Abstract:
Transient receptor potential melastatin 7 (TRPM7) is a widely expressed,
constituatively active, voltage independent ion channel known to be permeable to most
divalent and monovalent cations. TRPM7 may play an important role in Ca, Mgr, and
trace metal homeostasis. Our study examines the behavior of TRPM7 under
physiologically relevant conditions. Confocal laser microscopy and the indicator dye
fluo-3/AM allowed for visualization of intracellular Ca“ accumulation in control and
TRPM7 overexpressing HEK-293 cells when exposed to external solutions with varying
concentrations of Ca“ and Mg“. When compared with control cells, TRPM7
overexpressing cells show greater influx and efflux of Ca“ in response to changes in
external Ca“, indicating that TRPM7 functions as a calcium channel under
physiologically appropriate conditions. Our results also indicate that Mg“ interferes with
the movement of Ca“ through the TRPM7 channel. The data further suggest that the
percentage of TRPM7 channels blocked by Mg“ is proportional to the extracellular Mg
concentration. This implies a possible mechanism for ion permeation through TRPM7
that involves ion-ion interactions within the channel protein and implicates TRPM7
channels in Ca“ and Mg homeostasis.
Introduction:
Transient receptor potential (TRP) channels were first identified in a drosophila
mutant characterized by photoreceptors that produce abnormally short receptor potentials
in response to light (Montell 2002). The melastatin sub-family of TRP channels is
common in many mammalian cells including those found in the lymph nodes, thymus,
bone marrow, and brain (Wolf 2004). TRPM7 knockouts die unless given supplemental
magnesium, indicating that the ion channel is essential for normal cell functions (Schmitz
et al. 2003). Overexpression of the ion channel can also cause cell death possibly due to
apoptosis triggered by the influx of Ca through TRPM7 (Nadler 2001).
TRPM7 may be involved in Ca“, Mg“, and trace metal homeostasis. TRPM7 is
regulated by intracellular Mg- and Mg“-ATP, and the ion channel conducts several
essential and non-essential trace metals, including Zn2, Ni2, Ba2, Co*, Mn2, Sr2, and
Cd“ (Monteilh-Zoller et al. 2003). Imaging studies using the indicator dye fura 2/AM
have suggested that TRPM7 provides a pathway for Ca“ entry into the cell (Monteilh-
Zoller et al. 2003). Patch clamp and imaging studies have demonstrated Mn* influx in
TRPM7 expressing cells in the presence of Ca and Mg“, suggesting that Mn2
competes with Ca“ and Mg- for entry through TRPM7 (Thompson and Hermosura,
unpublished).
Homeostatic regulation of Ca“, Mg-, and trace metals is required for normal cell
function. Mg¬ is a cofactor for many enzymes involved in cellular metabolism. Mg¬
deficiency has been implicated in Chronic Fatigue Syndrome, and a variety of
cardiovascular and neurological problems, including stroke and hemorrhage (e.g. Altura
et al. 1998; Amighi et al. 2004; Van den Bergh et al. 2003). Some trace metals also serve
as cofactors for enzymes, but abnormal levels of trace metals have been correlated with
Alzheimer’s and Parkinson’s disease and related syndromes. Aluminum accumulation in
the amyloid plaques associated with Alzheimer’s disease has been assumed to play a role
in the etiology of the disease, although this assumption has recently been called into
question (Kasa et al. 1995). Öther research has shown that copper and zinc bind to
amyloid beta, which may explain accumulation of these trace metals in amyloid plaques
(Atwood 2000). Parkinson’s related cell death in the substantial nigra (SN) has been
correlated with high levels of free iron, low levels of the iron-binding protein ferritin,
high levels of zinc, and low levels of copper in SN tissue (Dexter et al. 1992). African
green monkeys with unilateral MPTP-induced parkinsonism showed high concentrations
of Fe“ and Fe" in the substantia nigra compacta on the MPTP-lesioned side, leading
researchers to conclude that high concentrations of unbound iron may be linked to cell
death in the SN (Temlett et al. 1994).
However, pathology associated with trace metal accumulation may be better
explained by high levels of trace metals coupled with low levels of Ca and Mg'
During an epidemic of Parkinson’s-ALS symptoms in Guam in the 1950s, many patients
developed severe dementia and most died within five years of the onset of symptoms;
these Parkinson’s ALS clusters occurred in regions of Guam with high levels of
Aluminum and Manganese coupled with low levels of calcium and magnesium in the soil
and river water (qtd. in Yasui and Garruto 1995). Laboratory studies have shown that
rats fed a diet low in Ca“ and Mg- with or without high aluminum showed increased
aluminum deposition in central nervous system tissue (Yasui and Garruto 1995).
Pathology related to disturbance in trace metal and earth metal homeostasis may
become more prevalent in the future because industrial use of trace metals is increasing.
For example, the fungicides Maneb and Mancozeb and the gasoline additive MMT
(antiknock 33x) all contain manganese. Better understanding of the structure and
function of TRPM7 may help in understanding Ca and Mg- homeostasis and the
pathology that results from disturbance of metal homeostasis.
Electrophysiological studies have concluded that TRPM7 is a nonselective cation
channel permeable to most mono and divalent cations (e.g. Kerschbaum et al. 2003),
however when divalent cations are present in the external solution TRPM7 only passes
divalent cations. We propose that TRPM7 may be a calcium channel that can be forced
to pass monovalent cations under the artificial conditions required for voltage clamp
studies. While voltage clamp studies have provided valuable information that has
ultimately led to the current four barrier, three well Eyring rate theory model of the
channel pore, little is known about the channel’s behavior under more physiologically
relevant conditions. Our study examines the behavior of TRPM7 in the presence of
physiologically appropriate levels of Ca and Mg. Our results indicate that TRPM7
functions as a calcium channel in solutions that approximate normal physiological
conditions. We further demonstrate that magnesium blocks Ca“ flux through TRPM7 in
a dose dependant manner.
Materials and Methods:
Cell Culture
Up-regulation of TRPM7 expression was achieved using the A10 cell line
generously provided by A. Sharenberg. A10 cells were created from human embryonic
kidney (HEK-293) cells stably transfected with murine TRPM7/pCDNA under the
control of a tetracycline-inducible promotor. Cells were cultured in Dulbecco’s modified
eagle’s medium (DMEM) with 10% Fetal Bovine Serum (FBS), 0.1% penicillin, 0.1%
streptomycin, 5ug/ml blastocidin, and 0.4 mg/ml zeocin. Cells were kept in a 30° C
incubator with 5% CO2 and passaged every 2-4 days when barely confluent.
Äfter each passage, cells were plated onto glass cover slips that had been coated
with high molecular weight poly-lysine and rinsed with nanopure water to increase cell
adhesion. 24-48 hours after plating, half the cells were induced with lug/ml tetracycline
(TET) so that they would begin to up-regulate expression of TRPM7. Sister cells from
the same plating were left uninduced as controls. Control and TET induced cells
received the same treatment in every other respect. TET induced and control cells show
morphological differences that aided in differentiating between the two groups (fig. 1).
Solutions
Cells on the glass coverslips were transferred from the tissue culture medium to a
buffered saline solution of the following composition (mM): 140 NaCl, 2.8 KCl, 10
Hepes-NaÖH, 2 CaClz. Experimental solutions containing different concentrations of
Ca“ and Mg“ were made using the concentrations listed in table 1. Solutions containing
0.2, 0.02, and 0.002 mM Mg“ were made from a OmM Ca“, OmM Mg stock solution
containing 2mM metal chelator, EGTA (table 1). The amount of MgClz added to the
stock solution was adjusted to account for EGTA binding to free Mg“. Calculations
were made using MaxChelator (Patton et al. 2004). All solutions were titrated with IN or
O.IN NaÖH to bring the pH to 7.2. Glucose (approximately.02g/5Oml) was added to all
solutions a few hours before use.
Imaging
The Ca“ indicator dye fluo3/AM (Molecular Probes) increases in fluorescence
when bound to intracellular Ca“. Cells in 2mM Ca“ buffered saline solution were
loaded with fluo-3 (2.5uL/ml external solution) and gently agitated for 40 min. Cells
were then washed with fresh 2mM Ca“ saline at least 20 min before the start of imaging.
The concentration of Ca“ inside the cell was visualized using an Olympus Fluoview 300
laser scanning microscope with a UPLFL 20x lens (fig. 2).
For viewing under the microscope, coverslips were transferred individually to a
plexiglass perfusion chamber that held Icc of saline solution. A perfusion system
consisting of a lcc syringe with attached microtubing delivered the experimental
solutions to the plexiglass chamber and excess solution was drawn off using a vacuum
pump. Solution changes were made over the course of 60 seconds. Data was collected
every 4 seconds for a total 240 seconds.
Data Analysis
Individual cells were defined as regions of interest (ROIs) using Fluoview
Physiology 4.1 (Tiempo Physiology). Isolated cells or cells with few confluent neighbors
were chosen in order to minimize complications due to cell-cell interactions. Typically
ten regions of interest were defined for each trial. Measurements of fluorescence
intensity over time in each ROI were made using Fluoview Physiology. We used Igor
Pro version 4.01 (Wave Metrics) to create graphs of fluorescence versus time for
individual ROIs and for the average of all ROIs in each trial. In order to compare graphs
from different trials and to account for differences in dye loading across trials, we
calculated a baseline initial fluorescence (Fo) by averaging all the data points collected
before perfusion with a new solution (baseline=O to 20 seconds). We then normalized the
wave by dividing fluorescence at each time point (F) by the initial fluorescence (Fo) to
obtain values of AF/ Fo. Trials in which the same treatment was applied (e.g. removal or
addition of extracellular Ca“) were averaged using Igor Pro. When applicable, the
results are reported as mean value +/- one standard error.
The percent block graphs were designed using the fluorescence intensity of cells
bathed in 0 mM Ca“ saline solution as the hypothetical maximum block of TRPM7.
Percent block was then calculated by comparing fluorescence intensity at the end of each
trial (Ffinal) and plotted on a semilog scale to accommodate the range of Mg?
concentrations studied.
Results:
Response to Changes in Extracellular [Cat 1:
For TET induced TRPM7 overexpressing cells in 2mM Ca2 buffered saline,
initial fluorescence (Fo) calculated from all data points before perfusion (from 0 to 20
seconds) was 0.90 +-.03 (n=12 trials). Values of AF/ Fo decreased to 0.69 +- 0.03
(n=12 trials) following perfusion with the OmM buffered saline solution (fig. 3). This
decrease in fluorescence indicates a decrease in internal [Ca“). We can infer that the
decrease in internal [Ca# is due to Ca“ efflux through TRPM7 because uninduced
control cells showed a much smaller decreased in fluorescence. For control cells, in
2mM Ca“ buffered saline, initial fluorescence (Fo) was 0.99 + 0.01 (n=12 trials).
Values of AF/ Fo decreased to 0.96 +-0.01 (n=12 trials) following perfusion with OmM
Ca“ buffered saline (fig. 3).
TET induced cells also show a greater change in internal [Ca“ in response to
addition of external calcium when compared with uninduced control cells. For TET
induced cells in OmM Ca“ buffered saline, initial fluorescence (Fo) was 1.00 +-0.00
(n—9 trials). Values of AF/ Fo increased to 1.42 +- SE= 0.06 (n-9 trials) following
perfusion with the 2mM buffered saline solution (fig. 4). This increase in fluorescence
indicates an increase in internal [Ca. We can infer that this increase in internal [Ca?
is due to Ca“ influx through TRPM7 because un-induced control cells did not show a
similar increase. For control cells, in OmM Ca“ buffered saline, initial fluorescence (Fo)
was 1.00 +- 0.01 (n=12 trials). Values of AF/ Fo did not change 1.01 +-0.01 (n=12
trials) following perfusion with OmM Ca“ buffered saline (fig. 4).
Response to the Addition of Mg
For TET induced TRPM7 overexpressing cells in 2mM Ca2/OmM Mg buffered
saline, fluorescence values of AF/ Fo increased .03 units (n=10 cells) following perfusion
with the 2mM Ca“7.002mM Mg“ buffered saline solution. The same cells showed a
decrease in AF/ Fo of.15 units (n=10 cells) following perfusion with 2mM Ca/02mM
Mg buffered saline, .17 units (n=10 cells) following perfusion with 2mM Ca2/.2mM
Mg“ buffered saline and .28 units following perfusion with 2mM Ca?/2mM Mg
buffered saline solution (fig. 5a). This decrease in fluorescence indicates a decrease in
internal [Ca“]. We can infer that the decrease in internal [Ca* is due to Ca* efflux
through TRPM7 because the uninduced control cells showed a much smaller decreased in
fluorescence. For control cells, in 2mM Ca“ buffered saline, values of AF/ Fo increased
1 units (n=10 cells) and .05 units (n=10 cells) following perfusion with 2mM
Ca“7.002mM and 2mM Ca“7.02 mM Mg buffered saline, respectively and decreased
02 units (n=10 cells) and .06 units (n=10 cells) following perfusion with 2mM
Ca“7.2mM and 2mM Ca“/2mM Mg buffered saline, respectively (fig. 5b)
Response to the Removal of Mg
For TET induced TRPM7 overexpressing cells in 2mM Ca21/.002mM Mg?
buffered saline, fluorescence values of AF/ Fo increased.07 units (n-10 cells) following
perfusion with 2mM Ca“/OmM Mg- buffered saline solution. The same cells showed an
increase in AF/Fo of.2 units (n=10 cells), .25 units (n=10 cells) and .22 units (n=10 cells)
when buffered in 2mM Ca“7.02 mM Mg“, 2mM Ca/.2mM Mg* and 2mM Ca21/2mM
Mg“ buffered saline, respectively, following perfusion with 2mM Ca/OmM Mg
buffered saline solution (fig. 6a). This increase in fluorescence indicates a increase in
internal [Ca“. We can infer that the increase in internal [Ca2 is due to Ca2 influx
through TRPM7 because the uninduced control cells do not show an much increase in
fluorescence. The control cells in this experiment produced values of AF/ Fo that did not
deviate beyond +-.03 (n—4 trials) (fig. 6b).
Response to the Removal of Ca“ from Solutions of Varied Mg Molarity
For TET induced TRPM7 overexpressing cells in 2mM Ca?/.002mM Mg?
buffered saline, fluorescence values of AF/ Fo decreased .40 units (n-10 cells) following
perfusion with 0 mM Ca“7.002mM Mg buffered saline solution. The same cells
showed a decrease in AF/ Fo of 21 units (n=10 cells), .23 units (n=10 cells) and .08 units
(n=10 cells) when buffered in 2mM Ca“/.02mM Mg“, 2mM Ca21/.2mM Mg* and 2mM
Ca“ /2mM Mg buffered saline, respectively, following perfusion with solutions
containing no Ca“ (fig. 7a). This decrease in fluorescence indicates a decrease in
internal [Ca“. We can infer that the decrease in internal [Ca2 is due to Ca2 efflux
through TRPM7 because the uninduced control cells do not show as much decrease in
fluorescence. For control cells, values of AF/ Fo decreased.14 units (n=10 cells), ,03
units (n=10 cells), .03 units (n=10 cells) and .2 units (n=10 cells) following perfusion
with 0 mM Ca“7.002mM Mg“ buffered saline, OmM Ca2/.02mM Mg buffered saline,
OmM Ca7.2mM Mg buffered saline and OmM Ca2/2mM Mg2 buffered saline,
respectively (fig. 7b).
Discussion:
This study uses change in fluorescence intensity to measure Ca“ accumulation in
the cell. The assumption that Ca“ accumulation is due to Ca“ flux through TRPM7 was
based on the historical reliability of the HEK 293 /AlO expression system as well as
correlative evidence provided by control experiments and the experimental design
(Monteilh-Zoller et al. 2003; Kerschbaum 2003; Thompson and Hermosura,
unpublished).
The experimental method employed throughout this study was one in which
extracellular conditions could be altered during fluorescence imaging. This paradigm
allowed us to address two major topics of interest: the second contingent on the first. By
altering the concentration of extracellular Ca“ in the absence of Mg“ we were able to
establish that TRPM7 is a viable means of Ca“ flux. Three subsequent experiments were
designed to probe the effect of extracellular Mg“ on Ca flux through TRPM7. Trials
involved a dose-gradient in order to best articulate the relationship between Ca“ flux and
extracellular Mg“ concentration. We found that Mg- blocks Ca* flux through TRPM7
in a concentration dependent fashion.
In the first experiment involving Mg“, we found that the fluorescence decreased
consistently with the addition of extracellular Mg“ and concluded that Mg" does block
the influx of Ca through TRPM7. Furthermore, the final fluorescence (Ffinal) was found
to decrease proportionally to the increase of Mg- concentration across trials. Uninduced
control cells also showed a decrease in Ffinal in response to the addition of Mg“, but on a
much smaller scale. The clarity of concentration dependence suggested the usefulness of
a graph depicting the relationship between Mg“ concentration and the percent blockage
experienced by the TRPM7 channels (fig. 8a).
The percent block graph provides some indication of the permeation mechanism
involving TRPM7, Ca flux and changing extracellular Mg- concentrations. If it is
assumed that the block is due to a single binding site on the channel, then Eyring rate
theory would predict an exponential curve reaching a horizontal asymptote as the
mechanism approaches 100% saturation. In this scenario, the Mg“ concentration that
causes a half-block, or 50% channel saturation, would be considered the dissociation
constant, or Kd, for that binding site. Although our study does not avail itself to
definitive results in this regard, we can begin to reveal an exponential trend in the percent
block graph and note a possible half-block at 100 uM Mg“. Using voltage clamp
techniques to study Na’ influx through TRPM7 in the absence of intracellular Mg?
possible Kd of 1 uM has been proposed (Kerschbaum et al. 2003). Although the current
study differs significantly in design, the two results are not in obvious contradiction.
In the second experiment of the series, the Mg“ block was removed and Ca was
better able to pass through TRPM7 channels. Values of Ffinal were seen to increase in a
Mg“ concentration dependent manner. A percent block graph was designed and the half-
block or possible Kd was determined to be 500 uM Mg (fig. 8b). Control trials reflected a
similar increasing trend on a smaller scale. Some deviation from the trend may be
explained by experimental error. If this deviation appears consistently, however, it may
be an indication of more complicated ion-ion interactions at the binding site. It is also
notable that trials involving Ca“ influx appear to show more inconsistency between cells
than trials involving Ca“ efflux (fig. 9). This inconsistency may be due to variability in
the cell cycle and the mechanisms that regulate internal stores and intracellular
concentrations of Ca and Mg.
In the third experiment of the series, in which various extracellular concentrations
of Mg“ were held constant while Ca“ was removed, results showed a sharp decrease in
fluorescence. In addition, the amount of change in fluorescence decreased as the
concentration of Mg“ was increased. That is to say, higher Mg- concentrations meant
more blocking and less intracellular Ca“ available for efflux through TRPM7 at the start
of the experiment. Again, Ca“ flux was found to be dependent on the concentration of
extracellular Mg¬t.
From this study alone we cannot define a permeation mechanism but we have
established a clear line of inquiry for future investigation. Exemplary trials should be
replaced by averages across many trials. An attempt to determine the value of half-block,
or Kd of the binding site, should be made by measuring over a greater range of Mg?
concentrations. A more detailed dose-dependent graph will more accurately depict the
nature of ion-ion interactions at the binding site. Also, voltage clamp measurement of
Ca“ current is necessary to validate our correlation of intracellular Ca accumulation
and Ca“ flux through TRPM7, to better elucidate the properties of individual channel
and to approximate the number of channels involved in each experiment.
Conclusions:
In the absence of Mg“, TRPM7 does act as a Ca“ channel. We can further
conclude that external Mg- blocks Ca" influx and efflux through TRPM7 channels in a
dose-dependent fashion. We have estimated that the half block or possible Kd is between
100 and 500 uM of Mg“. We can further hypothesize that it is the interaction of Mg* at
the binding site(s) of the channel that is slowing or blocking movement of Ca“ through
TRPM7. Most broadly, we can propose that TRPM7 play a role in Mg* and Ca
homeostasis in the cell.
Acknowledgements:
Our warmest thanks go to Chris Patton, Christian Reilly and Peggy Lynch with
special acknowledgement of Stuart Thompson for his oversight and enthusiasm.
Literature Cited:
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E. Minar, M. Schillinger. 2004. Low serum magnesium predicts neurological
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Monteilh-Zoller, M.K., M.C. Hermosura, Nadler, M.J.S., Scharenberg, A.M., Penner, R.,
and Fleig, A. 2003. TRPM7 provides an ion channel mechanism for cellular entry
of trace metal ions. Journal of General Physiology. 121: 49-60.
Montell, C., L. Birnbaumer, V. Flockerzi. 2002. The TRP channels, a remarkably
functional family. Cell. 108: 595-598.
Nadler, M.J., M.C. Hermosura, K. Inabe, A.L. Perraud, Q. Zhu, A.J. Stokes, T. Kurosaki,
J.P. Kinet, R. Penner, A.M. Scharenberg, and A. Fleig. 2001. LTRPC7 is a Mg¬
ATP-regulated divalent cation channel required for cell viability. Nature.
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Schmitz, C., A.L. Perraud, C.O. Johnson, K. Inabe, M.K. Smith, R. Penner, T. Kurosaki,
A. Fleig, A.M. Sharenberg. 2003. Regulation of vertebrate cellular Mg“
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Table 1. Extracellular solutions for all experiments (mM
Nacl
KCl
Hepes
Naoll
27
2 Ca 2Mg
2 Ca 12Mg
140
2 Ca 1O2Mg
140
2.8
10
2 Ca J.002Mg
140
2.8
2 Ca ONg
140
2.8
)
0 Ca pMg
140
10
0 Ca 12Mg
2.8
140
2.8
10
O Ca JO2Mg
140
0 Ca J.002Mg
145
2.8
O CaTOMgE

PH was adjusted to 7.2 with NaÖH
Cach
Mge
.02
002
.02
.002
0
EGTA
2
Figure Legends:
Fig. 1. Morphological Differences between TET induced TRPM7 overexpressing cells
and uninduced control cells. Uninduced HEK-293 cells show a spindly morphology
often with elongated processes. Following induction with tetracycline (TET) cells
become more rounded and often contain large vacuoles.
Fig. 2. TET induced TRPM7 overexpressing cells as they appear using confocal laser
microscopy. Ten individual cells defined as regions of interest (ROIs).
Fig. 3. Change in fluorescence over time in response to a change in external solutions
from 2mM Ca buffered saline to OmM Ca“ buffered saline. Perfusion with the OmM
Ca“ buffered saline occurred from 20 to 80 seconds. Red circles represent the response
ET induced TRPM7 overexpressing cells. Blue triangles represent the response of
uninduced control cells.
Fig. 4. Change in fluorescence over time in response to a change in external solutions
from OmM Ca“ buffered saline to 2mM Ca“ buffered saline. Perfusion with the 2mM
Ca“ buffered saline occurred from 20 to 80 seconds. Red circles represent the response
of TET induced TRPM7 overexpressing cells. Blue triangles represent the response of
uninduced control cells.
Fig. 5. Change in fluorescence over time in response to a change in external solutions,
from OmM Mg“ to one of four concentrations of Mg*. Ca concentration is maintained
at 2mM Ca“. (a) TET induced cells overexpressing TRPM7 (b) Control cells
Fig. 6. Change in fluorescence over time in response to a change in external solutions
containing one of four concentrations of Mg to external solutions containing no Mg
Ca“ concentration is maintained at 2mM Ca“. (a) TET induced cells overexpressing
TRPM7 (b) Control cells
Fig. 7. Change in fluorescence over time in response to the removal of Ca- from
external solution. In each trial, Mg“ concentration is maintained at one of four
concentrations. (a) TET induced cells overexpressing TRPM7 (b) Control cells
Fig. 8. Percent block of TRPM7 as a function of Mg concentration. (a) % block
calculated from experiment in which the concentration of Mg“ is increased while
external Ca“ concentration is maintained. (b) % block calculated from experiment in
which the concentration of Mg“ is decreased while external Ca“ concentration is
maintained.
Fig. 9. Individual cells show considerable variation in their response to a change in
external solution from OmM Ca“ to 2mM Ca
Fig. 1
Control Cells
TET Induced
)
Fig. 2
Fig. 3
g r eengeen
1.0
0.90 -
0.8:
— TET Induced
0.80 -
- Contro


ae g  ge e
0.70 -


100
150
200
Time (seconds)
Fig.
1.4 -
1.3-
+ 12-
1.1 -
1.0-
eeee


Contro
T Induced


100
150
200
Time (seconds)
Fig. 5
A
1.2
TET Induced Cells
1.1-
0 mM to.002 mM
10-

0.9-
0 mM to.02 mM


0.8-
0 mM to.2 mM
07-
0 mM to 2 mM
06-

O mM Mg2t, A Ca?
05+

100
50
150
200
Time (s)
Control Cells
0 mM to .002 mM
W
0 mM to.02 mM
1o Ae
0 mM to.2 mM
0 mM to 2 mM

07-
06+
150
200
Time (s)
Fig.
A

1.00

.002 mM Mg2t


096

0.90 -
02 mM Mg2
2 mM Mg2
085-
30.80

o D.75-
2 mM Mg2
0.70-
OmM Ca
TET Induced Cells
0.65-

50
100
150
200
Time (s)
B
2 mM Mg
.002 mM Mg2

1.0-

Wa

B
02 mM Mg
0.9-
2 mM Mg?
0.8
0.7-
Control Cells
150
50
100
200
Time (s)
Fig. 7
A
1.4-
113-
1.2
511
10
1.4
1.3-
1.2
11-
1.0
.002 mMMg?
02 mM Mg2t
2 mMMg?.
H
2 mM Mg

V
W
100
150
200
Time (s)
2 mM Mg?
002 mM Mg2

02 mMNg

K

V
W
2 mMMgt


50
100
150
200
Time (s)
Fig. 8
A
60
50 -

40-
30-
§ 20-
10-
0-1


2.3 4 5678
2 3 45678
0.01
Mg2 concentration (mM)
B
60 -
50-
40-
30-
20-
10-

2 3 45678
2 3 45678
0.01
Mg concentration (mM)

2 3 45678

2 3 45678
2
2
Fig.
350- ndividual Cells 550592 0O to 20

lo

3000

2500 -
V
2000





1500-



-
1000 -

a-
—

100
50
150
200
Time (seconds)