ABSTRACT:
The family of TRP ion channels constitutes a fairly recent set of cation channels found
in almost all cell types, with diverse roles ranging from mediating responses of nerve
growth factors to regulation of temperature and vasorelaxation of blood vessels. Such a
plethora of functions also reflect diverse permeability to ions. In this study, the specific
member TRPM7 is tested to understand its role in calcium regulation. As an initial test
of the possibility that TRPM7 channels contribute to regulation of Ca2 influx, [Ca2) was
measured by the fluorescent Ca“ indicator, Fluo-3-AM, using confocal laser microscopy
In cells in which TRPMT channel expression was induced and the cells were perfused
with Ca“ -free solution, restoration of extracellular Ca“ (2mM) caused marked
increases in [Ca“1; whereas non-induced cells did not show much change in [Ca2tl
From the proportion of Ca“ that is taken into the cell, it is plausible that TRPMT is a vital
mode of Ca“ entry. A second test looked at the effects of Gd" (20uM) on Ca2 influx.
The introduction Gd““ quenched fluo-3 fluorescence, indicating that Gd" blocked
conductance of Ca“ through the TRPMT channel. A final experiment was conducted to
analyze the effects of adding Mg“. Results indicate that the addition of extracellular
Mg“ (2mM) reduces Ca“ influx. This shows an interaction of both internal and external
Mg“ in the regulation of TRPM7 in blocking Ca2* influx and in allowing Mg2 influx.
Importance of magnesium regulation is indicative of a role for TRPMT in magnesium
homeostasis.
INTRODUCTION
TRPMT
Metal ion transporters play a major role in maintaining the proper concentrations
of important metal ions in the cell, including Ca“ and Mg““. Of recent interest is a newly
discovered group of ion channels, the TRP (transient receptor potential) ion channel
family, which was named after the TRP channel that was first discovered in a
spontaneously occurring Drosophila mutant. In comparison to the wild type, the mutant
expressed only a transient receptor potential to prolonged exposure to bright light. This
resulted from a defect in light-induced Ca“ influx (Montell et al., 2002). Similarity
between all TRP channels is the presence of a six-transmembrane polypeptide
structure that assembles together into tetramers that form cation-permeable pores
(Clapham, 2003). Of particular interest is the TRPM7 cation channels, a member of the
melastatin sub-family that have been shown to have a widespread distribution in many
types of cells, including those of the lymph nodes, thymus, bone marrow, and brain
(Wolf, 2004). TRPMT is unique as a "bifunctional" protein that forms both an ion
channel and a protein kinase that may be involved in mediating other proteins (Cahalan,
2001). Electrophysiological studies show that TRPMT is a nonselective cation channel
that conducts influx of divalent cations and efflux of monovalent (Cahalan & Kozak,
2003, Monteilh-Zoller et al., 2003, Nadler et al., 2001). However, there is partial
selectivity in the presence of certain physiological conditions. For instance, when both
external Ca“ and Mg“ are removed, there is an inward current that conducts
monovalent cations such as Na’ and K (Cahalan & Kozak, 2003). Also, TRPMT
activity has been shown to be modulated by internal Mg“, where -0.6 mM inhibits
TRPM7 currents (Nadler at al., 2003). Not only do the ubiquitous expression of these
channels reflect its vital role, but also the fact that overexpression results in cell death.
In addition, deletion of this channel also results in cell death in certain types of cells,
Therefore, presence of TRPMT serves a vital role in cellular processes, and cracking in
mechanisms of regulation of specific cations would aid in understanding its vital role.
Calcium
Divalent metal ions, such as Ca“ and Mg“, are essential for many metabolic
processes. Ca“ is a ubiquitous intracellular regulator of cellular function, serving as an
important secondary messenger in many cellular processes ranging from contraction
and motility to cell division and cell death. It is present at -100 nM in the intracellular,
which is much lower than the 2 mM concentration found extracellularly. The strict
regulation of intracellular Ca“ indicates that reduced levels, or prolonged exposure to
increased levels of Ca“, result in cell death. Previous studies using voltage clamp
experiments showed that TRPMT is a significant entry pathway for Ca“ (Monteilh-Zoller
et al., 2003). This implies that TRPM7 plays a role in Ca“ regulation. However, the
voltage clamp experiment looked at cation current through the channel, and not
specifically isolating Ca“. This experiment attempts to isolate Ca“ by using a calcium
fluorescent indicator, Fluo-3, in order to specifically analyze Ca2 influx.
Magnesium
Mg“ plays a unique role in the cell by attaching to ATP, thus providing a Mg-ATP
form that is used by most enzymes in important processes such as proliferation and
differentiation (Wolf, 2004). Therefore, proper levels of Mg“ is critical to cellular function.
Although studies have been conducted to measure Mg“' uptake into the cell, the
particular proteins involved in the process are not clearly understood. Understanding
the mechanisms involved in magnesium homeostasis will aid in the development of
pharmaceutical drugs that target symptoms of magnesium deficiency (such as chronic
fatigue syndrome) and diseases that may be treated with an extra boost of magnesium.
For instance, preeclampsia is a serious condition developed by women in late
pregnancy, and results in high blood pressure, edema, muscle contractions and even
death if the condition of the woman is not treated. Magnesium sulfate have been used
to provide patients with increased levels of magnesium, which gets taken up by muscle
cells and causes them to relax. But a more effective thereapy, as suggested by
Scharenberg, would be to directly increase the magnesium uptake into cells of the
smooth muscle. Understanding magnesium regulation through the TRPMT may aid in a
drug that activates TRPMT to allow for magnesium influx.
Importance of magnesium is also implicated in studies of TRPM6 channels,
which have similar channel and enzyme characteristics as TRPM7. Konrad et al (2004)
found that when the TRPM6 is mutated, there is both a defect in intestinal magnesium
absorption and renal magnesium conservation, which are characteristic of
hypomagnesemia with secondary hypocalcemia. This study stresses the important role
of TRPM6 in magnesium transport. Structural similarities between TRPMT and TRPMG
suggest that TRPMT may also have similar roles.
Considering the vital role of magnesium and possible links of magnesium
regulation in the TRPMT channel, recent interests in the TRPMT channel have been
shifted towards its possible role in magnesium homeostasis (Wolf, 2004). TRPMT have
been shown to have a clear role in mediating magnesium uptake into the cells. The
native conductance of TRPM7 was previously identified in Jurkat T Lymphocytes and
RBL Cells, and described as the MagNuM (magnesium-nucleotide-inhibited metal) and
MIC (magnesium-inhibited cation) (Nadler et al., 2001; Hermosura et al., 2002, Prakriya
and Lewis, 2002). Cahalan and Kozak (2003) show that in addition to internal free Mg?
other divalent cations, including Ba2’, Sr2*, Zn2', and Mn2', also inhibited current
through the TRPM7 channel. But considering the higher Mg2 concentration in the cell.
Mg“ has a larger inhibitory effect. Monteilh-Zoller et al (2003) showed that although
TRPMT is nonselective to metal ions, with affinities of Zn2tæ Ni2t »» Ba2t» co2 » Ma
2 Mn“ 2 Sr2 Cde2 Ca“, physiological conditions mainly permit the transport of Mg?
which is one of the main cations present in the extracellular fluid. This study attempts to
shed some light on the relation of TRPM7 and extracellular Mg2t. By studying
interactions of extracellular Mg“ and Ca2 through TRPM7 at physiological
concentrations, we are able to further understand how TRPMT regulates these cations.
Gadolinium
Gd“ is an important trivalent lanthanide that has been used extensively in a
diagnostic manner to study characteristics of ion channels. In particular, Gd" have
been shown to block many mechanically-stimulated ion channels as well as most Ca-
channels, most notably the L-type and N-type calcium channels (Biagi and Enyeart,
1990; Caldwell et al., 1998). It has been shown to prevent atrial fibrillation by blocking
stretch-activated ion channels in astrocytes (Bode et al., 2001). It has also been used
as a contrast enhancer in MRI. Although Gd“ have been shown to block TRPCI and
TRPM4, effects of Gd“ have not been tested with the other TRP sub-families (Clapham
et al, 2002). Furthermore, although TRPMT is not known to be mechanically stimulated.
it does have an affinity for Ca“. Therefore, it is important to study the effects of Gds
which may yield important implications on the characteristics of TRPM7, and its possible
regulatory mechanisms
It is plausible that deviations in TRPM7 function contribute to abnormal Ca?t
Mg- and other metal ion accumulation that may be associated with neurodegenerative
diseases such as Parkinson's and Alzheimers, and other diseases of Mg“ deficiency.
The study of ion permeability through these channels allows us to understand the
mechanisms through these channels and the important roles they play.
MATERIALS & METHODS
Cell Culture
HEK293 (Human embryonic kidney cell line) cells were transfected with the mTRPM7
gene, and grown on glass coverslips with DMEM medium supplemented with 10%
bovine serum, 10% FBS, blasticidin (5uglmL), and zeocin (0.4 mglmL). These cells
were chosen for their low or null basal expression of TRP genes. TRPM7 expression
was induced 1 day before experimental run by adding 1.5uglmL tetracycline in the
culture medium. Experimental runs were done 25-30 hours postinduction.
Solutions
• Measurements of intracellular changes in fluorescence (as an indicator of Ca2
influx) was carried out using the Ca“ sensitive indicator Fluo-3-AM under a laser
confocal microscope. Cells grown on glass coverslips were transferred to small
petri dish to be loaded with Fluo-3-AM (5uL). Cells are loaded with the dye for 40
minutes at room temperature, then washed for 5 minutes on a rotator. Bathing
solution is a standard solution of the following composition (mM): Nacl 140, KCI
2.8, HEPES NaÖH 10, Caclz 2, MgClz 2, Glucose 10, pH 7.2. Cacl2 and Mgolz
concentrations were adjusted based on the specific experimental test.
Adjustments are as indicated in the text and figure legends. In specific solutions
-2+
of O mM Ca270 mM Mg2, O mM Ca 1z mM Mg2t, 2 mM Ca2t/0 mM Mg2“, Nacl
concentration is adjusted to 145mM to correct for osmolarity
1mM GdCl stock solution was obtained from Dr. Bill Gilly's lab. This was
adjusted to 20uM using the specific Ca“/Mg“ solution in the experimental run.
lo obtain maximum fluorescence for calibration, 1mM ionomycin was added into
ImL of either 2 mM Ca270 mM Mg2 or 2 mM Ca27/2 mM Mg2* solution to obtain
2uM ionomycin solution, which was then added at the end of each experimental
run. Calibration to a maximum fluorescence allows us to look at compare
fluorescence changes between different experimental conditions and between
the induced and non-induced cells
Solution changes were performed through a hand pipetting (using an electric
pipetter) through one end of the chamber and a suctioning system on the other
end of the chamber. Chamber was kept at 30°C during the experimental run.
Fluo-3 measurements
Olympus Confocal Laser Microscope was used to visualize Fluo-3-loaded cells.
Cells are placed in perfusion chambers (made of plexy-glass). Images were
collected at 2.8 second intervals. Images were displayed on a computer monitor
and recorded during a 28 second baseline period, a 34 second perfusion period
and cells are let to sit in the test solution for the remainder of the time series (until
the end of the 210 second test run).
• Considering that the dissociation value (Ka) may vary in different cellular
environments such as pH, temperature, viscocity, presence of Mg“ and other
ions (Probes.com, 2004), [Ca“jis not calculated using the formula ([Ca2hee-
KalF-Fminl/IFmax-F1) (Kao et al, 1989). Rather, changes in [Ca2'1 (AJC:
24) were
estimated as % AFIFo, which provides a quantitative estimate of the proportion of
fluorescence changes relative to the initial basal fluorescence level that is
distinctive to each cell. To obtain this value, fluorescence at each point in time
(F) is divided by the maximum fluorescence (Fmax) of each specific cell, obtained
from adding ionomycin to the extracellular Ca* solution. This value is then
normalized to the average of the basal fluorescence level Fo (t=O to 28 seconds).
to give us the fluorescence change relative to the basal level. % AFIFo was
obtained for 6 individual cells per experimental run. The Igor Pro 4.09A program
is used for graph displays and data analysis.
To account for differences in cell biology, cells present at different stages of the
cell cycle and differences in dye load, we established a criterion from preliminary
analyses in which cells chosen were most similar in dye load, cellular
characteristics and cellular cycle. Individual cells chosen were based on
similarities in size, shape, basal fluorescence level, and cells were mostly those
that are isolated from other cells, or are only touching one or two cells (to avoid
cellular processes that may affect the cells).
Statistics
Unpaired t-test and single-factor ANÖVA were used to test for statistical
significance. Significance was considered to be less than p=0,05.
RESULTS
TRPMT provides a significant entry pathway for Ca“
To test whether there is a significant Ca“ influx through the TRPMT channel, we
ran an image scan of cells in an initial bathing solution of OmM Ca2*/OmM Mg2. Cells
were then perfused with a test solution of 2mM Ca2/OmM Mg* at 28 seconds into the
experimental run. Figure 1 shows a comparison of the fluorescence changes between
the induced and non-induced cells. In induced cells, the increase in intracellular Ca?
concentration in response to exposure to an external concentration of 2mM Ca2 is
characterized by a rapid fluorescence rise to a maximum of 222.4 + 12.1% (means +
SEM, n=24) that of the basal fluorescence level. By contrast, fluorescence increase in
the non-induced cells reach a maximum of 106.14 + 6.7% (n-24) that of the basal level
which is a mere 6.14% increase from the basal level. Therefore, the overexpression of
TRPMT in the induced cells significantly contributes to calcium influx (p-0.0002),
Considering the possibility of dye fade, the 6.14% fluorescence increase in the non¬
induced cells is significant to show that the other calcium channels present in the non¬
induced cells are also contributors to the intracellular Ca“ influx. The gradual decrease
in fluorescence rate of increase results from saturation of all channels. Evidence of a
gradual plateau in fluorescence level is present towards the end of the time series.
20 UM Gd“ blocks Ca2 influx
lo assess the possibility of an external block by Gd"", we exposed induced cells
to a series of experimental runs in a schematic order. First, we tested the fluorescence
change of induced cells from an initial bathing solution with OmM Ca2*/ OmM Mg2t/OuM
Gd“, to a testing solution of 2mM Ca2*/ OmM Mg2t/OuM Gd3. This fulfills one
experimental run. Next, the cells washed out with the OmM Ca2*/ OmM Mg27/OUM Gd¬
and tested with a solution of 2mM Ca2/ OmM Mg2720uM Gd“' to observe the Gd
effects. To control for variations between cells, we analyzed fluorescence changes
analysis on the same cells going through the different test solutions. With the
introduction of the 2mM Ca“ solution with O uM Gd“', fluorescence rose to a maximum
of 225.93+- 19.85% (n=6), which is consistent with the experiment testing ca influx
through the induced cells. However, when 20uM Gd“ was added to the Ca2* solution,
the maximum fluorescence was 87.48 + 2.51% (n-6) that of the basal level. This is an
approximately 10% less than the basal fluorescence. The decrease shows that not
much Ca“ entered the cells during the experimental run, and the drop in fluorescence is
indicative of dye fade. Gd“, therefore, blocks influx of Ca2' through the TRPMT
channels (p20.0001). Furthermore, In comparison to non-induced cells that have been
exposed to the 2mM ca solution (maximum of 106.15+- 7% (n-24)), the maximum
fluorescence value achieved with the Gd“' block is significantly less (p=0.0188). This
indicates that not only does Gd“ block the TRPM7 channels present in the
overexpressed cells, but may possibly block other Ca“ channels present in the HEK293
cells.
In a second experimental run, we reversed the order of exposure to the Gd¬
block to see if the block is reversible. This is also done to account for confounding
factors due to the order of the experimental manipulation, such as effects on cell
physiology due to prolonged experimental manipulations. Cells were first exposed to a
bathing solution of OmM Ca27 OmM Mg-/OuM Gd“ and then perfused with the testing
solution of 2mM Ca27 OmM Mg-720uM Gd“. Fluorescence increased to a maximum
value of 110+-4.07% (n=6). Thus, reversing the order of the experiment still shows that
Gd° blocks the TRPM7 channel (p50.0001). Compared to the non-induced cells
exposed to just the 2mM Ca solution, this increase is not statistically significant
(p=0.6408). Discrepancies between the significance levels of the Gd“ solution tested
first and the Gd““ solution tested second to the non-induced cells tested with 2mM Ca¬
indicate that further tests need to be conducted to understand how Gd“' may be
affecting other Ca channels in the HEK293 cells. Äfter this, the Gd“t and Ca2t solution
12
was washed out with OmM Ca“7OuM Gd“ solution, and fluorescence change was
recorded for the testing solution with just 2mM ca. Fluorescence increased to a
maximum of 221+-13.1% (n=6), indicating that Gd“ effects are reversible (p50.0001).
A comparison of difference in fluorescence changes between cells exposed to
Gd“ first in the experimental run, and cells exposed to Gd" after a test with just the
Ca“ solution has been conducted, reveal that there is significance in the order of Gd'
exposure (p=0.0214). There was a greater influx of Ca“ in cells first exposed with Gd¬
which can be explained by differences in cell biology. Cells exposed to a Gd"-free
solution first experienced a greater decrease in fluorescence from the basal value.
showing that the effects of Gd“ are stronger with earlier experimental manipulation. To
this end, we also analyzed the Ca“ tests in these experimental runs, and found that the
order of these tests were not significant (p-0.8386). Thus, calcium influx into the
TRPMT seems not to be affected by order of exposure to calcium, which indicates that
magnitude of Gd“ effects are fully reversible. We conclude that in a Mg?-free solution.
20 uM Gd° blocks Ca“ influx through TRPMT channel and this block is reversible.
Physiological Mg“ levels impede Ca“ permeation through TRPM7
To test the effects of external Mg“ on Ca influx through TRPM7, cells were
2+„
exposed to a testing solution of 2mM Ca 7zmM Mg“ solution. Fluorescence increased
to a maximum of 96.53 +-3.83% (n=12), in comparison with cells exposed to a solution
of just 2mM Ca“, where the maximum fluorescence increase is 222.4 + 12.1% (n=24).
Comparison between these two testing solutions shows that Mg* significantly blocks
Ca influx (p«O.0001).
Comparing significance of effects between Ca“ influx in the presence of Mg",
Gd“, and in non-induced cells
To assess whether the Mg“ and Gd“tis blocking other channels in the HEK293
cells, an ANOVA comparison was used to test significance of the maximum
fluorescence rise in the cells exposed to 2mM Cal2mM Mg* (n=12), 2mM Ca2 /20uM
Gd“ with Gd“ tested first in order (n=6), 2mM Ca2t /20uM Gd3t with Gd“ tested
second in order (n=6), and the non-induced cells exposed to just 2mM Ca2* (n-24),
Results show no significant effects (p=0.5) when the two Gd“ tests were pooled
together (n=12), indicating that Mg“ and Gd“ perhaps only blocks TRPMT channels.
However, the significance seen in cells that were exposed to Gd“ in the second order
indicate that further tests will need to be done to analyze this effect.
DISCUSSION
Results from this study hold important implications for Ca2 and Mg2t signaling
through the TRPM7 channel. It is shown that the mTRPMT gene, when overexpressed
in HEK293 cell line, is a Ca“ channel that allows Ca2* accumulation in Mg"-free
extracellular environment. Influx of Ca“ was also present in non-induced cells, although
in relatively low amounts. This reflects the presence of other Ca*' channels in the
HEK-293 cells, which also contribute to the increased fluorescence value seen in the
induced cells. However, there is a high significant difference between induced and non¬
induced cells, which indicates that TRPMT offers a substantial entry pathway for Cae in
14
the absence of extracellular Mg“. Our results contradict that of Monteilh-Zoller et al
2003, in which it was found that at extracellular concentrations of 2mM Ca2' and 2mM
Mg“, there is significant Ca“ influx through the overexpressing TRPMT.
We also demonstrated the inhibitory effects of both 2mM Mg* and 20 uM Gd3
on Ca“ influx through the TRPM7. Ca“ influx is blocked by Gd", which is a trivalent
cation that commonly blocks mechanoreceptors and Ca“ channels. Considering that
Gd“ has a similar ionic radius to Ca2 (0.938 A as compared to 0.99 A, respectively),
Gd“ perhaps serves as a cation competitor that mimics Ca“ in its binding to the pore of
ion channels (Hamill and McBride, 1996). The finding that Gd" shows a reversibility of
effect indicates that Gd“ does not permanently bind to the competing binding site in the
ion channel. The usage of Gd“ serves as a tool for characterizing the channels, and
for studying the permeability pathways. Our results open up a gateway for further
investigations into the mechanisms of the TRPM7 channel, which may hold broad
implications for the TRP family.
Similar to Gd“, we propose that there may be binding site competition, in which
Mg“ binds to a site in the channel where Ca“ normally binds to. Our results show that
the presence of 2 mM Mg“ significantly inhibits Ca* influx through the TRPM7 channel.
This is consistent with an electrophysiological study, where it was found that at above
micromolar concentrations of external Mg“, the I shape of the monovalent current
through the channel was altered, and inward currents were preferentially blocked
(Kerschbaum et al., 2003). Although Gd“' has a similar magnitude of blocking effect on
the TRPM7, the 2mM Mg“* in combination with 2mM Ca2 is the normal physiological
condition of the cell. Therefore, under physiological conditions, there may be
competitive interaction for absorption of Mg* and Ca2, such that Mg2" strongly
decreases the Ca“ influx. Monteilh-Zoller et al (2003) found the affinities of TRPM7 to
a number of divalent cations, with Mg“ affinity in the middle of the range, and Ca?
affinity at the lowest end. But considering the high concentration of both Mg2t and Ca?
in the extracellular, in comparison to miniscule amounts of trace metals, these two jons
probably out-compete the trace metals. The higher affinity of TRPMT to Mg2 indicates
that Mg“ does not necessarily block the TRPMT channel, but rather out-competes Ca¬
for binding site within the channel. Thus, under physiological conditions, the channel is
more specifically targeted towards magnesium, as well as allowing an entry way for
trace metals, since TRPM7-mediated trace metal entry were also found to occur at
physiological levels of Ca“ and Mg“ (Monteilh-Zoller et al, 2003). In addition,
considering that Gd“ inhibits Ca“ influx in a Mg“-free solution, it would be interesting
to conduct further research on the possibility of a Gd“ block on Mg* influx under normal
physiological conditions.
Previous research shows Ca““ influx is blocked by O.6mM internal Mg2t, during
which channel allows efflux of monovalent ions. Therefore, it seems that both internal
and external Mg“ levels act in a complementary manner to block Ca2* influx. In
addition, Schmitz showed that millimolar concentrations of internal Mg“ completely
blocked the inward current through TRPM7, which also blocks Mg“ influx. The negative
feedback loop therefore allows homeostatic regulation of Mg“ through TRPM7. The
importance of TRPMT in regulation of Mg“ influx is shown in knockout experiments
where the absence of TRPMT resulted in cell death. In addition, in the same study
knockout cells that were introduced with supplemental amounts of Mg“ were able to
survive (Schmitz et al, 2003). This further supports an important Mg2 regulatory
pathway in the TRPM7. In addition, overexpression of TRPMT in many different types of
cells is also lethal (Nadler, 2001). This perhaps indicates that the overexpressed
channels result in an imbalance in intracellular Mg2“. Further evidence of Ma?
homeostatic regulation in TRPM7 come from studies of the close relative TRPM6.
where it was found that TRPMG is vital in magnesium reabsorption. Patients who have
an inherited form of hypomagnesema were found to have mutations in TRPM6,
Chubanov et al. (2004) suggests an interaction of TRPM6 and TRPM7 to form a
functional complex at the cell surface for regulation of magnesium. Further studies are
needed to understand how heteromers of TRPM6 and TRPMT may affect regulation of
magnesium homeostasis in the cell.
Magnesium homeostasis is crucial to life. Understanding regulation of Mg“
through the TRPMT channel aids in studies of pharmaceutical drugs that may alter the
function of TRPMT to selectively allow for rapid Mg“ uptake into the cells. Drugs that
can inhibit or stimulate activity of this ion channel may be able to help to manage or
even stop certain diseases. The fact that TRPMT is a significant entry pathway for Ca¬
in a Mg“-free extracellular environment may imply mechanisms cellular dysfunction in
people suffering from magnesium deficiency. Considering the possible vital role that
TRPM7 plays in magnesium homeostasis, those suffering from magnesium deficiency
may experience an increase in Ca“ influx that is normally blocked by the presence of
Mg“. Both the decrease in intracellular Mg“ and the subsequent increase in Ca¬
above homeostatic levels may lead to negative effects in the cells.
CONCLUSIONS
Under Mg“-free environment, TRPMT shows high affinity to Ca2* influx. However,
rather than an entry pathway for Ca*', TRPMT is more important as a transporter of
Mg- at physiological levels of Ca“ and Mg“. Considering that TRPMT is ubiquitous in
many different types of cells and its role in Mg“ homeostasis, TRPMT may be a critical
to life. Role of TRPM7 in regulation of Mg“ implicates its importance in cellular
metabolism. The study of Mg“ regulation through the TRPMT is a promising avenue for
pharmaceutical drugs that target many types of diseases.
ACKNOWLEDGEMENTS
I wish to thank professor Stuart Thompson for his guidance, insightful teachings, and
support. I would also like to acknowledge Arjun Rustagi for his assistance in the
experiments and in using the Igor program, and Chris Patton for his help in fixing my
computer problems.
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FIGURES

200 -
Induced Cells (n=24)
180 -
160 -
140-
Non-Induced Cells (n-24)
120-
100

150
50
100
200
Time (seconds)
Fig 1. Comparison of Ca“ influx between induced and non-induced cells. Cells are initially bathed in a
OmM Ca“/OmM Mg“ solution (from t=0 to t-28). At 28 seconds into the experiment, cells are perfused
with the test solution 2mM Ca“/OmM Mg“. Induced cells show a maximum rise of 222.4 + 12.1% (means
+ SEM, n=24) in comparison to basal level (t=O to 28). Non-induced cells show a maximum of 106.14 +
6.7% (n=24). P=0.0002.
220 -
200-
OUM Gd3+ (n-6)
180
160-
140 —
O UM Gd3+ Non-induced (n-24)
120 -
20 UM Gd3+ (n=6

g
100
d gdeeg
2
100
150
200
Time (seconds
Fig 2. Effects of 20uM GC
on Ca“ influx through TRPM7 (induced cells). Order of experimental tes
2mM Ca2/ OmM Mg“/OUM Gd“ tested first, then 2mM Ca“/ OmM Mg“/20uM Gd“. Presence of Gd
shows significant block of Ca“ influx (ps0.0001). Comparison of significance of 20uM Gd“ in induced
cells (dotted line) with OuM Gd“ in non-induced cells (circles): p-0.0188.
OUM Gd34 (n-6)/
200 -
180 -
160
140 -
20 UM Gd3+ (n=6
120 -
O UM Gd3+ Non-Induced cells (n=24)

6
100 asas
dooog, o og
o o
100
150
200
Time (seconds)
Fig 3. Effects of 20uM Gd“ on Ca“ influx through TRPMT (induced cells). Order of experimental test:
2mM Ca2/ OmM Mg“/20uM Gd“ tested first, then 2mM Ca“/ OmM Mg“/OuM Gd“. Order of experiment
is reversed to show possibility of a reversibility of the Gd“ block (p-0.0214). Presence of Gd“ shows
significant block of Ca“ influx (p20.0001). Comparison of significance of 20uM Gd" in induced cells
(dotted line) with OUM Gd“ in non-induced cells (circles): p-0.6408.
200 -

O mM Mg2+ (n-24)
180 -
160 -
140-
2 mM Mg2+ (n=12)
120:
.--

100
50
100
150
20
Time (seconds)
Fig. 4. Comparison of effects of 2mM Mg on Ca“ influx through the TRP channel (p50.0001).
Tests are performed on induced cells.