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J Biol Chem, Vol. 274, Issue 11, 6827-6830, March 12, 1999

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

Anthony Persechini and Benjamin Cronk

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

	ABSTRACT

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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 (K_d  10 nM) are efficiently activated throughout the cell, but efficient activation of low-affinity targets (K_d  100 nM) occurs only where free Ca²⁺-calmodulin concentrations can be locally enhanced.

	INTRODUCTION

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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 (K_d  10 nM), intermediate (10 nM < K_d < 100 nM), and low (K_d  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, G, 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-CB_SM-38 and FIP-CA₃₇, R1Q, R2Q; FIP-CB_SM-39, R12Q, R16Q; and FIP-CB_SM-41, R2Q. In vitro cooperativity coefficients and K_d 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,

(Eq. 1)

where L is Ca²⁺ or (Ca²⁺)₄-CaM, K_d' is the ligand concentration when the emission ratio (R) is midway between its maximum (R_max) and minimum (R_min) possible values and n is the cooperativity coefficient for the response. K_d' is related to the true titration midpoint (K_d) according to the following equation,

(Eq. 2)

where s_f,2 and s_b,2 are the indicator emission intensities at 535 nm when ligand-free or ligand-saturated, respectively. The s_f,2/s_b,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-CB_SM-41 (K_d = 2 nM), FIP-CB_SM-38 (K_d = 45 nM), and FIP-CB_SM-39 (K_d = 400 nM), or the Ca²⁺ indicator, FIP-CA₃₇ (K_d = 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 R_max, the 480/535 emission ratio for fully liganded indicator, in cells expressing the CaM indicators, the cells were permeabilized with 50 µM -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 R_perm, 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 -toxin (-hemolysin) in a buffered saline solution containing 1.3 mM added CaCl₂ (Fig. 1B). There is no loss of indicator fluorescence from -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 R_perm are produced in cells incubated with 5 µM ionomycin and 5 mM CaCl₂. Because the FIP-CA₃₇ response is CaM-independent, R_perm is equivalent to R_max, and we refer only to the latter value (Fig. 1B). To determine R_min, the emission ratio for ligand-free indicator, cells were permeabilized with -toxin in 3 mM BAPTA (Fig. 1B). Indicator emission ratios identical to R_min are observed in intact resting cells. R_max/R_min values are consistently 2 for the CaM indicators and 1.6 for the Ca²⁺ indicator. This difference is because of a higher R_min 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).

View larger version (33K): [in this window] [in a new window]   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 -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 R_perm values as described under "Experimental Procedures." A value of 2.3 ± 0.5 nM was determined for cells expressing FIPCB_SM-41, and values of 23 ± 2 nM and 45 ± 4 nM were determined for cells expressing FIP-CB_SM-38 and FIP-CB_SM-39. A plot of these values versus the apparent K_d 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 R_perm corresponds to only 60% of the full indicator response in cells expressing FIP-CB_SM-41, which has a 2 nM K_d 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 K_d 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 -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 K_d 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-CB_SM-41 or FIP-CB_SM-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 K_d values of 1 and 1.1 µM, respectively. Because these indicators bind (Ca²⁺)₄-CaM, not Ca²⁺, the apparent K_d 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 K_d values determined in cells expressing them are similar, which points to a physiological value of ~1 µM for this parameter (Fig. 2C).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   The responses of expressed indicators in -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 () 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-CB_SM-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 K_d values derived from these data are 2.7 and 0.9 µM, essentially identical to the values derived from data measured in -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-CB_SM-38 is freely distributed in the cell.

View larger version (24K): [in this window] [in a new window]   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 (K_d  10 nM) are efficiently activated throughout the cell, but efficient activation of low-affinity targets (K_d  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.

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.

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				M. Kapustina, G. E. Weinreb, N. Costigliola, Z. Rajfur, K. Jacobson, and T. C. Elston Mechanical and Biochemical Modeling of Cortical Oscillations in Spreading Cells Biophys. J., June 15, 2008; 94(12): 4605 - 4620. [Abstract] [Full Text] [PDF]

				F. Edlich, M. Maestre-Martinez, F. Jarczowski, M. Weiwad, M.-C. Moutty, M. Malesevic, G. Jahreis, G. Fischer, and C. Lucke A Novel Calmodulin-Ca2+ Target Recognition Activates the Bcl-2 Regulator FKBP38 J. Biol. Chem., December 14, 2007; 282(50): 36496 - 36504. [Abstract] [Full Text] [PDF]

				L. E. Fridlyand, M. C. Harbeck, M. W. Roe, and L. H. Philipson Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic -cell: a computational approach Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1924 - C1933. [Abstract] [Full Text] [PDF]

				J. W. Ryder, K. S. Lau, K. E. Kamm, and J. T. Stull Enhanced Skeletal Muscle Contraction with Myosin Light Chain Phosphorylation by a Calmodulin-sensing Kinase J. Biol. Chem., July 13, 2007; 282(28): 20447 - 20454. [Abstract] [Full Text] [PDF]

				R. E. Simpson, A. Ciruela, and D. M. F. Cooper The Role of Calmodulin Recruitment in Ca2+ Stimulation of Adenylyl Cyclase Type 8 J. Biol. Chem., June 23, 2006; 281(25): 17379 - 17389. [Abstract] [Full Text] [PDF]

				T. J. Weber, H. S. Smallwood, L. E. Kathmann, L. M. Markillie, T. C. Squier, and B. D. Thrall Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1512 - C1520. [Abstract] [Full Text] [PDF]

				A. J. Crossthwaite, A. Ciruela, T. F. Rayner, and D. M. F. Cooper A Direct Interaction between the N Terminus of Adenylyl Cyclase AC8 and the Catalytic Subunit of Protein Phosphatase 2A Mol. Pharmacol., February 1, 2006; 69(2): 608 - 617. [Abstract] [Full Text] [PDF]

				Y. Fukunaga, M. Matsubara, R. Nagai, and A. Miyazawa The Interaction between PSD-95 and Ca2+/Calmodulin Is Enhanced by PDZ-Binding Proteins J. Biochem., August 1, 2005; 138(2): 177 - 182. [Abstract] [Full Text] [PDF]

				H. Sim, K. Rimmer, S. Kelly, L. M. Ludbrook, A. H. A. Clayton, and V. R. Harley Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal Mol. Endocrinol., July 1, 2005; 19(7): 1884 - 1892. [Abstract] [Full Text] [PDF]

				G. C. Castellani, E. M. Quinlan, F. Bersani, L. N. Cooper, and H. Z. Shouval A model of bidirectional synaptic plasticity: From signaling network to channel conductance Learn. Mem., July 1, 2005; 12(4): 423 - 432. [Abstract] [Full Text] [PDF]

				X. Yu, J. H. Byrne, and D. A. Baxter Modeling Interactions Between Electrical Activity and Second-Messenger Cascades in Aplysia Neuron R15 J Neurophysiol, May 1, 2004; 91(5): 2297 - 2311. [Abstract] [Full Text] [PDF]

				D. M. Bautista and R. S. Lewis Modulation of plasma membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells J. Physiol., May 1, 2004; 556(3): 805 - 817. [Abstract] [Full Text] [PDF]

				E. Isotani, G. Zhi, K. S. Lau, J. Huang, Y. Mizuno, A. Persechini, R. Geguchadze, K. E. Kamm, and J. T. Stull Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse PNAS, April 20, 2004; 101(16): 6279 - 6284. [Abstract] [Full Text] [PDF]

				M. X. Mori, M. G. Erickson, and D. T. Yue Functional Stoichiometry and Local Enrichment of Calmodulin Interacting with Ca2+ Channels Science, April 16, 2004; 304(5669): 432 - 435. [Abstract] [Full Text] [PDF]

				D. M. Greif, D. B. Sacks, and T. Michel Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis PNAS, February 3, 2004; 101(5): 1165 - 1170. [Abstract] [Full Text] [PDF]

				S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille Intracellular calmodulin availability accessed with two-photon cross-correlation PNAS, January 6, 2004; 101(1): 105 - 110. [Abstract] [Full Text] [PDF]

				D. W. O'Callaghan, A. V. Tepikin, and R. D. Burgoyne Dynamics and calcium sensitivity of the Ca2+/myristoyl switch protein hippocalcin in living cells J. Cell Biol., November 24, 2003; 163(4): 715 - 721. [Abstract] [Full Text] [PDF]

				R. I. Herzog, C. Liu, S. G. Waxman, and T. R. Cummins Calmodulin Binds to the C Terminus of Sodium Channels Nav1.4 and Nav1.6 and Differentially Modulates Their Functional Properties J. Neurosci., September 10, 2003; 23(23): 8261 - 8270. [Abstract] [Full Text] [PDF]

				Q.-K. Tran, D. J. Black, and A. Persechini Intracellular Coupling via Limiting Calmodulin J. Biol. Chem., June 27, 2003; 278(27): 24247 - 24250. [Abstract] [Full Text] [PDF]

				K. Strange From Genes to Integrative Physiology: Ion Channel and Transporter Biology in Caenorhabditis elegans Physiol Rev, April 1, 2003; 83(2): 377 - 415. [Abstract] [Full Text] [PDF]

				I. Paarmann, O. Spangenberg, A. Lavie, and M. Konrad Formation of Complexes between Ca2+{middle dot}Calmodulin and the Synapse-associated Protein SAP97 Requires the SH3 Domain-Guanylate Kinase Domain-connecting HOOK Region J. Biol. Chem., October 18, 2002; 277(43): 40832 - 40838. [Abstract] [Full Text] [PDF]

				J. M. Bradshaw, A. Hudmon, and H. Schulman Chemical Quenched Flow Kinetic Studies Indicate an Intraholoenzyme Autophosphorylation Mechanism for Ca2+/Calmodulin-dependent Protein Kinase II J. Biol. Chem., May 31, 2002; 277(23): 20991 - 20998. [Abstract] [Full Text] [PDF]

				P. Lane and S. S. Gross Disabling a C-terminal Autoinhibitory Control Element in Endothelial Nitric-oxide Synthase by Phosphorylation Provides a Molecular Explanation for Activation of Vascular NO Synthesis by Diverse Physiological Stimuli J. Biol. Chem., May 17, 2002; 277(21): 19087 - 19094. [Abstract] [Full Text] [PDF]

J. Hulvershorn, C. Gallant, C.-L. A. Wang, C. Dessy, and K. G. Morgan Calmodulin levels are dynamically regulated in living vascular smooth muscle cells Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1422 - H1426. [Abstract]