Journal of Physiology
J Physiol (2003), 550.1, pp. 83–101
© The Physiological Society 2003
DOI: 10.1113/jphysiol.2002.035782
www.jphysiol.org
The plasma membrane calcium-ATPase as a major
mechanism for intracellular calcium regulation in neurones
from the rat superior cervical ganglion
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
Patch-clamp recording combined with indo-1 measurement of free intracellular calcium
concentration ([Ca2+]i) was used to determine the homeostatic systems involved in the maintenance
of resting [Ca2+]i and in the clearance of Ca2+ transients following activation of voltage-gated Ca2+
channels in neurones cultured from rat superior cervical ganglion (SCG). The Ca2+ binding ratio
was estimated to be ~500 at 100 nM, decreasing to ~250 at [Ca2+]i ∆ 1 mM, and to involve at least two
buffering systems with different affinities for Ca2+. Removal of extracellular Ca2+ led to a decrease in
[Ca2+]i that was mimicked by the addition of La3+, and was more pronounced after inhibition of the
endoplasmic reticulum Ca2+ uptake system (SERCA). Inhibition of the plasma membrane Ca2+
pump (PMCA) by extracellular alkalinisation (pH 9) or intracellular carboxyeosin both increased
resting [Ca2+]i and prolonged the recovery of Ca2+ transients at peak [Ca2+]i ≤ 500 nM. For [Ca2+]i
loads > 500 nM, recovery showed an additional plateau phase that was abolished in
m-chlorophenylhydrazone (CCCP) or on omitting intracellular Na+. Inhibition of the plasma
membrane Na+ –Ca2+ exchanger (NCX) and of SERCA had a small but significant additional effect
on the rate of decay of these larger Ca2+ transients. In conclusion, resting [Ca2+]i is maintained by
passive Ca2+ influx and regulated by a large Ca2+ buffering system, Ca2+ extrusion via a PMCA and
Ca2+ transport from the intracellular stores. PMCA is also the principal Ca2+ extrusion system at low
Ca2+ loads, with additional participation of the NCX and intracellular organelles at high [Ca2+]i.
(Received 7 January 2003; accepted after revision 14 April 2003; first published online 30 May 2003)
Corresponding author N. Wanaverbecq: Institute of Neurology, Department of Clinical and Experimental Epilepsy, Queen
Square House, London WC1N 3BG, UK. Email: n.wanaverbecq@ion.ucl.ac.uk
In mammalian sympathetic neurones several membrane
ion channels are regulated by intracellular calcium (Ca2+).
Thus, the influx of Ca2+ during action potentials activates
two types of Ca2+-dependent K+ channel: one (the ‘BK’
channel) accelerates spike repolarisation and induces a fast
after-hyperpolarisation (Belluzzi & Sacchi, 1990; Marsh &
Brown, 1991; Davies et al. 1996), while the other (the ‘SK’
channel) induces a slow after-hyperpolarisation and spike
train accommodation (Kawai & Watanabe, 1986; Sacchi et
al. 1995; Davies et al. 1996). Elevation of intracellular Ca2+
can also activate two species of chloride current, a fast
current gated directly by Ca2+ (Sanchez-Vivas et al. 1994a)
and a delayed current triggered by activation of protein
kinase C (Marsh et al. 1995). Finally, M-type K+ channels
are inhibited by intracellular Ca2+ with an IC50 around
100 nM (Selyanko & Brown, 1996), and there is evidence to
suggest that the release of Ca2+ from internal stores following
activation of certain G-protein-coupled receptors may
contribute to M channel inhibition in these cells (Cruzblanca
et al. 1998; Bofill-Cardona et al. 2000).
For these reasons, it seems essential to understand what
processes regulate intracellular Ca2+ levels in mammalian
sympathetic neurones. To date, the only information
available derives from experiments on caffeine-induced
Ca2+ release (Thayer et al. 1988; Hernandez-Cruz et al.
1995, 1997). In contrast, very little is known regarding the
magnitude or duration of the Ca2+ transients, or the factors
that affect these transients, following the entry of Ca2+
through voltage-gated Ca2+ channels, which are most
pertinent to the physiological effects referred to above.
In the present experiments, therefore, we have measured
the Ca2+ transients in dissociated rat sympathetic neurones
following activation of voltage-gated Ca2+ channels, and
have investigated some of the processes that determine the
duration of, and recovery from, these transients. We provide
evidence that the plasma membrane Ca2+-ATPase (PMCA;
Carafoli, 1994) is primarily responsible for recovery following
modest elevations of [Ca2+]i but that it is supplemented by
other mechanisms (principally a Na+–Ca2+ exchanger
(NCX) and mitochondrial uptake) following larger
elevations. We also show that PMCA is active at rest,
extruding Ca2+ that enters through a lanthanum (La3+)sensitive ‘leak’ channel, and that these neurones have a
high Ca2+-binding capacity. Parts of this work have been
published in abstract form (Wanaverbecq et al. 2000,
2001).
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
J Physiol 550.1
Journal of Physiology
84
METHODS
Cell culture
Superior cervical ganglion (SCG) neurones were isolated from
Sprague-Dawley rats and cultured following a method described
by Fernandez-Fernandez et al. (1999). Briefly, SCGs were removed
from 15- to 17-day-old male rats which had been decapitated after
having been humanely killed by CO2 asphyxiation (in accordance
with the UK Animals Scientific Procedures Act 1986). Following
an enzymatic treatment (collagenase, 500 U ml_1 for 15 min and
trypsin type XIIs, 1 mg ml_1 for 30 min), the digested tissue fragments were mechanically dissociated using fire-polished Pasteur
pipettes. The dissociated cells, resuspended into a Leibowitz supplemented medium, were finally plated on a laminin substrate in the
recording chambers (Trouslard et al. 1993) and kept in 35 mm
Petri dishes at 37 °C, under a 5 % CO2–95 % O2 atmosphere for up
to 3 days after culture. All culture reagents were obtained from
Gibco BRL except laminin, collagenase, trypsin (Sigma) and nerve
growth factor (Tocris).
Solutions
On the days of the experiment the recording chambers were transferred onto the microscope (Nikon Diaphot) stage and continuously
superfused at 10 ml min_1 with a modified Hepes-based extracellular medium (see Table 1, solution A) maintained at 33 °C
using a heating device. During the course of the investigation, the
ionic composition or the pH of the extracellular solution was
modified according to the list in Table 1.
Electrophysiology
SCG neurones were voltage clamped using an Axopatch 200A
patch-clamp amplifier (Axon Instruments) either in the wholecell or perforated patch configuration. Recording electrodes were
pulled from borosilicate glass (150 TF, Clark Electromedical) and
had nominal resistances of 2–4 MV when filled with a caesiumbased intracellular solution (see Table 2). For whole-cell recording,
100 mM indo-1 acid was added to the intracellular solution F
(Table 2) and the series resistance was ~5 MV. For recordings in
the perforated patch configuration, electrodes were dip-filled
either in solution G or H (Table 2) and subsequently back-filled
with the same intracellular solution but containing amphotericin B
(final concentration of 0.1–0.15 mg ml_1); a final series resistance
of ~10 MV was achieved after ~15 min. In this condition, cells
were preloaded with indo-1 by incubation for 30 min at 37 °C in
5 mM (final concentration) of the acetoxymethyl (AM) ester form
of indo-1 (indo-1-AM).
To ensure the quality of the seal and the healthiness of the patched
cells, the indo-1 ratio value (see below) was always monitored
before and after the seal formation and only cells with similar ratio
value (± 0.01 units) were kept for the rest of the experiment.
Transient rises in [Ca2+]i were then elicited by depolarising voltage
steps from _60 to 0 mV, generally for 60 and 500 ms, unless
otherwise stated in the text. To determine the involvement of a
particular clearance system, the decay phase of depolarisationinduced Ca2+ transients was compared before and after bath
application of an inhibitor for a putative Ca2+ clearance system.
Whole-cell currents and ratiometric measurements were low-pass
filtered at 1 kHz and digitised at 1–5 kHz using a Digidata 1200
interface driven by pCLAMP 8 software installed on a Dell
personal computer.
Intracellular calcium recording
The [Ca2+]i was estimated from the indo-1 fluorescence using the
ratiometric method described by Grynkiewicz et al. (1985):
[Ca2+]i = K*D((Rmin _ R)/(R _ Rmax)),
(1)
2+
with K*D the apparent indo-1 dissociation constant for Ca , R the
recorded 407 nm/480 nm ratio, and Rmin and Rmax the ratio values
at zero [Ca2+]i and at saturating [Ca2+]i, respectively. The indo-1
fluorescence signals were acquired using two photomultiplier
tubes with input filters at 407 and 480 nm (bandpass filter
396–414 nm and 474–487 nm, respectively) connected to a homebuilt ratiometric amplifier (Mr N. Gill, UCL, UK) and the ratio
values converted on-line into [Ca2+]i from a calibrated signal. The
calibration procedure was carried out in situ using intracellular
solutions containing 100 mM indo-1 (acid form) and known free
Ca2+ concentrations (0–10 mM Ca-EGTA) as described by Trouslard
et al. (1993). The parameters from eqn (1) were subsequently
obtained from the best fit of the experimental data points with:
Rmin = 0.23, Rmax = 3.45 and K*D = 1400 nM.
Drugs and chemicals
All drugs and chemicals were purchased from Sigma except indo-1
acid, indo-1-AM and 5,6-succinylmidyl carboxyeosin (CE) (Molecular
Probes). Indo-1-AM, thapsigargin, m-chlorophenylhydrazone
J Physiol 550.1
85
Journal of Physiology
Ca2+ homeostasis in SCG neurones
(CCCP) and amphotericin B were dissolved in DMSO (final
concentration < 0.01 %).
difference in the clearance plots would represent the contribution
of the inhibited system.
Analysis
All averaged data are expressed as means ± S.E.M. and the statistical
significance was determined by paired Student’s t test (P < 0.05)
unless otherwise stated.
Reverse transcription PCR
mRNA was extracted from rat whole superior cervical ganglia,
cerebral cortex, heart tissue and skeletal muscle using RNAzol B
protocol (Biogenesis Ltd) and reverse transcribed using oligo-dT
and the Superscript II reverse transcriptase (Gibco-BRL). The
PCR reaction was performed using the AmpliTaq polymerase
(Promega) to detect the presence of mRNA sequences coding for
PMCA and NCX isoforms. To amplify sequences of PMCA genes,
we used a sense primer (pmca-s) common to all four isoforms and
isoform-specific antisense primers (pmca1-a to 4-a) with pmca3-a
and 4-a corresponding to those previously described by Keeton et
al. (1993). For NCX, isoform selective sense and antisense primers
were designed with ncx1 sense and antisense primers corresponding
to those previously described by Lee et al. (1994). The various
primers have been designed against sequences coding for the
major isoforms of each protein and since several splice variants
have been described for each isoform of the two proteins, multiples
band could be detected in the PCR reaction. The sequences of
these primers are as follows: pmca-s 5‚-TBGGMGGBAAACCYTTCAGCTG-3‚ (nt 3342–3660, rat PMCA1); pmca1-a 5‚-CTTCTATCCTAAACTCGGGGTG-3‚ (nt 3857–3878 rat PMCA1); pmca2-a
5‚-GTCAGGTTGATCCCGCTGTCG-3‚ (nt 4057–4076, rat PMCA2);
pmca3-a 5‚-GAGCTACGGAATGCTTTCAC-3‚ (nt 4158–4178);
pmca4-a 5‚-CAGCACCGACAGGCGCTTGG-3‚ (nt 3824–3844),
ncx1-s 5‚-TAAAACCATTGAAGGCACAGC-3‚ (nt 1713–1734);
ncx1-a 5‚-CACTTCCAGCTTGGTGTGTT-3‚ (nt 2120–2140); ncx2-s
5‚-GGAGCATCTTTGCCTATGTCTCTGGC-3‚ (nt 611–637);
ncx2-a 5‚-TCGATGCTCTTGGGCGGGTCT-3‚ (nt 863–881);
ncx3-s 5‚-GGAGCGTCTTTGCCTATATTTG-3‚ (nt 620–642) and
ncx3-a 5‚-GCGAGATTCATCTACCTCCTTTC-3‚ (nt 963–983).
The cycling conditions used for the PCR reaction were 94 °C for
2 min and 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 70 °C for
1 min followed by a final step of 72 °C for 10 min. Aliquots of the
reaction were visualised on a 2 % (w/v) Metaphor agarose gel
(FMC Bioproducts) and the size of the obtained products
determined using a DNA molecular ladder (1Kb plus, GIBCOBRL).
Fitting procedure of the recovery phase. The recovery phase
of each Ca2+ transient was fitted using exponential decay
functions of the first or second order:
y = y0 + Aexp(_(x _ x0)/t),
(2)
y = y0 + A1exp(_(x _ x0)/t1) + A2exp(_(x _ x0)/t2),
(3)
where x0, y0, t and A represent the time at the peak, the resting
[Ca2+]i, the decay time constant and the amplitude associated to
each exponential component, respectively. The fitting procedure
was carried out in Origin 5 (Microcal software) using the nonlinear least-squares methods with statistical weighting; the goodness
of fit was assessed from the correlation coefficient and visualised
from the residue plot (with A2 ≥ 10 % A1). In the case of a multiphasic recovery, the time necessary to reach 50 % (tÎ) of the peak
amplitude (Amax) was measured and the mean values compared
before and after the application of an inhibitor.
Determination of the clearance rate. To estimate and better
visualise the contribution of a particular homeostatic system in
the removal of free Ca2+ (i.e. the return to resting [Ca2+]i) the
clearance rate (_d[Ca2+]/dt) was calculated as previously described
by Fierro et al. (1998). Briefly, (1) Ca2+ transients were fitted using
exponential decay functions (eqns (2) or (3)) and the derivative
function (d[Ca2+]/dt) was calculated from the fit. (2) _d[Ca2+]/dt
was then plotted as a function of the [Ca2+]i values obtained from
the exponential fit. (3) Finally, _d[Ca2+]/dt vs. [Ca2+]i from Ca2+
transients of similar amplitude were pooled together in each
condition (control versus inhibitor) and then fitted with a linear
regression (monoexponential decay) or a polynomial function of
the second order (biexponential decay) using Prism 3 software
(Graphpad) to built the clearance plots. For more clarity only the
best fit was represented in the figures. Subsequently, subtracting
the clearance plot obtained in the presence of an inhibitor for a
particular Ca2+ regulatory system from that obtained in control
generated the inhibitor-sensitive component, i.e. the relative
contribution of a specific homeostatic system in the overall
clearance of the free Ca2+. Since only one parameter of the overall
Ca2+ clearance was inhibited in each condition, it can be assumed
that the remaining mechanisms were unaltered and that the
Immunocytochemistry
SCG neurones in culture for 1–3 days were fixed with 0.2 % glutaraldehyde–2 % paraformaldehyde in phosphate buffer solution
(PBS, Sigma) for 20 min at room temperature and subsequently
permeabilised with 0.1 % Triton X-100 (Sigma) in PBS for
10 min. After several washes in PBS, fixed culture were incubated
for 1 h in a blocking buffer (PBS with 10 mg ml_1 bovine serum
Journal of Physiology
86
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
albumin (BSA)). They were then incubated for 1 h at room
temperature or 12 h at 4 °C with a PMCA isoform non-specific
mouse monoclonal antibodies (1/50 5F10, a gift from Dr Filoteo,
Department of Biochemistry and Molecular Biology, Mayo
Clinic/Foundation, Rochester, MN 55905, USA), rabbit polyclonal
isoform-specific antibodies (1/50 PMCA1 to 4, SWant Antibody)
or rabbit polyclonal antibodies directed against NCX1 (1/50 RDINaCaExch-1 cardiac isoform, Research Diagnostic Inc.) to detect
the presence of PMCA and NCX, respectively. Swine anti-mouse
or anti-rabbit antibodies conjugated to TRITC or FITC (1/50,
DAKO) were used for immunolabelling. After several washes in
PBS the coverslips were mounted onto slides with a mounting
medium (DAKO) and examined using a confocal microscope
(Leitz) or an epifluorescence microscope fitted with a monochromator (TILL-optoelektroniks) and a high resolution computer
controlled digital (CCD) camera (Hamamatsu 4880 w) driven
with the Openlab 3 software (Improvision). When using the CCD
camera, confocal images (~40) were acquired with 0.2 mm steps
and images at a particular focal plane were subsequently digitally
deconvolved using the nearest neighbour algorithm and five
neighbours above and below the plane of focus.
RESULTS
Properties of depolarisation-induced Ca2+
transients
In this first series of experiments, we applied depolarising
steps of variable duration to cells voltage clamped at _60 mV
in the perforated patch configuration. To measure changes
in [Ca2+]i cells were loaded by preincubation with indo-1AM (see Methods). The final concentration of indo-1 was
estimated to be around 50 mM since, after full hydrolysis of
indo-1-AM, the fluorescence intensity emitted at 480 nm
was equivalent to that measured in the whole-cell configuration when 50 mM indo-1 (acid form) was added to the
intracellular solution. We used a standard extracellular
bathing solution (Table 1, solution A), but omitted Na+
from the pipette solution (Table 2, solution H) to minimise
Ca2+ release from mitochondria (Gunter & Pfeiffer, 1990;
Colegrove et al. 2000), which otherwise complicated recovery
from large Ca2+ transients (see further below). Under these
conditions, resting [Ca2+]i was 102 ± 4 nM (n = 83;
mean ± S.E.M.).
Figure 1A illustrates somatic cytosolic Ca2+ transients
recorded following step-depolarisations to 0 mV for periods
varying between 30 and 500 ms. The peak amplitude of the
depolarisation-induced Ca2+ transients increased linearly
with pulse duration (Fig. 1Aa) and the rising phase was
monophasic (Fig. 1Ab) suggesting a direct correlation
between Ca2+ entry through voltage-dependent Ca2+ channels
and the rise in [Ca2+]i. Calcium clearance rates were
obtained from the time course of Ca2+ transients’ recovery
to resting levels fitted with mono- or bi-exponential
components (see Methods). Figure 1B shows the best fit
for the recovery of a small (< 500 nM) and a large (> 500 nM)
Ca2+ transient (60 ms and 500 ms depolarisation, @ and
1, respectively as illustrated in Fig. 1A). For the large Ca2+
J Physiol 550.1
transient, the recovery was bi-exponential (see below) and
the two components are represented (dashed lines). The
time constants for the Ca2+ transients’ recovery were
pooled together and plotted against the net rise in [Ca2+]i
(D[Ca2+]i i.e. the difference between peak and resting [Ca2+]i;
Fig. 1C). Below 500 nM [Ca2+]i the recovery phase could be
well fitted by a mono-exponential function, the time
constant for which shortened from ~5 s at 100 nM D[Ca2+]i
to ~2.5 s at 500 nM (Fig. 1Cb). Above 500 nM, recovery took
place bi-exponentially. The faster component appeared to
represent a continuum with that for recovery from transients
< 500 nM, since it had a similar time constant (2–2.5 s) and
the amplitudes of both increased linearly with increasing
D[Ca2+]i (Fig. 1D). The slow component had a variable time
constant between 10 and 60 s and a constant amplitude
(Fig. 1Ca and D).
Thus, to a first approximation, there appeared to be two
recovery processes – a fast process with a maximum rate
constant of ~0.4–0.5 min_1 but with a capacity that increases
linearly with increasing Ca2+ loads from 100 nM up to at
least 1.8 mM, and a slower process of fixed capacity that
only becomes clearly evident at Ca2+ loads above 500 nM.
Hence, in further experiments, we investigated these two
processes separately, by applying depolarising pulses of
either 60 or 500 ms, to generate small or large Ca2+ transients,
respectively. For each we tested the contributions of three
potential clearance mechanisms to the recovery rates: the
plasma membrane Ca2+-ATPase (PMCA), a Na+–Ca2+
exchange (NCX) process, and the endoplasmic (sarcoplasmic) reticulum Ca2+ uptake mechanism (SERCA). In
further experiments, we also assessed the role of mitochondria in the clearance of Ca2+ loads, and determined
the extent of Ca2+ buffering; these are considered separately.
Recovery from small transient rises in [Ca2+]i
To induce a small increase in [Ca2+]i (< 500 nM) we used
short (60 ms) depolarising steps to 0 mV (see above). These
generated a peak [Ca2+]i of 288 ± 15 nM, which recovered
with a mean time constant of 3.9 ± 0.2 s (n = 121; mean ±
S.E.M.). The principal determinant of the recovery rate
following these small Ca2+ transients appeared to be the
plasma membrane Ca2+-ATPase (PMCA). This exchanges
intracellular Ca2+ for extracellular protons (Carafoli, 1994)
and hence can be inhibited by extracellular alkalinisation
(see Benham et al. 1992; Park et al. 1996). In the present
experiments, raising extracellular pH from 7.4 to 9 had
two clear and reversible effects (Fig. 2). First, resting
[Ca2+]i rose from 102 ± 5 nM to 254 ± 27 nM (n = 9). This
was not due to release from intracellular stores, since it did
not occur in a Ca2+-free solution (see further below). Second,
the superimposed Ca2+ transients declined nearly threefold more slowly (Fig. 2B, note that the changes in [Ca2+]i
are represented as net rise): the decay time course remained
mono-exponential, but the time constant lengthened from
4.2 ± 0.7 s to 11 ± 3 s (n = 9; Fig. 2C, inset). The clearance
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
rate was calculated, as described in Methods, from the
exponential fit of nine Ca2+ transients (in each condition)
of similar amplitude. The data were pooled together and
plotted against [Ca2+]i and the best fit was obtained from a
linear regression of the pooled data. The clearance rate plot
against [Ca2+]i (Fig. 2C) indicates that, up to ~300 nM,
most of the Ca2+ is cleared through this pH-sensitive
(presumed PMCA-mediated) process.
87
In further experiments, neurones were voltage clamped in
the whole-cell configuration (open-tip with 100 mM indo-1
added to the intracellular solution), and 50 mM 5,6-succinylimidyl carboxyeosin (CE), a potent membrane-impermeant
inhibitor of the PMCA (Gatto et al. 1993) was added to the
pipette solution (this did not affect the spectral properties
of indo-1). Under whole-cell conditions, and in contrast to
the perforated patch configuration, the Ca2+ signal was
only stable for a period of 20 min maximum _ almost
Figure 1. Properties of somatic Ca2+ transients induced by depolarising steps in rat
sympathetic neurones
Aa, rat SCG neurones were voltage clamped under the perforated patch configuration and in the absence of
intracellular sodium. Somatic rises in [Ca2+]i were induced by depolarising steps from _60 to 0 mV for
increasing durations ranging from 30 to 500 ms (DT: 30, 60, 125, 250 and 500 ms). Ab, same traces as in Aa
but on a faster time scale to show the rising phase of the Ca2+ transients. B, calcium transients from Aa
induced by depolarising steps for 60 (@) and 500 ms (1) with the superimposed fit (black line). For the large
Ca2+ transient the recovery was bi-exponential and the two components of the fit are represented (dashed
line). Ca, plot of the decay time constant(s) against the net rise in [Ca2+]i (D[Ca2+]i i.e. the difference between
peak amplitude and resting [Ca2+]i) for Ca2+ transients induced by depolarising steps of increasing duration
(t, 1, time constant of mono-exponential recoveries; n = 117; t1 and t2, • and ª, time constants for the fast
and slow component of bi-exponential recoveries, respectively; n = 52). Cb, plot of the decay time constant
against D[Ca2+]i for small rises in [Ca2+]i (< 500 nM) represented on a larger scale (from panel Ca, left).
D, plot of the amplitude associated with the exponential components of the decay plotted against D[Ca2+]i
(A, 1, amplitude associated to mono-exponential recoveries, n = 117; A1 and A2, • and ª, amplitudes
associated with the fast and slow component of bi-exponential recoveries, respectively; n = 52). In Ca and D,
the vertical dashed line at D[Ca2+]i = 0.5 mM represents the critical threshold [Ca2+]i above which the Ca2+
transient’s recovery switches from a mono-exponential to a bi-exponential decay.
Journal of Physiology
88
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
certainly because of the dialysis of intracellular metabolites
or mobile Ca2+ binding proteins. Therefore, in whole-cell
configuration, the recording of [Ca2+]i was only carried out
in the first 15–20 min after membrane rupture. Addition
of CE also increased resting [Ca2+]i (291 ± 24 nM; n = 4 vs.
115 ± 2 nM; n = 26, in controls) and increased the time
constant for Ca2+ transient recovery from 6.5 ± 0.4 s
(n = 12) to 9.5 ± 1.0 s (n = 8).
+
In contrast, no appreciable role for the Na -dependent
Ca2+ extrusion (presumably NCX) in assisting recovery of
small Ca2+ transients could be discerned. Thus, replacement of extracellular Na+ with N-methyl-D-glucamine
(Table 1, solution B; see Blaustein & Lederer, 1999) had no
J Physiol 550.1
significant effect on either resting [Ca2+]i (99 ± 2 nM in
controls vs. 97 ± 9 in Na+-free; n = 7) or on the recovery
phase of small Ca2+ transients (t = 3.8 ± 0.2 s in controls
vs. 4.2 ± 0.3 s in Na+-free; n = 14 Ca2+ transients; Fig. 3A).
A plot of clearance rate against [Ca2+]i suggests that, in
these neurones, NCX plays a marginal role, at most, in this
recovery process (Fig. 3A, right panel).
Likewise, the endoplasmic reticulum Ca2+ pump (SERCA)
contributed very little to the clearance of small Ca2+ transients,
since the SERCA inhibitor thapsigargin (TG) had no
significant effect on the decay time constant (t = 3.7 ± 0.3 s
in controls vs. 4.3 ± 0.2 s in TG; n = 14 Ca2+ transients;
Figure 2. Both a rise in resting [Ca2+]i and a prolongation of the recovery phase for small Ca2+
transients is observed after PMCA inhibition
A, small Ca2+ transients induced by depolarising steps for 60 ms before, during and after extracellular
alkalinisation (pH 9) to inhibit PMCA. B, superimposed traces from A on a faster time scale and represented
as the net rise in [Ca2+]i (D[Ca2+]i) to compare the recovery phase. C, clearance rate plot in control and after
PMCA inhibition for small Ca2+ transients. Raw data were pooled together from 9 Ca2+ transients both in
control (CTR) and at pH 9 and only the best fits are represented (continuous line). Subsequently the
clearance plot at pH 9 was subtracted from the one in the control to generate the pH-dependent component,
i.e. PMCA contribution (dashed line). The inset represents the effect of PMCA inhibition on the decay time
constant (black bar, control; open bar, pH 9; n = 9 Ca2+ transients; data as mean ± S.E.M., *** P < 0.001).
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
Fig. 3B). Notwithstanding, TG was clearly effective in
inhibiting SERCA since it induced a persistent rise in
resting [Ca2+]i (105 ± 10 nM in controls vs. 143 ± 7 nM in
TG; n = 14), as can be seen from the intercept of the
clearance plot in Fig. 3Bb and can be visualised in the inset
in Fig. 6A. The mechanism for this rise is discussed further
below.
Recovery from larger transient rises in [Ca2+]i
Larger Ca2+ transients were induced by applying longer
(500 ms) depolarising steps to 0 mV. These generated a
mean peak [Ca2+]i of 1079 ± 166 nM (n = 52) followed by a
bi-exponential decay: the fast-decaying component had a
mean time constant of 2.3 ± 0.1 s and a mean capacity of
862 ± 73 nM (i.e. comprised the bulk of the clearance),
89
whereas the slower component had an average time constant
of 23 ± 2.2 s, and a mean capacity of 122 ± 7 nM (n = 52).
Recovery from these transients continued to be strongly
dependent on the activity of the PMCA, but now additional
contributions of the NCX and SERCA transporters could be
clearly discerned. Thus, inhibition of the PMCA transporter
by extracellular alkalinisation to pH 9 clearly prolonged the
decay (Fig. 4A, note that the traces are represented as net rise in
[Ca2+]i) and increased both fast and slow time constants about
two-fold (Fig. 4C). Clearance plots (Fig. 4B) suggest that Ca2+
was extruded almost entirely by the PMCA transporter at
[Ca2+]i up to ~300 nM but that this transporter tended towards
saturation at concentrations ≥ 750 nM. Concordant results
were obtained using intracellular CE (see above): both fast and
Figure 3. Neither the sodium–calcium exchanger nor the intracellular stores are involved in
the recovery from small rises in [Ca2+]i
Left-hand records, small Ca2+ transients induced by 60 ms depolarising steps in control and after either
extracellular Na+ substitution with N-methyl-D-glucamine to inhibit the sodium–calcium exchanger
(Aa, Na+-free) or after bath application of thapsigargin (TG; 100 nM) to impair Ca2+ uptake into the stores via
SERCA (Ba, TG). Since TG application induced a persistent elevation in resting [Ca2+]i (see Figs 6A, middle
panel and 11C for more details on this particular aspect) and to enable a better comparison of the recovery
phase, the traces in panel Ba are represented as D[Ca2+]i. Right-hand graphs, plots of the clearance rate in
control and after extracellular Na+ substitution (Ab, data from 14 Ca2+ transients) and SERCA inhibition
(Bb, data from 14 Ca2+ transients), respectively. The dashed lines in Ab and Bb represent the Na+-dependent
and the TG-sensitive component, respectively. The inset shows the effect of NCX (Ab) and SERCA (Bb)
inhibition on the decay time constant (black bar, control (CTR); open bar, Na+-free in Ab and TG in Bb
n = 14 Ca2+ transients data as mean ± S.E.M.).
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
J Physiol 550.1
Journal of Physiology
90
Figure 4. The plasma membrane Ca2+-ATPase also participates in the Ca2+ clearance for larger
rises in [Ca2+]i
A, large Ca2+ transients induced by depolarising steps for 500 ms in control and after PMCA inhibition
(pH 9). Note that the superimposed traces are represented as D[Ca2+]i and that the scale is micromolar (mM).
B, plot of the clearance rate in control and at pH 9 for large Ca2+ transients with the PMCA contribution
(dashed line, n = 14 Ca2+ transients). Note that the clearance plot after alkalinisation is offset by ~0.3 mM
because of the induced rise in resting [Ca2+]i following PMCA inhibition. C, decay time constants for the fast
(Ca) and slow (Cb) component of the Ca2+ transients’ recovery in control and at pH 9 (black bar, control
(CTR); open bar, pH 9; n = 14 Ca2+ transients, data as mean ± S.E.M.; **, *** P < 0.01 and 0.001 respectively).
slow recovery time constants were increased, from 2.9 ± 0.3
and 17.3 ± 1.1 s in controls (n = 14) to 7.3 ± 2 and 44.9 ± 5.6 s
with 50 mM CE (n = 6), respectively. Likewise, the CE-sensitive
component was predominant below 300 nM and tended
toward saturation above 750 nM.
However – and unlike the situation following small Ca2+
transients – removal of extracellular Na+ also slowed
recovery from large Ca2+ transients (Fig. 5A). This resulted
primarily from a slowing of the fast time constant for
decay, from 2.2 ± 0.1 s to 4.3 ± 0.3 s (n = 14; Fig. 5C); the
Figure 5. The sodium–calcium exchanger is involved in the recovery from large Ca2+ transients
A, large Ca2+ transients induced by depolarising steps for 500 ms in control and after removal of the
extracellular Na+ to inhibit the Na+-dependent Ca2+ extrusion (Na+-free). B, plot of the clearance rate in
control and in the absence of extracellular Na+ with the Na+-dependent component (dashed line, n = 14 Ca2+
transients). C, effect of extracellular Na+ removal on the fast decay time constant of the Ca2+ transients’
recovery (black bar, control (CTR); open bar, Na+-free; n = 14 Ca2+ transients, data as mean ± S.E.M.;
*** P < 0.001).
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
slow time constant was highly variable in Na+-free solution,
but was not significantly changed overall (controls,
21.0 ± 4 s vs. Na+-free, 26 ± 17 s; n = 14). Clearance plots
suggested that Na+-dependent extrusion through the NCX
transporter was negligible below ~300 nM [Ca2+]i but
assumed increasing importance between 0.5 and 1.5 mM
(Fig. 5B). In the absence of extracellular Na+, the clearance
rate was decreased by ~50 % (Fig. 5B), especially for large
rises in [Ca2+]i, a result, which is in agreement with the
50 % decrease in the fast time constant of the recovery
(Fig. 5C).
91
Following TG application, resting [Ca2+]i increased rapidly
before decreasing to a higher level than in control (Fig. 6A,
middle panel). This effect was still observed in the absence
of extracellular Ca2+ but reached a lower peak amplitude
and had a slower onset. It is assumed to be due to the
depletion of the Ca2+ stores and the subsequent activation
of store-operated Ca2+ channels (SOCC, see Fig. 11C).
Subsequently, the recovery from large depolarisationinduced Ca2+ transients was prolonged (Fig. 6B) and the
fast time constant of decay was doubled (from 2.4 ± 0.1 to
5.0 ± 2.0; n = 12 Ca2+ transients; Fig. 6D), suggesting that a
Figure 6. Thapsigargin prolongs the recovery from a large rise in [Ca2+]i
A, large Ca2+ transients were induced by depolarising steps for 500 ms before and after bath application of
thapsigargin (TG, 100 nM) to inhibit SERCA. Thapsigargin application induced an increase in resting [Ca2+]i
that subsequently returned toward the resting value but remained at a higher level than in control (see inset
in panel A and Fig. 11C). B, calcium traces from A were superimposed to compare the recovery phase before
and after SERCA inhibition. Note that the traces are represented as D[Ca2+]i because of the persistent rise in
resting [Ca2+]i induced following SERCA inhibition. C, plot of the clearance rate in control and after SERCA
inhibition and subsequent store depletion with the thapsigargin-sensitive component (dashed line, n = 12
Ca2+ transients). D, effect of TG on the fast decay time constants of the Ca2+ transients’ recovery (black bar,
control (CTR); open bar, TG; n = 12 Ca2+ transients, data as mean ± S.E.M.; *** P < 0.001).
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
Journal of Physiology
92
Figure 7. SCG neurones express all four PMCA proteins and their localisation is isoform
specific
A, expression of PMCA was detected using an isoform non-specific mouse monoclonal antibody (5F10,
1/50) counterstained with swine anti-mouse polyclonal antibodies conjugated to TRITC (1/50). Images were
acquired using a confocal microscope in bright field (left panel in A) and to reveal TRITC fluorescence (right
panel in A; optical slice of 0.5 mm, scale bar 10 mm, w 63 oil immersion fluorescent objective). The arrows in
the right panel in A show the absence of labelling at the point of contact between two cells. B, PCR analysis for
the detection of mRNA coding for PMCA in whole SCG and cerebellar cortex (CB). Isoform non-selective
sense primers were used along with isoform-specific antisense primers: lanes 1–3, PCR amplification of
pmca1 sequence; lanes 4–6, of pmca2; lanes 7–9, of pmca3 and lane 10–12, of pmca4. For each PCR reaction a
negative control was used (_ve, lanes 3, 6, 9 and 12) along with a ‘mock cDNA template’ (not shown on this
particular agarose gel). C, determination of the PMCA isoforms expressed in SCG neurones using isoformspecific rabbit polyclonal antibodies (PMCA-1 to -4, 1/50) counterstained with swine anti-rabbit polyclonal
antibodies conjugated to FITC (1/50). Images were acquired using a CCD camera attached to a fluorescent
microscope and the images presented were digitally deconvolved using the nearest-neighbour algorithm
(scale bar 10 mm, w 40 oil immersion fluorescent objective).
J Physiol 550.1
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
component of Ca2+ uptake by SERCA contributes to the
clearance of large cytosolic Ca2+ loads. From the clearance
plots (Fig. 6C) it appears that SERCA uptake increased
linearly with increasing [Ca2+]i, up to at least 1.5 mM. In spite
of the fact that TG increased resting [Ca2+]i (see Figs 6A,
inset, and 11C), it had no significant effect on the net
amplitude of the Ca2+ rise induced by a depolarising step
(D[Ca2+] = 763 ± 101 in controls vs. 677 ± 51 nM in
thapsigargin; n = 12). This suggests that, in these neurones,
Ca2+ transients induced by activating voltage-gated Ca2+
channels are not amplified by Ca2+-induced Ca2+ release
(CICR) from the endoplasmic reticulum (ER) (see also
Trouslard et al. 1993 and Figs 1Ab and 12B). However, the
lack of amplification by CICR can not be attributed to Ca2+
stores in an empty state. Thus, pressure application of
caffeine (10 mM for 5 s at 10 p.s.i.) consistently elicited
93
transient rises in [Ca2+]i that were still observed in the
absence of extracellular Ca2+ but not after TG application.
Immunocytochemical and PCR identification of
calcium transporters
Immunocytochemical and molecular biological tests were
carried out to confirm the expression of both PMCA and
NCX, and to determine the expression of specific isoforms
and their cellular localisation in SCG neurones. PMCA
expression was detected in the plasma membrane of SCG
neurones with 5F10, an isoform non-selective antibody
(Fig. 7A, left). An interesting feature was the absence of
staining at the point of contact between two cells (arrows
in Fig. 7A, right) suggesting that the protein would be
targeted at sites in contact with the extracellular medium.
Four major isoforms for PMCA have been cloned with
Figure 8. All three NCX isoforms are expressed in the whole ganglion but only NCX-1 is
present in SCG neurones
A, PCR analysis for the detection of mRNA coding for NCX isoforms in whole SCG (lane 1), cerebellar cortex
(CB, lane 3), heart (Ht, lane 5) and skeletal muscle (SkM, lane 8) as described below the top panel. Specific
sets of primers were used for the detection of each isoform (ncx-1 to -3 from top to bottom). For each PCR
reaction a negative control (_ve, lanes 9 in each panel) and for each tissue a ‘mock cDNA template’ (labelled
with the letter ‘m’ for mock, lanes 2, 4, 6 and 7 in each panel) were used. B, rabbit polyclonal antibody (1/50)
raised against the cardiac isoform (NCX-1) was used to determine the expression of NCX in SCG neurones.
The primary antibody was counterstained with a swine anti-rabbit polyclonal antibody conjugated to TRITC
(1/50). Images were acquired using a CCD camera attached to a fluorescent microscope. The top panel
represents the image before digital deconvolution and the bottom panel corresponds to the same plane of
focus but after digital deconvolution (scale bar 10 mm, w 40 oil immersion fluorescent objective).
Journal of Physiology
94
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
numerous splice variants. PCR analysis (Fig. 7B) indicates
that, in the whole ganglion, mRNA for all four isoforms was
present as well as some splice variants (multiple bands).
Immunocytochemical tests using isoform-specific antibodies
confirmed this and further suggested an isoform-specific
localisation (Fig. 7C). Thus PMCA-1 appears to be mainly
expressed on the soma of neurones whereas PMCA-2, -3
and -4 were also expressed on neurites.
J Physiol 550.1
Several isoforms for NCX have also been cloned; PCR analysis
indicated that the three major NCX isoforms are expressed in
the whole ganglion (Fig. 8A) but immunocytochemical tests
suggested that NCX-1 was expressed at a low level on the
neurone soma, (Fig. 8B, bottom panel).
Role of mitochondria in Ca2+ clearance
In these experiments, we added 10 mM Na+ to the pipette
solution (Table 2, solution G), to better replicate the
Figure 9. Mitochondrial inhibition with CCCP affects [Ca2+]i regulation
A, small and large Ca2+ transients were induced by depolarisation steps from _60 to 0 mV for 60 and 500 ms,
respectively, before and after bath application of 2 mM m-chlorophenylhydrazone (CCCP) to uncouple
mitochondria and impair mitochondrial Ca2+ regulation. The neurone was voltage clamped in the perforated
patch configuration with 10 mM intracellular sodium. Small (B) and large (C) Ca2+ transients from A were
superimposed to compare the recovery phase following CCCP application. In B the inset represents the effect
of CCCP on the decay time constant of small Ca2+ transients (black bar, control (CTR); open bar, CCCP;
n = 8 Ca2+ transients, data as mean ± S.E.M., P = 0.04). In C the inset represents the effect of CCCP on tÎ
(measured as shown in A, left), the time necessary to decrease [Ca2+]i by 50 % for large Ca2+ transients (black
bar, control (CTR); open bar, CCCP; n = 12 Ca2+ transients, data as mean ± S.E.M., ** P < 0.01).
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
normal intracellular solution and enable Na+-dependent
Ca2+ release from mitochondria (Gunter & Pfeiffer, 1990;
Colegrove et al. 2000). Addition of Na+ had no effect on
resting [Ca2+]i (102 ± 5 nM, with 10 mM [Na+]i vs.
110 ± 8 nM, with zero [Na+]i; n = 12). Nor did it affect the
amplitude or decay rate of the small Ca2+ transients
produced by 60 ms depolarising pulses (t = 3.6 ± 0.5 s
with 10 mM [Na+]i vs. 3.5 ± 0.1 s with zero [Na+]i; n = 16
and 57, respectively). However, it had a strong effect on the
recovery of the larger (≥ 1 mM) Ca2+ transients produced
by 500 ms depolarising steps. Thus, in the presence of
10 mM intracellular Na+, these showed a multiphasic
recovery phase with an initial rapid decrease to a
[Ca2+]i ∆ 300 nM, a plateau phase (maximum amplitude of
217 ± 18 nM; n = 12) lasting for tens of seconds and finally
the [Ca2+]i slowly returned to its resting value (Fig. 9A, left).
Upon bath application of 2 mM m-chlorophenylhydrazone
(CCCP), to uncouple mitochondria and impair mitochondrial Ca2+ regulation (see Gunter & Pfeiffer, 1990),
resting [Ca2+]i transiently increased (peak amplitude of
183 ± 8 nM; n = 12) before returning to control levels after
~1 min (Fig. 9A, middle panel). This was due to intracellular Ca2+ release, and not Ca2+ influx, since CCCP still
produced a transient rise in [Ca2+]i in the absence of extracellular Ca2+. Furthermore, this Ca2+ was most certainly
Figure 10. Resting [Ca2+]i is maintained
through a passive, lanthanum-sensitive
Ca2+ influx and an active Ca2+ extrusion via
PMCA
Removal of extracellular Ca2+ induces a decrease in
resting [Ca2+]i (Aa, Ca2+-free) whereas PMCA
inhibition induces a rise in resting [Ca2+]i (Ba, pH
9). In the presence of extracellular Ca2+, bath
application of 100 mM lanthanum (La3+ in Ab)
reproduces the decrease in resting [Ca2+]i observed
in a Ca2+-free medium and blocks (Bb) the rise in
[Ca2+]i observed following PMCA inhibition (pH
9). Ac, effect of 100 mM La3+ (cross-hatched bar;
n = 10) on resting [Ca2+]i compared to control
(black bar; n = 31) and removal of extracellular
Ca2+ (open bar; n = 13). Bc, effect of 100 mM La3+
(cross-hatched bar; n = 6) on the [Ca2+]i rise
induced at pH 9 compared to control (black bar;
n = 31) and after PMCA inhibition (open bar;
n = 23). (data as mean ± S.E.M.; *, *** P < 0.05 and
0.001, respectively, independent Student’s t test).
95
released from mitochondria since a CCCP-induced transient
rise in [Ca2+]i was still observed after SERCA inhibition.
Thus, it appears that under resting conditions, mitochondria
would be able to sequester Ca2+.
m-Chlorophenylhydrazone did not affect the amplitude of
the depolarisation-induced Ca2+ transients (D[Ca2+]i =
157 ± 17 nM and 909 ± 106 nM in controls vs. 165 ± 11 nM
and 908 ± 105 nM in CCCP for small (n = 8) and large
(n = 12) rises in [Ca2+]i, respectively), but dramatically
affected the properties of the large Ca2+ transients’ recovery
phase (Fig. 9): it abolished the plateau phase, but delayed
the overall recovery (see superimposed traces in Fig. 9C).
Thus, the half-time for recovery was lengthened from
2.5 ± 0.1 s in controls to 3.5 ± 0.3 s in CCCP (n = 12; inset
in Fig. 9C). In contrast, CCCP failed to prolong the
recovery from small rises in [Ca2+]i (t = 2.7 ± 0.2 s vs.
2.2 ± 0.2 s in CCCP; n = 8; Fig. 9B). This suggests that,
following large Ca2+ loads, [Ca2+]i stayed elevated for a
longer period of time when the mitochondrial function
was inhibited. We interpret these results to suggest that
mitochondria act as both a Ca2+ sequestration system and
as a secondary Ca2+ release system. Uptake by mitochondria
accelerates the initial recovery from large (≥ 1 mM) Ca2+
transients. On the other hand, Na+-dependent secondary
release from mitochondria delays recovery when intra-
96
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
cellular [Ca2+] falls below ~300 nM, providing a large rise
in cytosolic [Ca2+]i had been previously elicited.
Journal of Physiology
Control of the resting calcium concentration
Extracellular Ca2+ removal induced a decrease in resting
[Ca2+]i from 99 ± 4 nM (n = 31) to 80 ± 4 nM (n = 13)
(Fig. 10Aa and Ac). When Ca2+ was reintroduced, [Ca2+]i
returned to control values and sometimes a small and
transient overshoot was observed. This implies a tonic
influx of Ca2+ in 2.5 mM external [Ca2+]. Application of
lanthanum (La3+, 100 mM) in the presence of Ca2+ replicated
the effect of removing Ca2+ (Fig. 10Ab), reducing resting
[Ca2+]i to 77 ± 5 nM (n = 10), a level similar to that
observed upon removal of extracellular Ca2+. Lanthanum
had no effect in the absence of external Ca2+ (and hence did
not affect the spectral properties of intracellular indo-1)
but its effect suggests that influx might take place through
channels related to the store-operated Ca2+ channels
(SOCC; Kwan & Putney, 1990). The presence of SOCCs,
and their possible involvement, was also suggested by the
observation that the rise in [Ca2+]i produced by TG (see
above Fig. 6A, inset) was reversed to a decline on removing
extracellular Ca2+, and subsequent recovery on re-admitting
external Ca2+ was prevented in the presence of La3+
(Fig. 11C).
If there is a tonic influx of Ca2+ at rest, what is responsible
for its removal? As noted earlier, the primary mechanism
for restoring small depolarisation-induced rises in [Ca2+]i
is the PMCA transporter, which is capable of operating at
low levels of Ca2+ (see Carafoli et al. 1994 for review). This
is active at resting [Ca2+]i, since raising the extracellular pH
to 9 produced a slow rise in [Ca2+]i (Fig. 10Ba), which was
prevented by La3+ (Fig. 10Bb). As noted previously, resting
[Ca2+]i was also higher when the PMCA transporter was
J Physiol 550.1
inhibited with intracellular CE. The involvement of
voltage-dependent Ca2+ channels could be ruled out since
they were reversibly blocked by both lanthanum (100 mM)
and cadmium (Cd2+; 100 mM) but Cd2+ was without effect
on resting [Ca2+]i, i.e. Cd2+ did not induce a decrease in
resting [Ca2+]i and did not block the rise in [Ca2+]i observed
following extracellular alkalinisation (data not shown).
The ER also appears to be involved in regulating cytoplasmic
Ca2+ levels at rest, since removal of extracellular Ca2+
produced a larger fall in [Ca2+]i in the presence of TG
(applied 5 min prior to extracellular Ca2+ removal, in
Fig. 11A compare panels Aa and Ab) than in its absence
(Fig. 11B). This implies that functional Ca2+ stores would
contribute to the regulation of resting [Ca2+]i with Ca2+
being released from or sequestered into intracellular stores
to compensate for a decrease or an increase in resting
[Ca2+]i, respectively. Subsequently, a too-strong depletion
of intracellular store would induce SOCC activation to
enable Ca2+ stores replenishment.
Calcium buffering capacity in the soma of SCG
neurones
Most of the calcium that enters a neurone through voltagegated Ca2+ channels is bound to intracellular Ca2+-binding
molecules, leaving only a small proportion in the free ionic
form (‘calcium-buffering’: see Neher, 1995). We attempted
to assess the Ca2+ binding ratio, as an indication of the
cytosolic ability to buffer Ca2+, in SCG neurones. Calcium
currents (Fig. 12Aa) and indo-1 signals (Fig. 11Ab) were
simultaneously recorded following voltage steps of varying
duration using open-tip patch pipettes filled with 100 mM
indo-1. For these experiments we used neurones with
small neurites (to improve clamp efficiency) and with a
measured cell radius of ~10 mm (volume ∆ 4.2 pl). Total
Figure 11. Following SERCA inhibition the
decrease in resting [Ca2+]i is larger and faster
when the extracellular Ca2+ is removed
Extracellular Ca2+ removal in a neurone voltage
clamped at _60 mV in the perforated patch
configuration induces a larger decrease in resting
[Ca2+]i after SERCA inhibition with 100 nM
thapsigargin (TG in Ab was applied 5 min prior to
Ca2+ removal) compared to control (Aa). B, effect of
100 nM TG (cross-hatched bar) on resting [Ca2+]i
compared to control (CTR, black bar) and to
extracellular Ca2+ removal (open bar) (n = 13, data
as mean ± S.E.M.; *** P < 0.001). C, SERCA
inhibition with TG induces a persistent rise in
resting [Ca2+]i that is abolished in a Ca2+-free
extracellular solution or in the presence of 10 mM
La3+.
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
charge transfer (QCa) during the current was calculated
from the integral of the current signal after subtraction of
the residual current recorded in the presence of 100 mM
Cd2+ (Trouslard et al. 1993). The resultant change in total
intracellular Ca2+ concentration (D[Ca2+]total) was then
estimated from the equation:
D[Ca2+]total = QCa/zFV,
(4)
where F is the Faraday constant (96, 485 C mol_1), z the
valency of Ca2+ ions (z = 2) and V the accessible cell volume
(∆ 4.2 pl). Figure 12B shows a plot of the peak increase in
free Ca2+ ion concentration (D[Ca2+]i), measured from the
indo-1 signal, against the total increase in cytosolic
calcium (D[Ca2+]total), measured from the integral of the
Ca2+ current (eqn (4)). The linearity of this plot suggests
that there was no incremental increase in [Ca2+]i from
Ca2+-induced Ca2+-release, at least within the time frame of
these current pulses (see also Trouslard et al. 1993). The slope
of the least-squares regression line (∆ 0.0015) implies that
only 0.15 % of the Ca2+ entering remains in the free ionic
form, the rest being bound to intracellular buffers.
D[Ca2+]bound
[EGTA]
= ———————
—,
kEGTA = —————
2+
D[Ca ]i
D[Ca2+]i+ KD(EGTA)
97
(9)
where KD(EGTA) is the dissociation constant for Ca2+ binding to
EGTA (5.54 w 10_8 M at 33 °C, pH 7.4 and 300 mosmol l_1).
The two EGTA curves encompass the observed values of ke
quite well up to log[Ca2+]i = _6.6 (~250 nM), above which
ke stays fairly constant at 250. Therefore, a constant Ca2+
binding ratio of 250 corresponding to a low affinity
buffering system (with a Ca2+ binding ratio constant and
defined by kbuffer = [Buffer]/KD(Buffer); see Neher, 1995) has
been added to the curve representing the EGTA Ca2+
binding ratio. Thus, endogenous Ca2+ buffering in SCG
neurones may be regarded as comprising a high-affinity
The total Ca2+ binding ratio (k) can then be expressed by
the following equation (Neher & Augustine, 1992; see
Neher, 1995, for review):
k = D[Ca2+]bound/D[Ca2+]i,
(5)
D[Ca2+]bound = D[Ca2+]total _ D[Ca2+]i,
(6)
where:
Since some of the calcium is bound to indo-1, the endogenous Ca2+ binding ratio (ke) is defined by:
ke = ktotal _ kindo-1,
(7)
and we estimated kindo-1 from the following equation
(Palecek et al. 1999):
[indo-1] w KD(indo-1)
kindo-1 = —————————————————,
(8)
2+
([Ca ]rest + KD(indo-1))([Ca2+]peak + KD(indo-1))
where [indo-1] is the total indo-1 concentration (100 mM,
added to the intracellular solution) at equilibrium (∆ 10 min)
and KD(indo-1) is the dissociation constant for Ca2+ binding to
indo-1 and was obtained from the calibration procedures
(93.3 nM = KD = K*D(Rmin/Rmax)). Both the total (ktotal) and
the indo-1 (kindo-1) Ca2+ binding ratio were calculated in 14
different cells for each depolarising pulse, and the endogenous Ca2+ binding ratio (ke) calculated from the difference
(eqn (7)).
Figure 12C shows a plot of the calculated endogenous
binding ratio ke against the logarithm of the changes in free
[Ca2+]i for differing amounts of charge transfer binned in
10 pC increments. For comparison, the curves show the
equivalent binding ratios (kEGTA) for two concentrations
(50 and 100 mM) of the Ca2+buffer EGTA calculated from:
Figure 12. Determination of the endogenous Ca2+
binding ratio and comparison with the EGTA Ca2+
binding ratio
Transient rises in [Ca2+]i (Aa) were induced in a neurone voltage
clamped in the whole-cell configuration (open-tip with 100 mM
indo-1) following activation of voltage-dependent Ca2+ channels
with depolarising steps from _60 to 0 mV for 10, 20, 40, 80 and
160 ms (Ab). The currents in Ab represent Ca2+ currents (ICa)
obtained after digital subtraction of the outward current remaining
in the presence of 100 mM cadmium. B, pooled data of D[Ca2+]i, the
net changes in [Ca2+]i (measured with indo-1), as a function of the
changes in [Ca2+]total (D[Ca2+]total)calculated from QCa, the Ca2+
charge transfer (data obtained from 14 cells). The continuous line
represents the best fit obtained using a linear regression. C, plot of
the endogenous Ca2+ binding ratio (ke, 0; n = 14; data as
mean ± S.E.M.) as a function of the logarithm of D[Ca2+]i. The Ca2+
binding ratio for EGTA was calculated using
(kEGTA = [EGTA]/(KD(EGTA) + [Ca2+]i)2 with [EGTA] the total EGTA
concentration and KD(EGTA) = 5.54 w 10_8 M at 33 °C, pH 7.4 and
300 mosmol l_1. The endogenous Ca2+ binding ratio (ke) was then
compared to the theoretical kEGTA calculated for 50 and 100 mM
EGTA to which the Ca2+ binding ratio for a low affinity Ca2+ buffer
was added (~250, see text for further details).
98
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
Journal of Physiology
buffer (KD ∆ 50–100 nM) broadly equivalent to 50–100 mM
EGTA and a maximum binding capacity (binding ratio) of
~1000, associated to a low affinity buffer (KD >> 1 mM)
with a fixed Ca2+ binding capacity of ~250.
DISCUSSION
In the present study we have attempted to obtain a
reasonably comprehensive overview of the mechanisms
that are responsible for maintaining resting intracellular
[Ca2+] in rat sympathetic neurones and for restoring
[Ca2+]i following its transient elevation.
Under our recording conditions, indo-1 did not appear to
saturate for large increases in [Ca2+]i, nor did Ca2+ unbinding
from indo-1 appear to significantly affect the kinetics of
the Ca2+ transient’s recovery. Hence, consecutive depolarisation steps (3 w 500 ms every 5 s) would induce an
incremental rise in [Ca2+]i up to 2.5 mM and the recovery
phase was not prolonged using a higher indo-1 concentration
(from 50 to 250 mM).
To produce transient elevations of [Ca2+]i, we induced
brief Ca2+ influxes through voltage-gated Ca2+ channels by
applying depolarising steps of varying duration. As noted
previously (Thayer et al. 1988; Trouslard et al. 1993), these
produced a monotonic increase in peak [Ca2+]i with
increasing duration and charge entry, but no clear supralinearity as might be expected where there is substantial
Ca2+-induced Ca2+-release (CICR; see, for example,
Usachev et al. 1993). This is rather surprising since these
cells have prominent caffeine-sensitive Ca2+ stores that
appear to be full under resting conditions, as can be seen
following pressure applications of caffeine (see also Thayer
et al. 1988; Hernandez-Cruz et al. 1995). The latter authors
obtained evidence that CICR contributed to the rise in
[Ca2+]i following brief depolarisations by K+ ions, and
Kawai & Watanabe (1989) showed that ryanodine shortened
the Ca2+-dependent after-hyperpolarisation following an
action potential. One reason why a more prominent
contribution by CICR to the rise in cytosolic [Ca2+] was
not apparent in our experiments may be that the Ca2+
stores responsible for CICR following Ca2+ channel
opening are located just under the plasma membrane
(Henkart, 1980), and that any submembrane rise in [Ca2+]i
is dissipated by extrusion and diffusion without contributing
to the global cytosolic concentrations that we have measured.
Another explanation might be the necessity for [Ca2+]i to
overcome the strong endogenous buffering capacity
before being able to trigger a release from intracellular
stores. This accords with the presence of microdomains
where Ca2+ stores and specific channel sets are in close
apposition and would only induce localised responses
(Delmas et al. 2002).
Only a very small proportion (~0.1–0.4 % at [Ca2+]i < 1 mM)
of the Ca2+ that entered the neurones was registered as free
J Physiol 550.1
[Ca2+]i. Our estimate of the Ca2+ binding ratio (ke) for very
small rises in [Ca2+]i (i.e. near resting levels) was ~1000.
This value for the Ca2+ binding ratio in SCG neurones is
higher than the buffering capacity of chromaffin cells
(k ∆ 40; Neher & Augustine, 1992) and approaches that of
Purkinje neurones (k ∆ 2000, Fierro & Llano, 1996; Maeda
et al. 1999). Also, as in Purkinje neurones (Maeda et al.
1999), the Ca2+ binding ratio diminished with increasing
Ca2+ loads, to ~250 at D[Ca2+]i ∆ 500 nM. We interpret this
to suggest the presence of a high affinity buffer (or buffers),
with an effective affinity constant near to that for EGTA,
and a buffer (or buffers) with a much lower affinity (i.e.
much greater than 500 nM) and a constant buffering
capacity (~250) in the physiological range of [Ca2+]i.
Although little is known about the nature of the Ca2+
buffers in these cells, Sanchez-Vives et al. (1994b) have
previously reported the presence of calbindin D28K as well
as its upregulation following axotomy. Calbindin has an
effective macroscopic dissociation constant of ~10_8 M
(Leathers et al. 1990) and might contribute to the highaffinity buffering since it was shown in SCG neurones that
its upregulation was associated with a slowing of the rise
and decay of action potential-induced Ca2+ transients
(Sanchez-Vives et al. 1994b). The involvement of Calbindin
D28K or another mobile Ca2+ binding protein is further
supported by the observation that, in the whole-cell
configuration, resting [Ca2+]i increased and the recovery
rate of the Ca2+ transients was prolonged after recording
for 20 min or more. Finally, because mitochondria are able
to sequester a large amount of Ca2+ during the rising phase
of large Ca2+ transients and because they appear to be
localised close to the membrane and therefore close to the
Ca2+ channels, these organelles might contribute to the
‘low-affinity’ buffering capacity of these cells (as suggested
for other cells: see e.g. Thayer & Miller, 1990; Park et al.
1996).
These sympathetic neurones appear to resemble embryologically homologous (though functionally quite different)
peripheral sensory neurones (see Benham et al. 1992;
Usachev et al. 2002) in that the predominant mechanism
for recovery of [Ca2+]i to resting levels following moderate
(≤ 500 nM) rises is through extrusion by the plasma
membrane Ca2+-ATPase (PMCA). Thus, the recovery rate
was reduced ~60 % by extracellular alkalinisation, and
~30 % by intracellular CE. Following extracellular alkalinisation or in the presence of intracellular CE the Ca2+ transient’s
recovery was strongly prolonged but not completely inhibited.
Incomplete inhibition by extracellular alkalinisation to pH
9 probably results from the fact that half-maximal inhibition
of PMCA occurs at pH ∆ 8.5 (i.e. [H+] ∆ 3.2 nM; see
Carafoli, 1987; Xu et al. 2000). Hence, at pH 9 ([H+] = 1 nM),
the Ca2+ pump would only be inhibited by ~80 %. In
contrast, inhibition of NCX or the SERCA pump had no
effect on the recovery rate. At these low [Ca2+]i, the absence
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
of an effect of inhibition of NCX or SERCA, in contrast to
the effect of inhibition of PMCA, could be attributed to the
difference in the Ca2+ affinity of these Ca2+ transporters.
Thus, the Ca2+ affinity for PMCA has been reported to be
~0.2–0.5 mM (Carafoli, 1994) whereas for SERCA and
NCX it is suggested to be ~1 (Pozzan et al. 1994) and
~1–10 mM (Blaustein & Lederer, 1999), respectively.
In keeping with a single rate-limiting recovery process,
these small Ca2+ transients showed a mono-exponential
decline, with a maximum rate constant of 0.4 s_1. This is
comparable with that reported following similarly brief
depolarisations of sensory neurones by Benham et al.
(1992), but appreciably faster than the values obtained by
Usachev and colleagues (2002) using brief trains of action
potentials. In our experiments, the recovery rate constant
appeared to increase with the rise in D[Ca2+]i up to
~250 nM, but thereafter stayed constant. This might be
attributable to the known Ca2+–calmodulin-induced increase
in PMCA pump rate (Bautista et al. 2002; see also Carafoli,
1994). Usachev et al. (2002) have also reported an
increased PMCA extrusion rate (up to 40 %) following
Ca2+-dependent phosphorylation of PMCA4b by protein
kinase C (induced, for example, by bradykinin or ATP).
However, it seems unlikely that this could be sufficiently
rapid to affect recovery from a single 60 ms depolarising
step. Since mRNAs for at least the four major PMCA
isoforms were detected in these cells, and their protein
products appropriately localised to the plasma membrane,
we are unable to specify which isoform(s) was(were) most
responsible for the extrusion process.
As noted in some other neurones such as Purkinje cells
(Fierro et al. 1998; Maeda et al. 1999), recovery from large
Ca2+ transients followed a bi-exponential time course, with
the second component about ten times slower than that
for recovery from small transients. The rate constant for
this component showed no clear dependence on D[Ca2+]i;
instead, it appeared to contribute a fixed amount of Ca2+
clearance. One possibility is that it might represent the
slow dissociation of Ca2+ from endogenous buffers acting
as the limiting step in Ca2+ extrusion (Helmchen et al.
1996; Lee et al. 2000), or the sequestration and slow release
from intracellular organelles (Park et al. 1996).
Following these larger (> 500 nM) rises in [Ca2+]i, extrusion
via the PMCA tends toward saturation, and NCX appears
to play an increasing role in Ca2+ extrusion. The apparent
threshold for NCX was ~250–300 nM and the Na+-sensitive
extrusion via NCX then increased progressively up to
1.5 mM where it contributed some 60 % to Ca2+ clearance,
in accordance with its lower affinity (KD ∆ 1–10 mM;
Blaustein & Lederer, 1999). The SERCA pump clearly
plays a role in cytosolic Ca2+ clearance following large Ca2+
transients since thapsigargin reduced the rate of Ca2+
clearance. However, even following these large rises in
[Ca2+]i and as already mentioned earlier, there was no
99
evidence for an amplification mechanism of the Ca2+
signal mediated by intracellular stores.
In the presence of intracellular Na+, mitochondrial transport
also strongly affected the recovery from large transients.
Under these conditions, recovery was multiphasic with an
initial rapid decay and a characteristic plateau phase
followed by a secondary slow decrease towards resting
[Ca2+]i. In agreement with numerous previous studies
(Thayer & Miller, 1990; Friel & Tsien; 1994; Park et al.
1996; Colegrove et al. 2000), mitochondrial uncoupling
with protonophores, such as CCCP, induced both a delay
in the initial fast decay phase and the abolition of the slow
secondary plateau phase. Since the initial recovery rate in
the presence of CCCP was similar to that for the fast
component of recovery in the absence of intracellular Na+
(data not shown), active mitochondrial uptake serves to
accelerate the initial decline in cytoplasmic [Ca2+]i. Then,
with the decrease in cytoplasmic [Ca2+]i, Ca2+ release from
the mitochondria becomes progressively dominant over
Ca2+ uptake leading to the generation of the plateau that
corresponds to the ‘set point’ at which mitochondrial
uptake and release are in equilibrium (Gunter & Pfeiffer,
1990). In SCG neurones the set point was estimated at
217 ± 18 nM [Ca2+]i (n = 12), which is similar to that
measured in chromaffin cells (180 ± 25 nM, Park et al.
1996), and bullfrog sympathetic neurones (~200 nM, Friel
& Tsien, 1994; Colegrove et al. 2000) but appreciably lower
than in rat dorsal root ganglion neurones (~500 nM,
Thayer & Miller, 1990). In frog sympathetic neurones, the
strategic location of mitochondria near to the plasma
membrane facilitates this ‘buffering’ role (Pivovarova et al.
1999; McDonough et al. 2000). In rat sympathetic neurones,
they appear to be similarly located (S. J. Marsh, unpublished
electron microscope observations) suggesting that mitochondria could play both a role in the buffering of Ca2+
entering the cytosol (see above) and in the acceleration of
its clearance following transient rises.
Finally, the present experiments provide some useful
information regarding the regulation of resting [Ca2+]i in
SCG neurones. It is clear from the rise in resting [Ca2+]i
following extracellular alkalinisation (i.e. PMCA inhibition)
that there is a continuous extrusion of Ca2+ at rest through
the PMCA. This rise observed at alkaline extracellular pH
does not appear to be due to changes in intracellular pH
and subsequent pH-dependent Ca2+ release, since at pH 9
no increase in resting [Ca2+]i was observed in the absence
of extracellular Ca2+, and changes in intracellular pH were
minimal (≤ 0.05 units, as measured using 2‚,7‚-bis-(2carboxyethyl)-5-(and -6)-carboxyfluorescein, BCECF; data
not shown). In contrast, inhibition of the Na+–Ca2+exchanger
did not affect resting [Ca2+]i, supporting our conclusion
that this only becomes a significant route for Ca2+
extrusion following large rises in [Ca2+]i. The rise in resting
[Ca2+]i after PMCA inhibition in turn implies a mechanism
Journal of Physiology
100
N. Wanaverbecq, S. J. Marsh, M. Al-Qatari and D. A. Brown
for tonic incrementation of cytosolic [Ca2+]i. One such
mechanism is an influx from the extracellular medium,
since removing extracellular Ca2+ induced a fall in resting
[Ca2+]i. This decrease in [Ca2+]i was replicated by bath
application of La3+, but not by inhibition of voltage-gated
Ca2+ channels, and is therefore probably mediated through
channels related to the store-operated Ca2+ channels
(SOCC) and/or Trp channels. Evidence that part, at least,
of this influx may be through SOCC is provided by the fact
that store-depletion with thapsigargin also produced a
sustained rise in [Ca2+]i, which was reversed to a reduction
in [Ca2+]i in a Ca2+-free solution or on adding La3+. One
could therefore suggest that SOCC-related channels might
be active and responsible for the passive Ca2+ influx at rest
and that these same channels would be further activated
following Ca2+ depletion of intracellular stores. However,
on the basis of the sensitivity of Ca2+ influx to lanthanum,
these two routes for Ca2+ entry would appear to be only
partly related, if not entirely different. Furthermore, these
results suggest a cooperative interaction between the Ca2+
regulatory mechanisms of the plasma membrane and of
intracellular stores to efficiently control both cytosolic and
luminal [Ca2+]i. Thus resting [Ca2+]i would be maintained
constant as a result of the fine tuning between Ca2+ entry
and/or extrusion through the plasma membrane and Ca2+
sequestration and/or release from stores. In this respect (as
in several other aspects), these sympathetic neurones
resemble peripheral sensory neurones of dorsal root
ganglia (Usachev & Thayer, 1999). Another prospective
source of cytosolic Ca2+ at rest might be mitochondria;
however, inhibition of mitochondrial uptake with CCCP
only produced a transient rise of [Ca2+]i, suggesting that
mitochondria do not contribute strongly to long-term
maintenance of resting Ca2+ levels.
In conclusion, the results presented in this study suggest
the following deductions. Firstly, the plasma membrane
Ca2+-ATPase (PMCA) is responsible for extruding Ca2+ at
rest and after moderate elevations (≤ 500 nM). Additional
mechanisms of extrusion and sequestration come into play
after larger rises in [Ca2+]i. These include extrusion by the
Na+–Ca2+ exchanger (NCX), uptake by the endoplasmic
reticulum Ca2+ pump (SERCA), and uptake and subsequent
release by mitochondria. Secondly, resting [Ca2+]i is the
result of a passive Ca2+ influx through a La3+-sensitive
pathway (possibly a store-operated pathway), counterbalanced by extrusion by the PMCA. And finally, these
cells also show very high intracellular Ca2+ buffering capacity,
probably mediated by more than one system, which helps
to maintain a low free Ca2+ ion concentration.
J Physiol 550.1
REFERENCES
Bautista DM, Hoth M & Lewis RS (2002). Enhancement of calcium
signalling dynamics and stability by delayed modulation of the
plasma-membrane calcium-ATPase in human T cells. J Physiol
541, 877–894.
Belluzzi O & Sacchi O (1990). The calcium-dependent potassium
conductance in rat sympathetic neurones. J Physiol 422, 561–583.
Benham CD, Evans ML & McBain CJ (1992). Ca2+ efflux
mechanisms following depolarization evoked calcium transients
in cultured rat sensory neurones. J Physiol 455, 567–583.
Blaustein MP & Lederer WJ (1999). Sodium/calcium exchange: its
physiological implications. Physiol Rev 79, 763–854.
Bofill-Cardona E, Vartian N, Nanoff C, Freissmuth M & Boehm S
(2000). Two different signaling mechanisms involved in the
excitation of rat sympathetic neurons by uridine nucleotides. Mol
Pharmacol, 57, 1165–1172.
Carafoli E (1987). Intracellular calcium homeostasis. Annu Rev
Biochem 56, 395–433.
Carafoli E (1994). Biogenesis: plasma membrane calcium ATPase: 15
years of work on the purified enzyme. FASEB J 8, 993–1002.
Colegrove SL, Albrecht MA & Friel DD (2000). Dissection of
mitochondrial Ca2+ uptake and release fluxes in situ after
depolarization-evoked [Ca2+]i elevations in sympathetic neurons.
J Gen Physiol 115, 351–370.
Cruzblanca H, Koh DS & Hille B (1998). Bradykinin inhibits M
current via phospholipase C and Ca2+ release from IP3-sensitive
Ca2+ stores in rat sympathetic neurons. Proc Natl Acad Sci U S A 95,
7151–7156.
Davies PJ, Ireland DR & McLachlan EM (1996). Sources of Ca2+ for
different Ca2+-activated K+ conductances in neurones of the rat
superior cervical ganglion. J Physiol 495, 353–366.
Delmas P, Wanaverbecq N, Abogadie FC, Mistry M & Brown DA
(2002). Signalling microdomains define the specificity of receptormediated InsP3 pathways in neurons. Neuron 14, 209–220.
Fernandez-Fernandez JM, Wanaverbecq N, Halley P, Caulfield MP
& Brown DA (1999). Selective activation of heterologously
expressed G protein-gated K+ channels by M2 muscarinic
receptors in rat sympathetic neurones. J Physiol 515, 631–637.
Fierro L, Dipolo R & Llano I (1998). Intracellular calcium clearance
in Purkinje cell somata from rat cerebellar slices. J Physiol 510,
499–512.
Fierro L & Llano I (1996). High endogenous calcium buffering in
Purkinje cells from rat cerebellar slices. J Physiol 496, 617–625.
Friel DD & Tsien RW (1994). An FCCP-sensitive Ca2+ store in
bullfrog sympathetic neurons and its participation in stimulusevoked changes in [Ca2+]i. J Neurosci 14, 4007–4024.
Gatto C & Milanick MA (1993). Inhibition of the red blood cell
calcium pump by eosin and other fluorescein analogues. Am J
Physiol 264, C1577–1586.
Grynkiewicz G, Poenie M & Tsien RY (1985). A new generation of
Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem 260, 3440–3450.
Gunter TE & Pfeiffer DR (1990). Mechanisms by which
mitochondria transport calcium. Am J Physiol 258, C755–786.
Helmchen F, Imoto K & Sakmann B (1996). Ca2+ buffering and
action potential-evoked Ca2+ signaling in dendrites of pyramidal
neurons. Biophys J 70, 1069–1081.
Journal of Physiology
J Physiol 550.1
Ca2+ homeostasis in SCG neurones
Henkart M (1980). Identification and function of intracellular
calcium stores in axons and cell bodies of neurons. Fed Proc 39,
2783–2789.
Hernandez-Cruz A, Diaz-Munoz M, Gomez-Chavarin M, CanedoMerino R, Protti DA, Escobar AL, Sierralta J & Suarez-Isla BA
(1995). Properties of the ryanodine-sensitive release channels that
underlie caffeine-induced Ca2+ mobilization from intracellular
stores in mammalian sympathetic neurons. Eur J Neurosci 7,
1684–1699.
Hernandez-Cruz A, Escobar AL & Jimenez N (1997). Ca2+-induced
Ca2+ release phenomena in mammalian sympathetic neurons are
critically dependent on the rate of rise of trigger Ca2+. J Gen Physiol
109, 147–167.
Kawai N & Watanabe M (1986). Blockage of Ca-activated Kconductance by apamin in rat sympathetic neurones. Br J
Pharmacol 87, 225–232.
Kawai N & Watanabe M (1989). Effects of ryanodine on the spike
after-hyperpolarization in sympathetic neurones of the rat
superior cervical ganglion. Pflugers Arch 413, 470–475.
Keeton TP, Burk SE & Shull GE (1993). Alternative splicing of exons
encoding the calmodulin-binding domains and C termini of
plasma membrane Ca2+-ATPase isoforms 1, 2, 3, and 4. J Biol
Chem 268, 2740–2748.
Kwan CY & Putney JW (1990). Uptake and intracellular
sequestration of divalent cations in resting and methacholinestimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and
Ba2+ of agonist-stimulated divalent cation entry from the refilling
of the agonist-sensitive intracellular pool. J Biol Chem 265,
678–684.
Leathers VL, Linse S, Forsen S & Norman AW (1990). CalbindinD28K, a 1 alpha, 25-dihydroxyvitamin D3-induced calciumbinding protein, binds five or six Ca2+ ions with high affinity. J Biol
Chem 265, 9838–9841.
Lee SH, Schwaller B & Neher E (2000). Kinetics of Ca2+ binding to
parvalbumin in bovine chromaffin cells: implications for [Ca2+]
transients of neuronal dendrites. J Physiol 525, 419–432.
Lee SL, Yu AS & Lytton J (1994). Tissue-specific expression of
Na+/Ca2+ exchanger isoforms. J Biol Chem 269, 14849–14852.
McDonough SI, Cseresnyes Z & Schneider MF (2000). Origin sites of
calcium release and calcium oscillations in frog sympathetic
ganglia. J Neurosci 20, 9059–9070.
Maeda H, Ellis-Davies GC, Ito K, Miyashita Y & Kasai H (1999).
Supralinear Ca2+ signaling by cooperative and mobile Ca2+
buffering in Purkinje neurons. Neuron 24, 989–1002.
Marsh SJ & Brown DA (1991). Potassium currents contributing to
action potential repolarization in dissociated cultured rat superior
cervical sympathetic neurones. Neurosci Lett133, 298–302.
Marsh SJ, Trouslard J, Leaney JL & Brown DA (1995). Synergistic
regulation of a neuronal chloride current by intracellular calcium
and muscarinic receptor activation: a role for protein kinase C.
Neuron 15, 729–737.
Neher E (1995). The use of fura-2 for estimating Ca2+ buffers and
Ca2+ fluxes. Neuropharmacol 34, 1423–1442.
Neher E & Augustine GJ (1992). Calcium gradients and buffers in
bovine chromaffin cells. J Physiol 450, 273–301.
Palecek J, Lips MB & Keller BU (1999). Calcium dynamics and
buffering in motoneurones of the mouse spinal cord. J Physiol 520,
485–502.
Park YB, Herrington J, Babcock DF & Hille B (1996). Ca2+ clearance
mechanisms in isolated rat adrenal chromaffin cells. J Physiol 492,
329–346.
101
Pivovarova N, Hongpaisan J, Andrews SB & Friel DD (1999).
Depolarization-induced mitochondrial Ca2+ accumulation in
sympathetic neurons: spatial and temporal characteristics.
J Neurosci 19, 6372–6384.
Pozzan T, Rizzuto R, Volpe P & Meldolesi J (1994). Molecular and
cellular physiology of intracellular calcium stores. Physiol Rev 74,
595–636.
Sacchi O, Rossi ML & Canella R (1995). The slow Ca2+-activated K+
current, IAHP, in the rat sympathetic neurone. J Physiol 483, 15–27.
Sanchez-Vives MV & Gallego R (1994a). Calcium-dependent
chloride current induced by axotomy in rat sympathetic neurons.
J Physiol 475, 391–400.
Sanchez-Vives MV, Valdeolmillos M, Martinez S & Gallego R
(1994b). Axotomy-induced changes in Ca2+ homeostasis in rat
sympathetic ganglion cells. Eur J Neurosci 6, 9–17.
Selyanko AA & Brown DA (1996). Intracellular calcium directly
inhibits potassium M channels in excised membrane patches from
rat sympathetic neurones. Neuron 16, 151–162.
Thayer SA, Hirning LD & Miller RJ (1988). The role of caffeinesensitive calcium stores in the regulation of intracellular free
calcium concentration in rat sympathetic neurons in vitro. Mol
Pharmacol 34, 664–673.
Thayer SA & Miller RJ (1990). Regulation of the intracellular free
calcium concentration in single rat dorsal root ganglion neurones
in vitro. J Physiol 425, 85–115.
Trouslard J, Marsh SJ & Brown DA (1993). Calcium entry through
nicotinic receptor channels and calcium channels in cultured rat
superior cervical ganglion cells. J Physiol 468, 53–71.
Usachev YM, Demarco SJ, Campbell C, Strehler EE & Thayer SA
(2002). Bradykinin and ATP accelerate Ca2+ efflux from rat
sensory neurons via protein kinase C and the plasma membrane
Ca2+ pump isoform 4. Neuron 33, 113–122.
Usachev YM, Shmigol A, Pronchuk N, Kostyuk P & Verkhratsky A
(1993). Caffeine-induced calcium release from internal stores in
cultured rat sensory neurons. Neuroscience 57, 845–859.
Usachev YM & Thayer SA (1999). Ca2+ influx in resting rat sensory
neurones that regulates and is regulated by ryanodine-sensitive
Ca2+ stores. J Physiol 519, 115–130.
Wanaverbecq N, Marsh SJ & Brown DA (2000). Role of the plasma
membrane calcium ATPase in calcium clearance in rat
sympathetic neurones. Eur J Neurosci 12, 371.
Wanaverbecq N, Marsh, SJ & Brown DA (2001). Maintenance of
resting intracellular calcium concentration in rat sympathetic
neurones. J Physiol 536.P, 103P.
Xu W, Wilson BJ, Huang L, Parkinson EL, Hill BJ & Milanick MA
(2000). Probing the extracellular release site of the plasma
membrane calcium pump. Am J Physiol Cell Physiol 278,
C965–972.
Acknowledgements
This work was supported by the Wellcome Trust and the UK
Medical Research Council. We would like to thank Dr A. Filoteo
for the kind gift of the 5F10 antibodies and Dr P. Delmas for useful
discussion throughout the work.
Author’s website
www.ecclescorner.org