Journal of Neurochemistry, 2005, 94, 452–458
doi:10.1111/j.1471-4159.2005.03213.x
Developmental change in the calcium sensor for synaptic vesicle
endocytosis in central nerve terminals
Karen J. Smillie, Gareth J. O. Evans and Michael A. Cousin
Membrane Biology Group, Division of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Edinburgh, UK
Abstract
Synaptic vesicle endocytosis is stimulated by calcium influx in
mature central nerve terminals via activation of the calciumdependent protein phosphatase, calcineurin. However, in different neuronal preparations calcineurin activity is either
inhibitory, stimulatory or irrelevant to the process. We
addressed this inconsistency by investigating the requirement
for calcineurin activity in synaptic vesicle endocytosis during
development, using vesicle recycling assays in isolated nerve
terminals. We show that endocytosis occurs independently of
calcineurin activity in immature nerve terminals, and that a
calcineurin requirement develops 2–4 weeks after birth. Calcineurin-independent endocytosis is not due to the absence of
calcineurin activity, since calcineurin is present in immature
nerve terminals and its substrate, dynamin I, is dephosphorylated on depolarization. Calcineurin-independent endocytosis is calcium-dependent, since substitution of the divalent
cation, barium, inhibits the process. Finally, we demonstrated
that in primary neuronal cultures derived from neonatal rats,
endocytosis that was initially calcineurin-independent developed a calcineurin requirement on maturation in culture. Our
data account for the apparent inconsistencies regarding the
role of calcineurin in synaptic vesicle endocytosis, and we
propose that an unidentified calcium sensor exists to couple
calcium influx to endocytosis in immature nerve terminals.
Keywords: calcineurin, calcium, endocytosis, FM2-10, nerve
terminal, synaptic vesicle.
J. Neurochem. (2005) 94, 452–458.
Endocytosis of synaptic vesicles (SVs) is essential for the
maintenance of neurotransmission in central nerve terminals.
This is because endocytosis retrieves SVs from the neuronal
plasma membrane, allowing them to become available for
exocytosis after their subsequent recycling and refilling with
neurotransmitter. To ensure the efficiency of this retrieval
process, SV exocytosis and endocytosis are closely coupled
both temporally and spatially in nerve terminals. This high
degree of coupling originates from the influx of extracellular
calcium (Ca2+) into nerve terminals on their depolarization
(Cousin 2000). The vast majority of this Ca2+ influx occurs at
active zones. Active zones have a high density of voltagedependent Ca2+ channels and on nerve terminal depolarization, intracellular free Ca2+ levels can reach as high as 1 mM
(Lin and Scheller 2000). Such high levels are required since
the proposed Ca2+ sensor for exocytosis, synaptotagmin, has
a very low affinity for Ca2+ (Yoshihara et al. 2003). After SV
fusion with the plasma membrane, the same Ca2+ influx
stimulates SV endocytosis. SV endocytosis occurs in regions
surrounding the active zone called ‘endocytosis zones’.
However, the levels of Ca2+ required to stimulate the process
are approximately 100-fold lower (Cousin 2000). The Ca2+
sensor for SV endocytosis in mature central nerve terminals
is the Ca2+-dependent protein phosphatase calcineurin (CaN)
(Marks and McMahon 1998; Cousin and Robinson 2001;
Cousin et al. 2001).
CaN was identified as the Ca2+ sensor in isolated central
nerve terminals (synaptosomes) using fluorescent assays of
SV recycling (Marks and McMahon 1998; Cousin et al.
2001). However, in other neuronal preparations, the CaN
dependence of SV endocytosis differs dramatically. For
example, in primary neuronal cultures no requirement for
CaN in SV endocytosis has ever been demonstrated, even
though the process is Ca2+-dependent (Sankaranarayanan
and Ryan 2001). Furthermore, at neuromuscular junctions in
Drosophila larvae, CaN inhibits rather than stimulates SV
endocytosis (Kuromi et al. 1997; Kuromi and Kidokoro
452
Received January 12, 2005; revised manuscript received March 8, 2005;
accepted March 10, 2005.
Address correspondence and reprint requests to Michael A. Cousin,
Membrane Biology Group, Division of Biomedical and Clinical
Laboratory Sciences, George Square, University of Edinburgh, Edinburgh, UK, EH8 9XD. E-mail: [email protected]
Abbreviations used: Ba2+, barium; Ca2+, calcium; CaM, calmodulin;
CaN, calcineurin; CGN, cerebellar granule neurone; CsA, cyclosporin A;
SV, synaptic vesicle.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 452–458
Endocytosis: developmental demand for calcineurin
1999). Conflicting results on the CaN dependency of
endocytosis in chromaffin cells have been reported, with
some forms demonstrating an essential requirement for CaN
whereas in others, CaN is inhibitory or has little effect
(Artalejo et al. 1996; Engisch and Nowycky 1996; Chan and
Smith 2001; Chan et al. 2003). Taken together, these results
suggest that there are either multiple forms of SV endocytosis
that have differential CaN requirements, or that other as yet
unidentified Ca2+ sensors are responsible for this disparity.
We assessed whether the differing requirement for CaN in
SV endocytosis between neuronal preparations could result
from a change in the coupling of Ca2+ influx to the
endocytosis sensor during development. We used a system
that has an essential requirement for CaN activity (synaptosomes) and tested whether this was present throughout
development, using fluorescent assays of SV recycling. We
show that CaN activity only becomes essential for SV
endocytosis between the ages of 2 and 4 weeks after birth.
The same developmental pattern was also observed in
primary neuronal culture. Our study highlights the fact that
Ca2+ influx couples to a different Ca2+ sensor to stimulate SV
endocytosis in immature and adult animals.
Materials and methods
Materials
Cyclosporin A and cypermethrin were from CN Biosciences
(Nottingham, UK). FM2-10 was from Invitrogen (Paisley, UK).
Dynamin I antibody (C-16) was from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Calcineurin antibody was from BD
Biosciences (Oxford, UK). Coverslips were from Raymond Lamb
(Eastbourne, UK). All tissue culture plastics were obtained from
either Falcon (Plymouth, UK) or Greiner (Dursley, UK). Penicillin/
streptomycin, phosphate-buffered salts, foetal calf serum and
Minimal Essential Medium were obtained from Invitrogen (Paisley,
UK). All other reagents were from Sigma (Poole, UK).
Synaptosome FM2-10 exocytosis and endocytosis assays
Synaptosomes were prepared from rat cerebral cortex by centrifugation on discontinuous Percoll gradients (Dunkley et al. 1986). SV
exocytosis was measured using the loading and unloading of FM2-10,
as previously described (Cousin and Robinson 2000). Briefly,
synaptosomes (0.6 mg protein in 2 mL) were incubated for 5 min
at 37C in plus Ca2+ Krebs-like solution (118.5 mM NaCl, 4.7 mM
KCl, 1.18 mM MgCl2, 0.1 mM K2HPO4, 20 mM HEPES, 1.3 mM
CaCl2, 10 mM glucose, pH 7.4). FM2-10 (100 lM) was added
1 min before a standard stimulation with 30 mM KCl (S1). After
2 min of KCl stimulation, synaptosomes were washed twice in plus
Ca2+ solution containing 1 mg/mL bovine serum albumin to remove
non-internalized FM2-10. Washed synaptosomes were resuspended
in either plus or minus (minus 1.3 mM CaCl2, supplemented with
1 mM EGTA) Ca2+ Krebs-like solution at 37C in the presence or
absence of cyclosporin A (CsA), placed inside a fluorimeter (Spex
FluoroMax, Edison, NJ, USA) and stimulated with 30 mM KCl.
Unloading of accumulated FM2-10 was measured as the decrease in
453
fluorescence upon release of the dye into solution by exocytosis
(excitation 488 nm, emission 540 nm).
SV endocytosis was monitored in an identical manner to that
described above apart from being loaded with FM2-10 in the
presence or absence of either CsA or Ca2+ (Cousin et al. 2001).
Accumulated FM2-10 was quantified by subsequent unloading with
a standard addition of 30 mM KCl. Results are presented as the
difference between unloading in plus and minus Ca2+ solutions for
identical stimulation conditions for both exocytosis and endocytosis.
Glutamate release assay
The glutamate release assay was performed using enzyme-linked
fluorescent detection of glutamate (Nicholls and Sihra 1986; Cousin
and Robinson 2000). Synaptosomes were stimulated with either
1.3 mM CaCl2 or 1.3 mM BaCl2. Increases in fluorescence due to
production of NADPH were monitored in a fluorimeter at 340 nm
excitation and 460 nm emission. Experiments were standardized by
the addition of 4 nM of glutamate. Data are presented as barium
(Ba2+)-dependent glutamate release, calculated as the difference
between release in BaCl2- and CaCl2-stimulated samples.
SV recycling assays in cerebellar granule neurones (CGNs)
Primary cultures of CGNs were prepared from the cerebella of
7-day-old Sprague–Drawley rat pups as previously described (Cousin
and Nicholls 1997). SV exocytosis and endocytosis were monitored
with FM2-10 using an S1/S2 protocol, which internally controls for
responses from individual nerve terminals. This is necessary due to
the high degree of variability in dye loading and unloading between
individual nerve terminals in culture (Murthy et al. 1997). Cells
were removed from culture medium and left to repolarize for 10 min in
incubation medium {170 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4,
20 mM TES (N-tris[hydroxy-methyl]-methyl-2-aminoethane-sulfonic
acid), 5 mM NaHCO3, 5 mM glucose, 1.2 mM Na2SO4, 1.2 mM
MgCl2, 1.3 mM CaCl2, pH 7.4}. The total recycling pool of SVs
was loaded with FM2-10 (100 lM) by evoking SV recycling with
50 mM KCl (50 mM NaCl removed to maintain osmolarity) for 2 min.
This corresponds to S1 loading (Fig. 5a). CGNs were washed and after
15 min, FM2-10 was unloaded by two sequential 30 s KCl challenges
at 20 s and 120 s (S1 unloading, Fig. 5a). This S1 protocol provides an
estimate of the total number of SVs turned over during KCl
stimulation. This was repeated for CGNs pre-incubated with 40 lM
CsA for 15 min before and during S2 loading (for SV endocytosis) or
S2 unloading (for SV exocytosis). FM2-10 unloading was quantified
by monitoring the total decrease in fluorescence after normalizing
fluorescence in individual nerve terminals to an arbitrary value.
FM2-10 unloading was visualized using a Nikon (Kingston-UponThames, UK) epifluorescence microscope (Diaphot-TMD) and · 20
objective at 480 nm excitation and > 510 nm emission. Fluorescent
images were visualized using a Hamamatsu Orca-ER CCD digital
camera (Welwyn Garden City, UK) and offline imaging software
(Compix Imaging Systems, PA, USA). Data from at least three
independent experiments, each containing at least 70 nerve terminals,
were collated and presented as the ratio of the total unloading of
FM2-10 at S2 and S1, respectively (S2/S1).
Western blotting
The amount of dynamin I, calcineurin or phosphorylated dynamin I
in synaptosomes was monitored by western blotting using antibodies
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 452–458
454 K. J. Smillie et al.
directed against either dynamin I, calcineurin, or phospho-serine-774
and phospho-serine-778 [both on dynamin I (Tan et al. 2003)]. Blots
were scanned and analysed using GeneSnap and GeneTools
(SynGene, Cambridge, UK) software.
Results
SV endocytosis is CaN-dependent in adult, but not young
animals
SV endocytosis in mature central nerve terminals is dependent upon CaN activity (Marks and McMahon 1998; Cousin
et al. 2001). To determine whether CaN activity is also
essential for SV endocytosis in immature nerve terminals, we
performed exocytosis and endocytosis assays using the styryl
dye FM2-10 (Cousin and Robinson 2000). Figure 1a
displays the protocol for monitoring the effect of the CaN
antagonist, CsA, on both SV exocytosis and endocytosis.
When CsA was applied to adult synaptosomes that received a
standard load of FM2-10, it had no significant effect on KClevoked unloading and thus, exocytosis (Fig. 1b). This
confirms previous studies where CsA had no effect on
KCl-evoked Ca2+-dependent glutamate release in adult nerve
terminals (Nichols et al. 1994; Marks and McMahon 1998;
Cousin et al. 2001; Baldwin et al. 2003). When CsA or a
different CaN antagonist, cypermethrin, was applied prior to
FM2-10 loading, a significant inhibition of KCl-evoked
unloading was observed (Fig. 1c and data not shown).
Previous studies have confirmed that this inhibition is a direct
result of a block in SV endocytosis and not subsequent
recycling (Marks and McMahon 1998). Thus, CsA inhibits
the accumulation of FM2-10 by endocytosis, since exocytosis is unaffected by the drug.
To determine whether CaN was essential for SV endocytosis through development, we repeated these assays using
synaptosomes derived from 2-week-old animals. CsA produced a small, equivalent and significant inhibition of both
FM2-10 unloading and loading in these immature nerve
terminals (Figs 1d and 1e). Cypermethrin also had a similar
effect on FM2-10 loading in immature nerve terminals (data
not shown). This small inhibition of FM2-10 loading in the
endocytosis assay was a direct result of an upstream
inhibition of SV exocytosis and not SV endocytosis, since
the CsA inhibited FM2-10 unloading (SV exocytosis) to the
same extent. CsA had similar effects on Ca2+-dependent
glutamate release from immature synaptosomes (data not
shown). Since SV endocytosis is dependent on the prior
amount of exocytosis, the decrease in the number of SVs
loaded with FM2-10 was due to a decrease in availability of
SVs to retrieve. Thus, at 2 weeks, SV endocytosis is
independent of CaN activity.
To investigate when a requirement for CaN activity
develops, we examined the effect of CsA on SV exocytosis
and endocytosis in a range of animal ages (Fig. 2a). To
Fig. 1 CsA inhibits SV endocytosis, but not exocytosis, from mature
nerve terminals. (a) Scheme of SV exocytosis and endocytosis assays
using FM2-10 in synaptosomes. (b) and (d) Synaptosomes were
loaded with 100 lM FM2-10 during stimulation with 30 mM KCl. After
washing, synaptosomes were pre-incubated with or without extracellular Ca2+ for 5 min and FM2-10 unloading was stimulated with 30 mM
KCl. (c) and (e) Synaptosomes were pre-incubated with or without
extracellular Ca2+ for 5 min and then loaded with 100 lM FM2-10
during stimulation with 30 mM KCl. After washing, synaptosomes were
pre-incubated in extracellular Ca2+ and FM2-10 unloading was stimulated with 30 mM KCl. Ca2+-dependent unloading of accumulated
FM2-10 is displayed in (b)–(e). Where indicated, synaptosomes were
pre-incubated with 40 lM CsA for 5 min before and during FM2-10
unloading (b and d) or before FM2-10 loading (c and e). Synaptosomes were prepared from either 8-month (b and c) or 2-week
(d and e) animals. Upper bar indicates period of KCl stimulation,
n ¼ 3 ± SEM. Lower bar indicates ***p < 0.001, 2-way ANOVA.
obtain an accurate estimate of SV endocytosis, we used the
retrieval efficiency calculation (endocytosis/exocytosis; Cousin and Robinson 1998), since the amount of SV endocytosis
is dependent on the prior amount of exocytosis. We
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 452–458
Endocytosis: developmental demand for calcineurin
determined that SV endocytosis becomes CaN-dependent
4 weeks after birth (Fig. 2b). By 16 weeks, the requirement
for CaN starts to plateau until maturity is reached, where
almost half of all SV endocytosis is dependent on CaN
activity. Thus, immature nerve terminals do not require CaN
activity for SV endocytosis, and this requirement develops
with age.
SV endocytosis in young animals is Ca2+-dependent
One reason for the lack of CaN dependence of endocytosis in
immature nerve terminals could be that the enzyme is either
not present or not active. To test this possibility we probed
lysates from both immature and mature nerve terminals for
the presence of CaN. Figure 3 shows that there is no
difference in the amount of CaN present between immature
and mature nerve terminals. To test whether the CaN present
in immature nerve terminals is active, we examined the
phosphorylation status of one of its substrates, dynamin I.
Dynamin I is co-ordinately dephosphorylated by CaN in
mature nerve terminals at two sites on nerve terminal
depolarization (Tan et al. 2003; Larsen et al. 2004). When
probed with phopho-specific antibodies directed against
these sites, dynamin I was dephosphorylated on both during
depolarization of immature nerve terminals, and this dephosphorylation was abolished by CsA and cypermethrin
(Fig. 3, data not shown). Thus, CaN is present and active in
immature nerve terminals.
SV endocytosis may not require CaN activity in immature
nerve terminals because the process may not be
Ca2+-dependent at this stage of development. To test this
possibility we stimulated SV recycling with the divalent
Fig. 2 SV endocytosis develops a requirement for CaN with age. (a)
Effect of CsA on FM2-10 unloading (SV exocytosis) and FM2-10
loading (SV endocytosis). Results are expressed as a percentage of
control (i.e. % remaining after CaN inhibition). The difference in the
effect of CsA on exocytosis and endocytosis becomes significantly
different at 4 weeks (p < 0.001, Student’s t-test). (b) Retrieval efficiency (endocytosis/exocytosis) from synaptosomes derived from rats
of increasing age. For both (a) and (b), n ‡ 3 ± SEM.
455
cation, barium (Ba2+). Ba2+ inhibits a presynaptic voltagesensitive K+ channel, inducing transient plasma membrane
depolarizations similar to action potentials (Cousin and
Robinson 1998; Marks and McMahon 1998). It also
stimulates SV exocytosis by entering nerve terminals through
voltage-dependent Ca2+ channels and substituting for Ca2+.
However, Ba2+ does not support SV endocytosis in mature
nerve terminals, due to its inability to activate calmodulin
(CaM)/CaN (Cousin and Robinson 1998; Marks and
McMahon 1998). Ca2+ cannot inhibit K+ channels and is
therefore unable to evoke SV exocytosis independently. We
assayed Ba2+-evoked release of endogenous glutamate, since
(a)
(b)
Fig. 3 CaN is present and active in immature nerve terminals. (a)
Synaptosome lysates derived from either 2-week-old or 8-month-old
rats were blotted for either CaN, phosphorylated (P-774 and P-778) or
total (DynI) dynamin I. Lysates were prepared from either basal (B) or
depolarized (30 mM KCl) synaptosomes with or without pre-incubation
with 40 lM CsA (5 min) as indicated. (b) Relative intensity of signal
from two independent experiments displayed as a percentage of the
basal/control condition for both 2-week and 8-month samples. Open
bars denote total dynamin I (Dyn I), cross-hatched bars denote
phosphorylated (P-774) dynamin I and hatched bars denote CaN.
Error bars denote the range of intensities.
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 452–458
456 K. J. Smillie et al.
Ba2+ has complex effects on FM2-10 unloading (data not
shown). Ba2+ efficiently stimulated glutamate release
(Fig. 4a) but could not evoke SV endocytosis in immature
nerve terminals (Fig. 4b). Thus, SV endocytosis in immature
nerve terminals is robustly Ca2+-dependent, suggesting that a
different Ca2+ sensor must activate the process.
(a)
SV endocytosis develops CaN-dependency with age in
primary neuronal cultures
To demonstrate the developing requirement for CaN in SV
endocytosis, we used primary cultures of CGNs. Primary
neuronal cultures are usually derived from embryonic or
neonatal tissue, possibly explaining why a requirement for
CaN in SV endocytosis has never been reported. We
would predict that SV endocytosis would be initially
CaN-independent and then develop a requirement for CaN
on maturation in culture. We devised a protocol to test this
hypothesis (Fig. 5a). CGNs are loaded with FM2-10 using
KCl stimulation and the accumulated dye is then unloaded
with two KCl challenges (S1). The same nerve terminals
are loaded with CsA present either before (endocytosis) or
after (exocytosis) the S2 loading stimulus. Inhibition of
exocytosis or endocytosis by CsA is estimated as the ratio
of total FM2-10 unloaded at S2 to that at S1.
Figure 5(b–d) shows a CGN field after S1 loading
(Fig. 5b) and S2 loading (Fig. 5c) and unloading data from
a typical control experiment (Fig. 5d). Note that the total
amount of unloading for both S1 and S2 are equal,
reflecting the reproducibility of loading and unloading.
CsA had no effect on the S2/S1 ratio for either SV
exocytosis or endocytosis in CGNs after 10 days in vitro
(b)
Fig. 4 SV endocytosis in immature nerve terminals is Ca2+-dependent. (a) Glutamate release from 2-week-old animals was evoked by
either 1.3 mM CaCl2 or 1.3 mM BaCl2. Ba2+-dependent glutamate
release is displayed (BaCl2 minus CaCl2, n ¼ 3 ± SEM). Bar denotes
period of BaCl2 addition. (b) FM2-10 was loaded into 2-week-old
synaptosomes using an S1 stimulation of either 1.3 mM CaCl2 or
1.3 mM BaCl2. The amount of accumulated FM2-10 was quantified
with an unloading stimulus of 30 mM KCl (bar). Graph displays
Ba2+-dependent FM2-10 loading (BaCl2 minus CaCl2, n ¼ 3 ± SEM).
(d)
(c)
(e)
Fig. 5 SV endocytosis develops a CaN requirement with age in culture. (a) Protocol for quantifying the effect of CsA on SV exocytosis
and endocytosis in CGNs. Image of CGN neurite field from a 10 days
in vitro (DIV) culture loaded with FM2-10 at S1 (b) and S2 (c). Inset
displays the reproducible loading of nerve terminals during repetitive
stimuli (arrows). Scale bar denotes 1 lm. (d) Example of FM2-10
unloading from 10 DIV nerve terminals loaded at S1 and S2. The
fluorescence decrease from two repetitive 50 mM KCl stimuli (bars)
are summed and taken as the total amount of accumulated FM2-10 at
S1 or S2, respectively. (e) S2/S1 ratio of the effect of CsA (40 lM for
15 min) on either SV exocytosis (open bars) or endocytosis (hatched
bars) in young (10 DIV) or mature (> 30 DIV) cultures. Experiments
were ‡ 3 (10 DIV: control n ¼ 221, CsA exocytosis n ¼ 345, CsA
endocytosis n ¼ 246. > 30 DIV: control n ¼ 252, CsA exocytosis n ¼
351, CsA endocytosis n ¼ 215. All ± SEM).
(Fig. 5e). Thus, in immature CGN cultures both SV
exocytosis and endocytosis is CaN-independent. To determine if a requirement for CaN in SV endocytosis develops
with age, we performed the same protocol using CGNs
older than 30 days in vitro. A robust and reproducible
inhibition of SV endocytosis by CsA was observed, with
no effect on SV exocytosis (Fig. 5e). Thus, in both
primary neuronal cultures and acutely isolated nerve
terminals a requirement for CaN activity in SV endocytosis
develops on nerve terminal maturation.
Discussion
Ca2+ influx stimulates both SV exocytosis and endocytosis
in mature nerve terminals (Cousin and Robinson 1998;
Marks and McMahon 1998; Cousin 2000). The protein
phosphatase CaN is activated by this influx and the
subsequent dephosphorylation of a group of proteins,
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 452–458
Endocytosis: developmental demand for calcineurin
called the dephosphins, is essential for SV endocytosis
(Cousin and Robinson 2001). We demonstrate that the
requirement for CaN activity develops 2–4 weeks after
birth using nerve terminals prepared from both immature
and adult animals. The lack of CaN dependence of SV
endocytosis in immature nerve terminals is not explained
by an absence or inactivation of CaN, since dynamin I is
still dephosphorylated on nerve terminal depolarization.
Likewise, it is not because of a lack of Ca2+ dependency,
since SV endocytosis is abolished by substituting the
divalent cation Ba2+ for Ca2+ (Cousin and Robinson 1998;
Marks and McMahon 1998). Finally, we have shown that
SV endocytosis is CaN-dependent in mature, but not
immature primary neuronal cultures.
A developmental change in the CaN dependence of SV
endocytosis
A developmental requirement for CaN activity in SV
endocytosis explains the apparent contradictions in the
published literature. The only other experimental system to
demonstrate an obligatory requirement for CaN activity is
the bovine chromaffin cell (Chan and Smith 2001; Chan
et al. 2003). Two forms of endocytosis were observed in
cells derived from adult animals during stimulation with
action potential waveforms: a CaN-independent form that
predominated at basal frequencies (termed type I endocytosis) and a CaN-dependent form that was activated by
higher firing frequencies (termed type II endocytosis)
(Chan and Smith 2001). In a different study using adult
chromaffin cells, CaN antagonists abolished the
Ca2+-dependency of endocytosis evoked by square waveform depolarizations (termed compensatory retrieval)
(Engisch and Nowycky 1996). This loss of Ca2+-dependency was explained by a selective inhibition of type II
endocytosis, since compensatory retrieval is likely to be a
mixture of both type I and type II retrieval (Chan and
Smith 2001). In contrast, chromaffin cells derived from
immature animals have no requirement for CaN. Various
approaches to disrupt CaN function all resulted in an
acceleration of a rapid and excess retrieval of membrane
that was stimulated by high intensity square waveform
pulses (Artalejo et al. 1996). This suggests that CaN
activity may inhibit endocytosis in younger animals. A
similar inhibitory role for CaN is apparent at the
neuromuscular junction of Drosophila larvae, where CaN
antagonists greatly increase FM1-43 uptake into nerve
terminals (Kuromi et al. 1997; Kuromi and Kidokoro
1999). This form of SV endocytosis was strictly
Ca2+-dependent, as was the excess retrieval pathway
reported in calf chromaffin cells (Artalejo et al. 1996).
When these results are considered with our data it appears
that SV endocytosis is Ca2+-dependent at all ages of
animal, from pre to postnatal. However, during development the requirement for CaN changes from a putative
457
inhibitory role in immature nerve terminals to a stimulatory role in mature nerve terminals.
An unidentified Ca2+ sensor for SV endocytosis in
immature nerve terminals
We suggest that the lack of CaN dependency in immature
nerve terminals is due to a switch in the Ca2+ coupling of
SV endocytoisis during development. In other words, Ca2+
influx is still required to stimulate SV endocytosis, but the
sensor that transduces its effect is different. A requirement
for this unidentified sensor may continue into adulthood,
since CaN antagonists do not always abolish SV endocytosis (Cousin et al. 2001, but see Marks and McMahon
1998). One possibility is that Ca2+ influx controls essential
interactions between endocytosis proteins, such as that
observed for dynamin I with both endophilin and synaptophysin (Daly and Ziff 2002; Chen et al. 2003). Another
is that the Ca2+ binding protein CaM, is the Ca2+ sensor,
since excess membrane retrieval in calf adrenal chromaffin
cells has an absolute requirement for its activity (Artalejo
et al. 1996). Possible downstream targets are still unclear.
However, CaM may act via the release of a Ca2+dependent binding partner such as GAP-43, rather than the
activation of a Ca2+-dependent target. In cortical neurones
overexpression of GAP-43 mutants that constitutively bind
CaM disrupted SV endocytosis, whereas GAP-43 mutants
unable to bind CaM enhanced the process (Neve et al.
1998). Thus, the release of a Ca2+-independent CaMinteracting protein may be a possible mechanism to control
SV endocytosis. This has previously been observed in
yeast, where the release of the Ca2+-independent binding
partner, unconventional myosin I (Geli et al. 1998), and
the Ca2+-dependent activation of the actin binding protein,
Arp2/3, are both essential for endocytosis (Kubler et al.
1994; Schaerer-Brodbeck and Riezman 2000). Thus, CaM
may play a dual role in the control of SV endocytosis by
activating CaN and releasing a Ca2+-independent binding
partner.
We have uncovered a developmental switch in the Ca2+
coupling of SV endocytosis in central nerve terminals. In
immature animals, SV endocytosis has no requirement for
CaN activity. However, this requirement develops from
4 weeks after birth through to adulthood. Our data explain
previous inconsistencies in the published data and indicate
that a different Ca2+ sensor must stimulate the process in
immature nerve terminals.
Acknowledgements
We thank Prof. Phil Robinson for the gift of the phospho-specific
dynamin I antibodies. This work was supported by grants from
The Wellcome Trust (Ref: GR070569), the Cunningham Trust
and a University of Edinburgh Medical Faculty Scholarship (to
KJS).
2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 452–458
458 K. J. Smillie et al.
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