 |
INTRODUCTION |
Parathormone (PTH)1
secretion from the parathyroid gland is suppressed by high
extracellular calcium and magnesium (1). The calcium-sensing receptor
(CaSR) is responsible for the calcium-dependent inhibition
of PTH secretion (2). Direct binding of calcium or magnesium activates
the CaSR (3). Activation of the CaSR triggers
G
q/G
i-mediated signaling pathways (4).
Several mutations have been identified with increased activation of
this receptor (5, 6). CaSR mutants with increased affinity/potency for the agonist calcium and in part enhanced constitutive activity led to permanent inhibition of PTH secretion (7). Therefore, patients
with activated CaSR mutants suffer from hypoparathyroidism. A similar
phenotype of blunted PTH secretion is seen in patients with severe
magnesium deficiency (8-10). This finding is unexpected since the
effects of high magnesium on parathyroid hormone secretion are similar
to those of calcium, and therefore, low magnesium should be expected to
result in increased PTH secretion. And indeed, in contrast to patients,
rats respond to severe hypomagnesemia with increased secretion of PTH
(11, 12). It is known that hypomagnesemia reflects intracellular
magnesium deficiency (9). Thus, the site of magnesium action has been
assumed to lie intracellularly (9). However, causality between blunted
PTH secretion and magnesium deficiency is not established, although the
magnesium paradox has been known for more than 20 years (8). In search
for the mechanism we investigated the relationship between magnesium
deficiency, PTH secretion, and CaSR-mediated signaling. We present
evidence that increased activity of G
subunits leads to enhanced
signaling mediated by constitutive activation of the human CaSR.
Enhanced CaSR-mediated signaling may thus constitute the link between
severe hypomagnesemia and blunted PTH secretion.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
Human embryonic kidney cells
(HEK-293) were cultivated and transfected with plasmids encoding the
wild-type and the different CaSR mutants (7, 13-15) under the control
of the cytomegalovirus promotor as described (16).
Construction of G
i and CaSR Mutants--
For
mutagenesis of G
i we used the Escherichia
coli expression vector pQE60 containing the
G
i1-cDNA with an internal nucleotide sequence after
amino acid 121 encoding a hexahistidine tag. The codon coding for
arginine 209 of G
i1 was exchanged to cysteine by
site-directed mutagenesis (16). For mutagenesis of the human CaSR, an
additional restriction site for XhoI was introduced (silent mutation of leucine 276). The mutated cDNAs were sequenced entirely to confirm the identity of the mutants.
Expression and Purification of G
s and
G
i1--
The G
s and G
i1
proteins (wild-type and R209C) were expressed in E. coli
BL21(DE3) and purified by Ni2+-agarose according to the
manufacturer's protocol (Qiagen). Purified proteins were desalted and
concentrated by centrifugation through a Centricon concentrator with an
exclusion limit of 30,000 Da (Amicon). After the addition of 20% (v/v)
glycerol, the purified proteins were frozen in liquid nitrogen and
stored in aliquots at
80 °C at a protein concentration of 2-10
µg/µl.
Purification of G
o from Bovine
Brain--
G
o from bovine brain was prepared as
described (17).
Expression of G
q in Sf9 Cells--
G
q was expressed in Sf9 cells together
with G
1
2 using recombinant baculoviruses.
Forty-eight hours after infection, cells were harvested, and membranes
of baculovirus-infected cells were prepared as described (16).
Membranes were directly assayed for [35S]GTP
S binding
in comparison to control membranes expressing G
1
2 alone, since G
q was
not stable during further purification.
Preparation of HEK-293 Membranes--
Membranes of HEK-293 cells
transfected with the cDNA encoding the human CaSR were prepared at
4 °C by sucrose density gradient centrifugation as described (18).
The membrane pellet was treated with 5 M urea, washed, and
stored at a protein concentration of 0.1-0.5 µg/µl at
80 °C
.
Determination of [35S]GTP
S Binding to G
Subunits--
Basal and receptor-stimulated binding of
[35S]GTP
S to G
i, G
s,
G
o, and G
q was determined as described
(18, 19). Briefly, membranes of HEK-293 cells (control or
CaSR-transfected, 5-10 µg of protein/assay in 50 µl) were
reconstituted with the indicated G
subunits (40 nM) and
bovine brain G
(100 nM) for 30 min on ice in reaction
buffer (25 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, 160 nM GDP, and
MgCl2 as indicated). Similar results were obtained with
MgSO4. For determination of basal guanine nucleotide binding to G
, membranes and G
were omitted. The experiment was
started by the addition of [35S]GTP
S (60 nM; 2 × 106 cpm/50 µl). The reaction
tubes were placed at 25 °C, and at different time points, samples
(50 µl) were withdrawn and passed over nitrocellulose filters
followed by three washes with ice-cold washing buffer (20 mM Tris, 100 mM NaCl, 25 mM
MgCl2, pH 7.4), and filter-bound radioactivity was determined.
Determination of Cellular Inositol Phosphate
Levels--
Inositol phosphate levels of dispersed parathyroid or of
HEK-293 cells were determined as described (19) with minor
modifications. Before the experiment, cells were washed with buffer
(138 mM NaCl, 0.5 mM CaCl2, 5 mM KCl, 20 mM Na+-HEPES, pH 7.4)
containing the indicated concentration of MgCl2 and stored
in the same buffer for 15 min to equilibrate the cells with the
Mg2+ concentration tested. Then 10 mM LiCl was
added. After 20 min at 37 °C, cellular inositol phosphates were
extracted and determined.
Determination of Cellular Levels of Cyclic AMP--
Cellular
cAMP levels were determined in dispersed parathyroid cells or in
HEK-293 cells 48 h after transfection. Before the experiment the
cells were washed with buffer (138 mM NaCl, 0.5 mM CaCl2, 5 mM KCl, 20 mM Na+-HEPES, pH 7.4) containing the indicated
concentration of MgCl2. Cells were allowed to equilibrate
with the indicated Mg2+ concentration for 15 min. Then
buffer with the specified Mg2+ concentration and
supplemented with 0.2 mM isobutylmethylxanthine was added,
and the experiment was started by the addition of forskolin (10 µM), isoproterenol (100 nM), or buffer as a
control. After 20 min at 37 °C, cellular cAMP was extracted and
determined by radioimmunoassay (Immunotech).
PTH Release from Dispersed Human Parathyroid Cells--
Human
adenomatous or primary hyperplastic parathyroid glands removed during
surgery from patients with hyperparathyroidism were immediately placed
in ice-cold RPMI-medium. Dispersed parathyroid cells from parathyroid
tissue were prepared by digestion with collagenase and DNase similarly
as described (20, 21). For determination of PTH release, dispersed
parathyroid cells (1 × 105) were equilibrated for
1 h in incubation buffer (138 mM NaCl, 5 mM KCl, 20 mM Na+-Hepes, pH 7.4)
supplemented with CaCl2 and MgCl2 as indicated. After washing, PTH release was performed for 10 min at 37 °C in the
specified buffer. The concentration of immunoreactive PTH in the
incubation medium was measured by immunoradioactive assay determining
intact human parathyroid hormone (Intact PTH, Nichols Institute
Diagnostics). Inositol phosphate and cAMP levels were determined in
dispersed parathyroid cells similarly as described above.
Measurement of the Intracellular Free Mg2+
Concentration, [Mg2+]i--
For determination of
changes in [Mg2+]i, HEK-293 cells in incubation
buffer (138 mM NaCl, 5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 20 mM Na+-Hepes, pH 7.4) were loaded with 5 µM mag-fura-2/AM dissolved in dimethyl sulfoxide with
0.02% pluronic acid for 30 min at 37 °C in a humidified incubator.
Cells were washed three times with warmed incubation buffer and
incubated for 30 min at 37 °C to ensure complete deesterification.
Cells were finally washed once with fresh incubation buffer
supplemented with CaCl2 and MgCl2 as indicated.
Fluorescence was recorded with a PerkinElmer fluorescence photometer
(LS50B) at an emission wavelength of 520 nm and an excitation
wavelength alternating between 340 and 380 nm.
[Mg2+]i was determined from the ratio between 340 and 380 nm as described previously (22).
 |
RESULTS |
Effect of Magnesium Deficiency on PTH Release from Human
Parathyroid Cells--
In patients with severe hypomagnesemia, PTH
secretion is blocked (8-10). To analyze whether this in
vivo paradox is related to the release of PTH from parathyroid
cells, we determined the effect of low magnesium on PTH secretion
in vitro on dispersed parathyroid cells. As a control for
calcium-dependent stimulus-secretion coupling of the
parathyroid tissue from patients with hyperparathyroidism, stimulation
of the CaSR by calcium was measured. Calcium as a CaSR agonist blocked
the release of PTH with an EC50 of 1.5 ± 0.2 mM (n = 4) when the Mg2+
concentration was 1 mM (Fig.
1A). This EC50
value is in close correlation with the set-point values reported
previously for human adenomas or primary hyperplasias (23, 24). Severe
magnesium deficiency was simulated by decreasing the extracellular
Mg2+ to 0.1 mM. When Mg2+ was
decreased, PTH secretion was blocked independently of the extracellular
Ca2+ concentration (Fig. 1A). These findings
resemble the in vivo situation in patients; magnesium
deficiency blocks PTH release, leading to concomitant hypocalcemia
(8-10), and calcium replenishment cannot overcome this inhibition of
PTH secretion (9). The IC50 value of magnesium for the
inhibition of PTH release was 0.25 ± 0.04 mM, and
inhibition of PTH release is not seen before the Mg2+
concentration falls below 0.5 mM (Fig. 1B). This
finding correlates again with in vivo data demonstrating
that the magnesium levels in patients that induce PTH secretion block
are generally very low, with concentrations varying between 0.4 and 0.1 mM (8-10).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of magnesium on PTH release from
dispersed parathyroid cells. A and B,
dispersed parathyroid cells (1 × 105) were incubated
for 10 min in incubation buffer with the indicated concentration of
Mg2+ and Ca2+, and PTH release was determined
by radioimmunoassay. Basal PTH release at 0.5 mM
Ca2+ and 1 mM Mg2+ was 4.9 ± 1.5 ng/105 cells/10 min. C, inhibition of basal
cAMP-levels (=100%) in dispersed parathyroid cells incubated for 10 min in buffer with 0.5 mM Ca2+ and the
indicated concentration of Mg2+. D, inositol
phosphate levels of dispersed parathyroid cells labeled with
myo[3H]inositol for 12 h. Cells were incubated in
buffer with 0.5 mM Ca2+ and the indicated
concentrations of Mg2+ for 20 min at 37 °C. Maximum
stimulation (100%) was determined with 5 mM
CaCl2. Results are the means ± S.D.
(n = 4).
|
|
Enhanced Second Messenger Generation under Magnesium
Deficiency--
PTH secretion is inhibited by higher concentrations
(>1 mM) of Ca2+ and Mg2+, which
are agonists of the CaSR. Two different signaling pathways are
activated by the CaSR, generation of inositol phosphates and inhibition
of cAMP. Under magnesium deficiency, both signaling pathways were
enhanced in dispersed parathyroid cells (Fig. 1, C and
D). Generation of cAMP was inhibited to about 80-85% that of the maximum inhibition observed after stimulation with the CaSR
agonist calcium (Fig. 1C and not shown), and basal inositol phosphates were increased to 30-35% that of the maximum stimulation by magnesium or calcium (Fig. 1D). The IC50
values of the magnesium deficiency-dependent signal
enhancement were similar as for the PTH secretion, i.e.
0.19 ± 0.03 mM for the cAMP pathway and 0.2 ± 0.02 mM for the inositol phosphate production (Fig. 1,
C and D). These findings demonstrate that
magnesium deficiency affects similar signaling pathways as those
activated by the CaSR.
Desensitization of CaSR-mediated Signaling Affects PTH Release
under Magnesium Deficiency--
Is the CaSR necessary for mediating
the effect of magnesium deficiency on PTH release? To address this
question, we desensitized the CaSR on parathyroid cells by prolonged
calcium stimulation. Desensitization of the CaSR was visible by a
substantially decreased response to the agonist magnesium (6 mM) and calcium (2 mM), i.e. after
desensitization, magnesium and calcium inhibited PTH release by only
10% (Fig. 2, lower panel)
compared with a
60% inhibition of PTH release in nondesensitized
control cells (Fig. 2, upper panel). Interestingly, the
effect of magnesium deficiency on PTH release was suppressed similar to
the response to the CaSR agonists (Fig. 2, lower panel).
This finding strongly suggests that active cell surface-localized CaSRs
are a prerequisite for the effect of magnesium deficiency on PTH
release.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Desensitization of the CaSR. The CaSR of
human parathyroid cells was desensitized by incubation of the cells for
6 h in medium containing 5 mM Ca2+. PTH
release from parathyroid control cells (upper panel) or from
cells pretreated for 6 h with 5 mM Ca2+
(lower panel) was determined in cells incubated in buffer
with 0.5 mM Ca2+ and the indicated
concentration of Mg2+ or in buffer with 2 mM
Ca2+ and 1 mM Mg2+. Results are the
means ± S.D. (n = 4).
|
|
Effects of Low Magnesium on Inositol Phosphate Generation Mediated
by Basal Activation of Recombinantly Expressed Human CaSR--
Since
the previous experiments suggested that the site of magnesium action
lies within the axis CaSR-G-protein-effector, we further analyzed the
mechanism of the magnesium paradox in a transfected cell system. Direct
effects of magnesium on the signaling of the CaSR were analyzed
compared with control cells without this receptor. Basal inositol
phosphate levels of HEK-293 cells expressing the CaSR increased up to
2.8-fold when the Mg2+ concentration in the buffer was
decreased from 1 to 0.1 mM, whereas mock-transfected cells
without CaSR expression did not show any significant increase in basal
inositol phosphate levels under these conditions (Fig.
3A). The IC50
value of magnesium was 0.18 ± 0.02 mM. The CaSR was
responsible for the increase in basal inositol phosphate levels under
magnesium deficiency, since basal inositol phosphate levels increased
with increasing CaSR expression levels (Fig. 3B). Thus, the
effect of magnesium deficiency on basal activation of recombinantly
expressed human CaSR paralleled the effects of magnesium deficiency on
parathyroid cell signaling.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of magnesium on the human CaSR
expressed in HEK-293 cells. A, HEK-293 cells (1 × 106) were transfected with 10 µg of a plasmid encoding
the human CaSR. Mock-transfected cells served as a control. The
increase in basal inositol phosphate levels compared with the mock
control was determined in buffer containing 0.5 mM
Ca2+ and the indicated concentration of Mg2+.
B, HEK-293 cells were transfected with increasing amounts of
a plasmid encoding the human CaSR, and basal inositol phosphate levels
were determined in buffer containing 0.5 mM
CaCl2 and either 0.1 mM MgCl2 ( )
or 1 mM MgCl2 ( ). The total amount of DNA
was held constant with plasmid pcDNA3. C, CaSR-mediated
inhibition of cAMP levels. Concentration-response relationship of
MgCl2 for the CaSR-mediated inhibition of
forskolin-stimulated (10 µM) cAMP levels in HEK-293 cells
expressing the human CaSR ( ) or in mock-transfected control cells
( ). Cells were kept in buffer containing 0.5 mM
CaCl2 and the indicated concentrations of
MgCl2. Results are the means ± S.D.
(n = 6).
|
|
CaSR-mediated Inhibition of Adenylyl Cyclase--
CaSR-mediated
inhibition of adenylyl cyclase activity was determined. In HEK-293
cells, forskolin-stimulated adenylyl cyclase activity was inhibited by
the CaSR. Maximal inhibition was obtained with 5 mM
Ca2+ in buffer with 0.5 mM or 0.1 mM Mg2+ (not shown). Low magnesium alone
resulted in partial inhibition (Fig. 3C). The CaSR-mediated
inhibition of cAMP levels at 0.1 mM Mg2+ was
64 ± 8% that of the maximum inhibition by 5 mM
Ca2+ (not shown). The IC50 value of magnesium
was 0.25 ± 0.05 mM (Fig. 3C). In contrast,
the forskolin-stimulated cAMP levels of mock-transfected control cells
were barely affected by a decrease in magnesium (Fig. 3C).
Together these findings demonstrate that the G
i- and G
q-mediated pathways triggered by constitutive activity
of the CaSR were affected by magnesium deficiency in HEK-293 cells
similarly as in dispersed parathyroid cells (cf. Fig.
1).
Effect of Magnesium on the Activation of a CaSR Mutant with Altered
Ion Binding Properties--
Magnesium is an agonist of the CaSR.
Therefore we asked whether the extracellular magnesium binding site(s)
of the CaSR is (are) involved in mediating the signal enhancement under
magnesium deficiency. Different CaSR receptor mutants were expressed
(Fig. 4A). Similar expression
levels of the different CaSR mutants were verified in immunoblot (Fig.
4A). Although CaSRR185Q has a nearly 10-fold
decreased potency for calcium (data not shown and Ref. 15) and
magnesium (Fig. 4B, panel II, and not shown), the
IC50 value of magnesium in inhibiting basal receptor activation was similar between cells expressing mutated
CaSRR185Q and the wild-type CaSR (Fig. 4B,
panel I). This finding strongly suggests that the
extracellular ion binding site of the CaSR for calcium and magnesium is
not involved in mediating the signal enhancement under low
magnesium.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of magnesium deficiency on CaSR
mutants with altered affinity for the extracellular binding of
magnesium and calcium or with altered basal activity. Immunoblot
(IB) of the wild-type human CaSR (lane 2),
CaSRR185Q (lane 3), CaSRF128L
(lane 4), CaSRR795W (lane 5), and the
rat CaSR (lane 6) detected in membranes of transfected
HEK-293 cells. Mock-transfected HEK-293 cells served as a control
(lane 1). B, inositol phosphate levels of HEK-293
cells expressing the indicated CaSR incubated in buffer containing 0.5 mM Ca2+ and the indicated concentrations of
Mg2+. Data are expressed as % of maximum, i.e.
the increase in inositol phosphate levels after stimulation of the
wild-type CaSR by 5 mM CaCl2. Results are the
means ± S.D. (n = 4).
|
|
Magnesium Deficiency Increases Signaling Mediated by the Basal
Activity of Different CaSR Mutants--
CaSR mutants with altered
basal activity were analyzed. Basal activity of CaSRF128L
was increased 1.6-fold compared with the wild-type receptor (Fig. 4B, panel IV). The maximum signal enhancement
observed under magnesium deficiency (0.1 mM) in
CaSRF128L-expressing cells was also increased 1.5-1.8-fold
compared with the wild-type receptor (Fig. 4B, panel
III), whereas the IC50 value of magnesium in
inhibiting basal CaSRF128L activation was similar between
the wild-type CaSR and the mutated CaSRF128L (Fig.
4B, panel III). As a control, the
EC50 value for the activation of CaSRF128L by
magnesium was 3.5 ± 0.2 mM (Fig. 4, panel
B, panel IV). Thus, signaling mediated by the basal
activity of the wild-type CaSR and of CaSRF128L is enhanced
under magnesium deficiency with similar IC50 values, but
the absolute extent of this enhancement is different and depends on the
basal activity of the respective CaSR.
To further analyze whether basal CaSR activity determines the absolute
extent of the signal enhancement by low magnesium, we coexpressed
wild-type CaSR together with mutant CaSRR795W, resulting in
receptor heterodimers with altered functional properties (18, 25),
e.g. decreased basal activity. CaSRR795W
displays defective G-protein-coupling. Coexpression of
CaSRR795W with wild-type CaSR decreased the
affinity/potency for calcium (not shown) or magnesium about 2-fold
(Fig. 4B, panel IV). In parallel, the signal
generated by the basal activity of CaSR-CaSRR795W
heterodimers was suppressed to 20-25% that of the signal of the wild-type receptor at 0.5 mM and at 0.1 mM
Mg2+ (Fig. 4B, panel III). By
contrast, the IC50 value of the magnesium decrease-dependent signal enhancement was not altered.
Thus, a decrease in the basal activity of the CaSR is accompanied by a decrease in the absolute extent of the signal enhancement under magnesium deficiency but no change in the IC50 value of
magnesium. Together these findings confirm that magnesium deficiency
enhances signaling mediated by the basal activity of the CaSR.
Comparison of the Signals Generated by the Basal Activity of the
Rat and the Human CaSR--
Is the basal activity of the CaSR related
to the magnesium paradox of blunted PTH secretion in vivo?
Since the paradox of blunted PTH secretion under severe magnesium
deficiency has been observed in patients but not in rats (11, 12), we
compared the basal activity of the rat and the human CaSR. With 0.5 mM Mg2+, the increase in basal inositol
phosphate levels of rat CaSR-expressing cells was only 15-20% that of
the increase of cells expressing the human CaSR (Fig. 4B,
panels V and VI). Interestingly, a decrease in
the extracellular magnesium also led to an enhancement of the signaling
mediated by basal rat CaSR activity, with an IC50 value for
magnesium similar to that of the human CaSR (Fig. 4B,
panel V). However, the absolute extent of the signal
enhancement of the rat CaSR at 0.1 mM Mg2+ was
again only 15-20% that of the human CaSR (Fig. 4B,
panel V). Equally effective levels of the human and the rat
CaSR were expressed in these experiments, as determined by maximum
stimulation with 10 mM Mg2+ (Fig.
4B, panel VI) and by similar concentration
response relationships with EC50 values of 2.8 ± 0.3 and 5.6 ± 1 mM for calcium (not shown) and magnesium
(Fig. 4B), respectively. Together these experiments demonstrate that differences in PTH secretion under magnesium deficiency reported for patients and rats correlate with differences in
the basal activity of the respective CaSRs and with differences in the
absolute extent of the signal enhancement under magnesium deficiency.
The findings that the IC50 values of magnesium for the
signal enhancement were similar between the human and the rat CaSR and
between different CaSR mutants with different basal activity or with
different affinity for extracellular magnesium binding suggest a common
magnesium binding site different from the extracellular ion binding
site of the CaSR.
CaSR-mediated Activation of G-proteins--
Since the
extracellular magnesium binding site of the CaSR did not seem to be
involved in the magnesium paradox, we asked whether magnesium acted on
the intracellular side of the CaSR, at the CaSR- G-protein interface.
To address this question, CaSR-mediated activation of G-proteins was
determined by the receptor-stimulated enhancement of
[35S]GTP
S binding to recombinant
G
i-protein. A decrease of the Mg2+
concentration from 1 to 0.1 mM or 0.01 mM
increased the rate of CaSR-mediated binding of
[35S]GTP
S nearly 2-fold (Table
I and Fig.
5A). The IC50
value for the magnesium-dependent inhibition of
CaSR-stimulated GTP
S binding was 0.18 ± 0.04 mM
(Table I). Thus, magnesium suppresses the CaSR-mediated G-protein
activation and the CaSR-stimulated activation of G
q- and
G
i-coupled pathways with similar concentration-response relationships. These findings also demonstrate that magnesium acts
within the axis receptor-G-protein.
View this table:
[in this window]
[in a new window]
|
Table I
IC50 [Mg2+] and t1/2 of the CaSR-stimulated
binding of [35S]GTP S to wild-type G i and to
G reconstituted with membranes
from CaSR-expressing HEK-293 cells as described under "Experimental
Procedures."
±S.E.; n = 6.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
CaSR-stimulated
[35S]GTP S binding.
Membranes of HEK-293 cells transfected with the CaSR-cDNA were
prepared and reconstituted with wild-type G i
(A) or mutant G
(B). CaSR-stimulated GTP S binding was determined at
t = 5 min in the absence of Ca2+ in buffer
containing the indicated concentrations of MgCl2. The
CaSR-stimulated increase in [35S]GTP S binding is
given, i.e. the difference between the
[35S]GTP S bound to CaSR-transfected cell membranes and
control membranes. Results are the means ± S.E.
(n = 6).
|
|
CaSR-mediated G-protein Activation of
G
--
Is the magnesium
binding site responsible for this inhibition of basal CaSR activation
localized on the CaSR or on G
? Since the IC50 value of
magnesium for the inhibition of basal CaSR activation was not affected
by CaSR mutants with increased or decreased affinity for extracellular
magnesium or calcium (cf. Fig. 4), we focused on the
magnesium binding site of the G
subunit (26, 27). Although magnesium
effects on the activation of G
subunits have not been detected on
G
i/o isolated from bovine brain (28), the fact that
magnesium suppresses the guanine nucleotide exchange of small
GTP-binding proteins (29) suggests the possibility of a similar
mechanism for G
subunits. And indeed, on wild-type G
i, magnesium inhibited basal guanine nucleotide
exchange, with an IC50 of 0.18 mM (Table
II). By contrast, basal guanine
nucleotide exchange of a G
i mutant,
G
with impaired magnesium
binding (30) was not affected by 1 mM Mg2+
(Table II). The mutant had a decreased affinity for magnesium, with an
IC50 value of 2.8 ± 0.4 mM for the
magnesium-dependent inhibition of basal
[35S]GTP
S binding (Table II).
View this table:
[in this window]
[in a new window]
|
Table II
IC50 and Imax (%) determined at 1 mM
Mg2+ of the magnesium-induced inhibition of basal
[35S]GTP S binding to different purified G subunits
determined as described under "Experimental Procedures."
±S.E.; n = 6.
|
|
CaSR-stimulated [35S]GTP
S binding was determined also
for the G
mutant. In contrast
to the wild-type G
i, CaSR-stimulated
[35S]GTP
S binding to
G
was not significantly suppressed by 1 mM Mg2+ compared with 0.1 mM Mg2+ (Fig. 5B). Also, the
t1/2 were similar at 0.1 and 1 mM
Mg2+ (4.8 ± 0.3 and 4.5 ± 0.4 min, Table I).
Together these data are compatible with the concept that the magnesium
binding site responsible for the enhancement of CaSR-mediated G-protein
activation under low magnesium is localized on the G-protein.
Determination of the Intracellular Free Mg2+
Concentration at Different Extracellular Mg2+
Concentrations--
Since the previous experiments suggested that the
magnesium binding site responsible for the enhancement of CaSR-mediated G-protein activation was localized intracellularly on the G
subunit, we asked whether a decrease in the extracellular free Mg2+
concentration, [Mg2+]e, was followed by a
decrease in the intracellular free Mg2+ concentration,
[Mg2+]i. HEK-293 cells were loaded with
mag-fura-2, and [Mg2+]i was determined. The
[Mg2+]i of resting HEK-293 cells incubated in
buffer with 1.0 mM Mg2+ was 0.7 ± 0.06 mM (Fig. 6A). This
value is in good agreement with the [Mg2+]i of
resting vascular smooth muscle cells (22). By contrast, when the
HEK-293 cells were incubated for 20 min in buffer with 0.1 mM Mg2+, the [Mg2+]i was
reduced to 0.15 ± 0.03 mM (Fig. 6B). This
finding demonstrates that a decrease in [Mg2+]e
leads to a concomitant decrease in [Mg2+]i. Upon
the addition of 1.0 mM extracellular Mg2+, the
[Mg2+]i of magnesium-depleted HEK-293 cells
increased to the initial value within 15 min (Fig. 6C).
These experiments are in agreement with previous data demonstrating
that alterations in [Mg2+]e are accompanied by
concomitant alterations in [Mg2+]i (31).
Considering that effects of magnesium on guanine nucleotide exchange
were observed with a Mg2+ concentration below 1 mM (cf. Fig. 5, Tables I and II), the observed
decrease in [Mg2+]i upon a decrease of
[Mg2+]e is sufficient to mediate effects on
guanine nucleotide exchange of G-proteins in intact cells.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Measurement of [Mg2+]i
at different extracellular Mg2+ concentrations.
[Mg2+]i of HEK-293 cells loaded with mag-fura-2
was determined with cells incubated in buffer supplemented with 0.5 mM CaCl2 and 1.0 mM
Mg2+ (A) or 0.1 mM Mg2+
(B and C). In panel C, the cells
received 1.0 mM Mg2+ at the time point
indicated by an arrow.
|
|
Inhibition of G
i and the Release of PTH from
Parathyroid Cells--
To further analyze whether G-proteins are
indeed involved in mediating the magnesium paradox of blunted PTH
secretion, we determined the effect of G
i/o-protein
inhibition on parathyroid cells (Fig. 7).
Pertussis toxin treatment abolished the effect of high magnesium (6 mM) in suppressing PTH release (Fig. 7, B versus A). Pertussis toxin also abolished the
effect of low magnesium (0.1 mM) on PTH release (Fig.
7B). These findings demonstrate that G
i/o
proteins are essentially involved in mediating the magnesium paradox of
PTH release.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of pertussis toxin pretreatment on PTH
release from dispersed human parathyroid cells. PTH release was
determined in buffer containing 0.5 mM Ca2+ and
the indicated concentrations of Mg2+ in the absence
(A and B) or presence (C and
D) of 100 nM isoproterenol. In panels
B and D, cells were pretreated with pertussis toxin
(100 ng/ml) for 8 h. Results are means ± S.E.
(n = 6) (*p < 0.05;
**p < 0.01).
|
|
Pertussis Toxin Treatment Reveals Magnesium Effects of
G
s-mediated Signaling--
The magnesium binding site
on the G
subunit that seems responsible for the enhancement of
CaSR-mediated signaling under magnesium deficiency is conserved between
various G
subunits (26). In accordance with this fact, GTP
S
binding to various G
subunits was inhibited by magnesium with
similar concentration response relationships (Table II). However, in
parathyroid cells, magnesium deficiency selectively enhanced
CaSR-mediated inhibitory signaling, whereas the stimulation of PTH
secretion by isoproterenol was not enhanced under magnesium deficiency
(Fig. 7C). Since G
i/o proteins are the most
abundant G
subunits in the cell, we asked whether inhibition of
G
i/o proteins could reveal effects of low magnesium on
G
s-mediated signaling. And indeed, when
G
i/o proteins were blocked by pertussis toxin,
isoproterenol-stimulated PTH release was slightly but significantly
enhanced under 0.1 mM Mg2+ compared with 1 mM or 6 mM Mg2+ (Fig.
7D). Thus, in parathyroid cells the enhancement of
G
s-mediated signaling under magnesium deficiency is
overcome by the increased activity of G
i/o proteins.
Effect of Magnesium Deficiency on Signals Generated by Activation
of Different G
q/i-coupled Receptors--
If the
magnesium binding site responsible for the enhancement of CaSR-mediated
signaling under magnesium deficiency is localized on the G
subunit,
signals mediated by the basal activity of receptors other than the CaSR
should be similarly enhanced under magnesium deficiency. To analyze
whether magnesium deficiency enhances the basal activity of
G
q/i-coupled receptors in intact cells, we determined
the effect of magnesium deficiency on signals generated by the basal
activity of two different G
q/i-coupled receptors, the
angiotensin II AT1 and the bradykinin B2
receptor. Both receptors were expressed at high levels in HEK-293 cells
to increase signaling mediated by basal receptor activity. Inositol
phosphate levels stimulated by basal receptor activity of the
AT1 and the B2 receptor were increased
2-3-fold under low magnesium (0.1 mM) compared with 1 mM Mg2+ (Table
III). Since extracellular magnesium is
not an agonist of the AT1 and the B2 receptor
as of the CaSR, we finally asked whether signals generated by
agonist-mediated receptor activation were enhanced similarly under
magnesium deficiency as were the signals generated by constitutive
receptor activity. And indeed, signaling of angiotensin II and
bradykinin was enhanced under magnesium deficiency (0.1 mM
Mg2+) compared with 1 mM Mg2+ as
detected by a 2-3-fold decrease in the EC50 values of
angiotensin II and bradykinin (Table III). Together these experiments
demonstrate that signaling mediated by activation of different
G
q/i-coupled receptors is enhanced under magnesium
deficiency. Furthermore, these data are in agreement with the newly
identified mechanism of magnesium suppressing the guanine nucleotide
exchange of G
subunits in intact cells.
View this table:
[in this window]
[in a new window]
|
Table III
Magnesium effects on G q-stimulated signaling
The basal or agonist-stimulated increase in inositol phosphate levels
of HEK-293 cells expressing the angiotensin II AT1 or the
bradykinin B2 receptor was determined in buffer supplemented
with 1 or 0.1 mM Mg2+. Constitutive receptor
activity is expressed as % of maximum agonist stimulation with 1 µM angiotensin II or bradykinin and was similar at 1 and
0.1 mM Mg2+.
±SD; n = 3.
|
|
 |
DISCUSSION |
Cloning of a CaSR-cDNA (3) and the identification of several
CaSR mutants from patients with hypo- and hyperparathyroidism (1, 5-7)
clearly established the mechanistic relationship between serum calcium
and magnesium and PTH secretion. Despite this progress, the paradox of
blunted PTH secretion under magnesium deficiency is still an open
question. Previous work suggested that the target of magnesium action
was intracellular (9). Since PTH synthesis was not affected by
magnesium deficiency (32) and PTH levels rose within minutes after
parenteral magnesium replacement (33), the defect was pinpointed to the
level of PTH secretion.
And indeed, we found that in vitro PTH secretion from
parathyroid cells was blocked under low magnesium similarly to that reported in patients. Since the effect of low magnesium was dependent on the axis CaSR-G-protein, i.e. desensitization of the CaSR
or inhibition of G
i-proteins by pertussis toxin also
suppressed the effect of magnesium deficiency on PTH secretion, we
reconstituted this system of CaSR-G-protein in vitro in a
cell line other than the parathyroid cell. The characteristics of the
magnesium deficiency-mediated signal enhancement of basal CaSR activity
were similar in parathyroid and in CaSR-expressing HEK-293 cells. The
magnesium binding site was localized on the G
subunit; the presence
of a G
i mutant with decreased affinity for magnesium
abolished the effect of magnesium on CaSR-mediated G-protein
activation, whereas on CaSR mutants with increased or decreased
affinity for magnesium, the IC50 value of the magnesium
deficiency-mediated signal enhancement did not change. Together these
findings let us conclude that the enhancement of G-protein activation
under magnesium deficiency enhances signals mediated by constitutive
CaSR activity. The absolute extent of this enhancement is greater for
the human CaSR than for the rat CaSR because the human receptor has a
higher constitutive activity. This difference between the rat and the
human CaSR leads to blunted PTH secretion under severe hypomagnesemia
in patients but not in rats. Although our data suggest that the
magnesium binding site responsible for this effect is located on the
G
, we cannot rule out the possibility that the human CaSR differs from the rat CaSR by an additional yet unknown magnesium binding site
acting in concert with the magnesium binding site on the G
subunit.
Magnesium-dependent inhibition of guanine nucleotide
exchange by stabilizing guanine nucleotide binding is a common feature of small GTP-binding proteins (29). Our finding that magnesium suppresses the release of GDP on G
subunits parallels the role of
magnesium on c-Ras mechanistically (34). In contrast to GDP-bound Ras,
which has a more than 10-fold higher affinity for magnesium than G
(34), the affinity of magnesium for GDP-bound G
subunits is in the
submillimolar range. Therefore pathophysiological alterations in the
magnesium homeostasis will affect selectively the activity of
heterotrimeric G-proteins. Guanine nucleotide exchange of
G
i, G
o, G
s, and
G
q was similarly affected by magnesium. Therefore the
detected mechanism of magnesium stabilizing the GDP-bound G
may
define a novel function of magnesium on heterotrimeric G-proteins.
Why does magnesium deficiency in vivo selectively enhance
signaling mediated by the CaSR although low magnesium enhances guanine nucleotide exchange of many different G
-proteins. Several
characteristics of receptor-mediated signaling may be responsible for
this phenotype. In vivo, G
i/o proteins are
the most abundant G-proteins. Therefore the enhancement of
G
i/o-mediated signaling will be predominant. This
suggestion is true for parathyroid cells, where magnesium effects on
G
s-mediated signaling are not detectable unless
G
i/o proteins are inhibited. The absence of magnesium
effects on G
s-mediated signaling has also been found for
G
s-dependent secretion processes other than
PTH (35). Concerning G
q/11-mediated signaling, minor or
absent effects of magnesium deficiency on
G
q/11-stimulated responses in vivo may also
be due to enhanced activation of G
i/o because many
G
q/11-coupled receptor systems are functionally antagonized by G
i-mediated responses and vice
versa (36-38). Therefore magnesium
deficiency-dependent signal enhancement of
G
q/11-coupled receptor systems may only become apparent
in vitro when compensatory mechanisms are not effective (39)
and/or under conditions of overexpression (Table III). By contrast,
CaSR-mediated inhibition of PTH release is one of the few physiological
systems that needs simultaneous stimulation of G
i and
G
q proteins. Since there exists no functional antagonism
of CaSR-mediated signaling, as demonstrated by the phenotype of
activating CaSR mutations from patients with hypoparathyroidism (7),
magnesium deficiency in vivo may selectively affect
CaSR-governed PTH release due to an increase in
G
i/o/q/11 activation.