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 |
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 |
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, 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-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,
|
(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,
|
(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
-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
-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
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
-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 -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
-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
-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-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
-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.
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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