4-CaM concentration in the cell is 20-fold less than the concentration required to half-saturate pure CaM in vitro (28). This demonstrates that thermodynamic coupling between Ca2+ and target binding in CaM-target complexes is of crucial importance in the cell as it keeps free (Ca2+)4-CaM concentrations low and shifts their dependence on free Ca2+ into the physiological concentration range (29-31). Furthermore, our results suggest that, if resting global intracellular free Ca2+ concentrations are maintained below 0.2 µM little or no global activation of CaM targets should occur.

Our observations indicate that high-affinity calmodulin targets (Kd <=  10 nM) are efficiently activated throughout the cell, but efficient activation of low-affinity targets (Kd >=  100 nM) occurs only where free (Ca2+)4-CaM concentrations can be locally enhanced. Interestingly, most low-affinity CaM targets that have been identified are permanently or transiently associated with the plasma membrane, including the CaM-dependent adenylate cyclases (15, 32). We hypothesize that there are at least two mechanisms that could produce a local enhancement in the free (Ca2+)4-CaM concentrations in this region: 1) diffusional recruitment of CaM because of local increases in free Ca2+, and 2) concentration of CaM by plasma membrane-associated CaM-binding proteins like neuromodulin or neurogranin, which have been proposed to function as CaM sinks (33-35). Recent evidence suggests that store-operated Ca2+ entry, which appears to produce local elevations in the free Ca2+ concentration at the plasma membrane, is necessary to activate expressed CaM-dependent adenylate cyclase activities in HEK-293 cells, consistent with a requirement for diffusional recruitment of CaM (36-38). And CaM-dependent cyclases in neurons are associated with neurogranin and/or neuromodulin, implying that the putative CaM sink proteins may help to produce the free (Ca2+)4-CaM concentrations needed to activate cyclase activity (15, 39). Mechanisms similar to these may operate in other regions of the cell. For example, Deisseroth et al. (40) have recently reported that translocation of CaM into the nucleus correlates with Ca2+-dependent phosphorylation of CREB in hippocampal neurons, suggesting that enhanced free (Ca2+)4-CaM concentrations may be required to activate a necessary protein kinase activity in the nucleus. Our observations suggest that spatial variations in the free (Ca2+)4-CaM concentrations that can be produced in different regions of the cell may play an important role in shaping the functional response to a Ca2+-mediated signal.

    ACKNOWLEDGEMENTS

We acknowledge Dr. Shey-Shing Sheu, in our department, for generously allowing us the use of his microscope photometry system for this study, and Dr. Margaret Shupnick (University of Virginia), for supplying the HEK-293 cell line stably expressing the receptor for thyrotropin releasing hormone.

    FOOTNOTES

* This work was supported by Public Health Service Grant DK44322 (to A. P.) and a grant from Small Molecule Therapeutics, Inc. (to A. P.).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 To whom all correspondence should be addressed: Dept. of Pharmacology & Physiology, 601 Elmwood Ave., Box 711, University of Rochester Medical Center, Rochester, NY 14642. Tel.: 1-716-275-3087; Fax: 1-716-461-3259; E-mail: ajp2o{at}crocus.medicine.rochester.edu.

    ABBREVIATIONS

The abbreviations used are: CaM, calmodulin; FRET, fluorescence resonance energy transfer; FIP-CA, Fluorescent Indicator Protein-CAlcium binding; FIP-CB, Fluorescent Indicator Protein-Calmodulin Binding; GFP, green fluorescent protein; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; TRH, thyrotropin releasing hormone.

    REFERENCES
Top
Abstract
Introduction
References
  1. Huang, C. Y., Chau, V., Chock, P. B., Wang, J. H., and Sharma, R. K. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 871-874[Abstract]
  2. Blumenthal, D. K., and Stull, J. T. (1980) Biochemistry 19, 5608-5614[Medline] [Order article via Infotrieve]
  3. Persechini, A., McMillan, K., and Leakey, P. (1994) J. Biol. Chem. 269, 16148-16154[Abstract/Free Full Text]
  4. Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866[Medline] [Order article via Infotrieve]
  5. Bauer, P. J. (1996) J. Physiol. 494, 675-685[Abstract]
  6. Faux, M. C., and Scott, J. D. (1997) J. Biol. Chem. 272, 17038-17044[Abstract/Free Full Text]
  7. Pronin, A. N., Satpaev, D. K., Slepak, V. Z., and Benovic, J. L. (1997) J. Biol. Chem. 272, 18273-18280[Abstract/Free Full Text]
  8. Cruzalegui, F. H., and Means, A. R. (1993) J. Biol. Chem. 268, 26171-26178[Abstract/Free Full Text]
  9. Nicol, S., Rahman, D., and Baines, A. J. (1997) Biochemistry 36, 11487-11495[CrossRef][Medline] [Order article via Infotrieve]
  10. Medvedeva, M. V., Kolobova, E. A., Wang, P., and Gusev, N. B. (1996) Biochem. J. 315, 1021-1026[Medline] [Order article via Infotrieve]
  11. Bjork, J., Lundberg, S., and Backman, L. (1995) Eur. J. Cell Biol. 66, 200-204[Medline] [Order article via Infotrieve]
  12. Cali, J. J., Parekh, R. S., and Krupinski, J. (1996) J. Biol. Chem. 271, 1089-1095[Abstract/Free Full Text]
  13. Choi, E. J., Xia, Z., and Storm, D. R. (1992) Biochemistry 31, 6492-6498[Medline] [Order article via Infotrieve]
  14. Choi, E. J., Wong, S. T., Hinds, T. R., and Storm, D. R. (1992) J. Biol. Chem. 267, 12440-12442[Abstract/Free Full Text]
  15. Cooper, D. M., Karpen, J. W., Fagan, K. A., and Mons, N. E. (1998) Adv. Second Messenger Phosphoprotein Res. 32, 23-51[Medline] [Order article via Infotrieve]
  16. Katada, T., Kusakabe, K., Oinuma, M., and Ui, M. (1987) J. Biol. Chem. 262, 11897-11900[Abstract/Free Full Text]
  17. Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381[Medline] [Order article via Infotrieve]
  18. Persechini, A., Lynch, J. A., and Romoser, V. A. (1997) Cell Calcium 22, 209-216[Medline] [Order article via Infotrieve]
  19. Romoser, V. A., Hinkle, P. M., and Persechini, A. (1997) J. Biol. Chem. 272, 13270-13274[Abstract/Free Full Text]
  20. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Nature 388, 882-887[CrossRef][Medline] [Order article via Infotrieve]
  21. Bers, D., Patton, C., and Nuccitelli, R. (1994) Methods Cell Biol. 40, 3-29[Medline] [Order article via Infotrieve]
  22. Johnson, J. D., Snyder, C., Walsh, M., and Flynn, M. (1996) J. Biol. Chem. 271, 761-767[Abstract/Free Full Text]
  23. Shupnik, M. A., Weck, J., and Hinkle, P. M. (1996) Mol. Endocrinol. 10, 90-99[Abstract]
  24. Gough, A. H., and Taylor, D. L. (1993) J. Cell Biol. 121, 1095-1107[Abstract]
  25. Tansey, M. G., Luby-Phelps, K., Kamm, K. E., and Stull, J. T. (1994) J. Biol. Chem. 269, 9912-9920[Abstract/Free Full Text]
  26. Kakiuchi, S., Yasuda, S., Yamazaki, R., Teshima, Y., Kanda, K., Kakiuchi, R., and Sobue, K. (1982) J. Biochem. 92, 1041-1048[Abstract]
  27. Chafouleas, J. G., Bolton, W. E., Hidaka, H., Boyd, A. E. d., and Means, A. R. (1982) Cell 28, 41-50[Medline] [Order article via Infotrieve]
  28. Persechini, A., Stemmer, P. M., and Ohashi, I. (1996) J. Biol. Chem. 271, 32217-32225[Abstract/Free Full Text]
  29. Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482[Medline] [Order article via Infotrieve]
  30. Olwin, B. B., Edelman, A. M., Krebs, E. G., and Storm, D. R. (1984) J. Biol. Chem. 259, 10949-10955[Abstract/Free Full Text]
  31. Olwin, B. B., and Storm, D. R. (1985) Biochemistry 24, 8081-8086[Medline] [Order article via Infotrieve]
  32. Xia, Z., and Storm, D. R. (1997) Curr. Opin. Neurobiol. 7, 391-396[CrossRef][Medline] [Order article via Infotrieve]
  33. Gamby, C., Waage, M. C., Allen, R. G., and Baizer, L. (1996) J. Biol. Chem. 271, 26698-26705[Abstract/Free Full Text]
  34. Liu, Y., Fisher, D. A., and Storm, D. R. (1993) Biochemistry 32, 10714-10719[Medline] [Order article via Infotrieve]
  35. Gerendasy, D. D., Herron, S. R., Watson, J. B., and Sutcliffe, J. G. (1994) J. Biol. Chem. 269, 22420-22426[Abstract/Free Full Text]
  36. Fagan, K. A., Mahey, R., and Cooper, D. M. (1996) J. Biol. Chem. 271, 12438-12444[Abstract/Free Full Text]
  37. Nakahashi, Y., Nelson, E., Fagan, K., Gonzales, E., Guillou, J. L., and Cooper, D. M. (1997) J. Biol. Chem. 272, 18093-18097[Abstract/Free Full Text]
  38. Marsault, R., Murgia, M., Pozzan, T., and Rizzuto, R. (1997) EMBO J. 16, 1575-1581[Abstract/Free Full Text]
  39. Mons, N., Harry, A., Dubourg, P., Premont, R. T., Iyengar, R., and Cooper, D. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8473-8477[Abstract]
  40. Deisseroth, K., Heist, E. K., and Tsien, R. W. (1998) Nature 392, 198-202[CrossRef][Medline] [Order article via Infotrieve]
  41. Blumenthal, D. K., and Krebs, E. G. (1987) Methods Enzymol. 139, 115-126[Medline] [Order article via Infotrieve]


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