Vm regulates mGlu5-induced Ca2+release
To investigate the effect of Vm on mGlu5 function and
determine the optimal experimental conditions, we examined mGlu5-induced
Ca2+ release from intracellular stores. mGlu5
stimulation is known to induce a transient increase of
Ca2+ oscillations in various cell types, including
cultured astrocytes , hippocampal neurons , midbrain neurons , as well
as in heterologous expression systems such as Chinese Hamster Ovary
cells and Human Embryonic Kidney 293T (HEK293T) cells . In line with
these findings, in HEK293T cells expressing the
GCaMP6s fluorescent
Ca2+ sensor (Figure 1A ), mGlu5 agonist
(RS)-3,5-dihydroxyphenylglycine (DHPG, 100 µM) induced a
Ca2+ increase exclusively in cells expressing mGlu5,
as evidenced by GCaMP6s fluorescence fluctuations measured in the cell
population using a fluorimeter (Figure 1B ). The mGlu5
agonist-induced Ca2+ increase in mGlu5-expressing
cells was inhibited by application of MPEP (10 µM), a mGlu5-specific
negative allosteric modulator (NAM), confirming the requirement for
mGlu5 receptor activation (Figure 1B ).
In order to determine the effect of Vm on mGlu5-induced
Ca2+ increase, we treated cells with equimolar
solutions containing increasing concentrations of KCl (from 3 mM, a
physiological concentration referred to as “Vrest”, up
to 100 mM) to induce a systematic, well-controlled, and long-lasting
membrane depolarization (as previously demonstrated in neurons ).
Interestingly, KCl 100 mM had no effect on basal intracellular
Ca2+ concentrations (Figure Supp. 1A ), but
significantly reduced mGlu5 agonist-induced Ca2+increase by 22.3 ± 7.1% compared to KCl 3 mM (Figure 1C ). We
measured that KCl 100 mM application increased the membrane potential by
23.6 ± 0.9 mV (Figure 1D ), referred to as
“Vdepol”. Whole cell patch clamp recordings indeed
reported a reversible switch from Vrest = -28.4 ± 3.5 mV
at physiological KCl concentration (3 mM) to Vdepol =
-4.8 ± 0.9 mV with KCl 100 mM (Figure 1D ). The dose response
curve of DHPG-induced Ca2+ release further revealed a
reduction of the agonist efficacy at Vdepol compared to
Vrest (Figure 1E ). Additionally, saturating
concentrations of another mGlu5 agonist, quisqualate (10µM), triggered a
Ca2+ increase at Vrest, which was
reduced by 45 ± 9.5% at Vdepol (Figure 1F ).
These initial findings indicate that the ability of the mGlu5 receptor
to increase intracellular Ca2+ is dependent on
Vm. To investigate whether the effect of
Vm was specific to the mGlu5 receptor, we examined
another Gq-coupled receptor, the angiotensin II type 1
receptor (AT1), and did not observe any influence of Vmon AT1 receptor-mediated Ca2+ release, thereby
supporting a specific effect of Vdepol on mGlu5
signaling in this context (Figure 1G ).
To investigate the effect of Vm on mGlu5-induced
Ca2+ increase at a cellular level, we performed live
cell imaging of GCaMP6s fluorescence (Figure 2A andsupplemental Video1 ). Initially, large recordings in the entire
field of view showed a global increase in Ca2+ induced
by the mGlu5 agonist, quisqualate (10µM), at Vrest. This
increase was significantly reduced at Vdepol (30 ± 10 %
reduction compared to Vrest at the peak of fluorescence,Figure 2B and supplemental Video2 ). The global
Ca2+ increase was an average of the single cell
Ca2+ fluctuations, which were classified into 3
distinct populations of cells depending on the type of responses: silent
cells, cells displaying 1 single Ca2+ spike, and
Ca2+ oscillating cells (Figure 2C ), as
reported previously . Vdepol modified the distribution
of cells in each of these categories, with a global shift towards less
oscillating populations (Figure 2C ), and a significant
reduction in the proportion of cells with an oscillating
Ca2+ pattern (10.6% reduction of the proportion of
oscillating cells compared to Vrest, Figure
2D ). Subsequently, for each oscillating cell, we studied the global
frequency of Ca2+ oscillations induced by mGlu5
agonist. Quisqualate induced a mean of 0.49 ± 0.02 oscillations/min per
cell at Vrest, but only 0.39 ± 0.02 oscillations per
cell at Vdepol. Thus, Vdepol induced a
20.3 ± 4.8% reduction in the frequency of Ca2+oscillations triggered by mGlu5 stimulation (Figure 2E ).
Given that depolarization in neurons can also occur following receptor
stimulation, we investigated the impact of Vdepolsubsequent to mGlu5 activation. Specifically, we measured the effect of
Vdepol compared to Vrest on oscillating
cells that had undergone Ca2+ oscillations at resting
membrane potential as a result of mGlu5 stimulation. To do so, we
determined the instantaneous frequency, which represents the average
inter-oscillation frequency between the first and last oscillations. Our
findings consistently revealed a significant reduction in the
instantaneous frequency, from 1.18 ± 0.11 at Vrest to
0.85 ± 0.04 oscillation/min at Vdepol, representing a 28
± 4.7% decrease (Figure 2F ). These results suggest that a
membrane depolarization of approximately 20-25mV, whether it occurs
before or after mGlu5 activation, can diminish the receptor’s ability to
induce Ca2+ release from intracellular stores, in few
seconds. Specifically, Vdepol reduced the number of
agonist-responsive cells and the efficiency of the responding cells,
resembling the characteristics of a NAM .