(Received for publication, July 6, 1995)
From the
Ca-dependent neurotransmitter release consists
of at least two components: a major fast component that is insensitive
to Sr
and a minor slow component that is potentiated
by Sr
(Goda, Y., and Stevens, C. F.(1994) Proc.
Natl. Acad. U. S. A. 91, 12942-12946). These results suggest
that at least two Ca
sensors act in synaptic vesicle
fusion with distinct Ca
and Sr
binding properties. We have now investigated the relative
Ca
and Sr
binding activities of
synaptotagmins to evaluate their potential roles as
Ca
sensors for the fast and slow components. Our
results demonstrate that the first C
domains of
synaptotagmins I, II, III, V, and VII have very similar Ca
requirements for phospholipid binding (range of EC
= 2.6 µM to 5.0 µM), but
distinct Sr
requirements (EC
range
= 23 µM to 133 µM); synaptotagmins I
and II had the lowest Sr
affinity, and synaptotagmin
III the highest Sr
affinity. Purified synaptotagmin I
from bovine brain exhibited similar properties as its recombinant first
C
domain, suggesting that the first C
domain
fully accounts for its Ca
-dependent phospholipid
binding properties. Sr
was unable to trigger syntaxin
binding by synaptotagmin I at all concentrations tested, whereas it was
effective for synaptotagmin III. These results suggest that different
C
domains have distinct Sr
binding
properties. They support the hypothesis that synaptotagmins localized
on the same vesicle perform distinct functions, with synaptotagmins I
and II serving as candidate Ca
sensors for the fast
component in release and synaptotagmin III for the slow component.
Synaptotagmins constitute an abundant family of
Ca-binding proteins of synaptic vesicles.
Structurally, synaptotagmins are composed of a short intravesicular
sequence, a single transmembrane region, and two copies of an
cytoplasmic repeat homologous to the C
domain of protein
kinase C (reviewed in Südhof(1995)). Synaptotagmins
are highly conserved in evolution (Perin et al., 1991a;
Bommert et al., 1993; Nonet et al., 1993) and
expressed in multiple isoforms with at least 9 genes in mammals (Perin et al., 1990; Geppert et al., 1991; Mizuta et
al., 1994; Hilbush and Morgan, 1994; Ullrich et al.,
1994; Li et al., 1995; Craxton and Goedert, 1995).
Ca
binds to purified synaptotagmin I at micromolar
concentrations and triggers phospholipid binding by synaptotagmin I
(Brose et al., 1992). Ca
-dependent
phospholipid binding by synaptotagmin I is at least partly mediated by
its first C
domain since the isolated first C
domains of synaptotagmin I and other synaptotagmins, when
produced as recombinant proteins, also bind phospholipids as a function
of Ca
(Davletov and Südhof, 1993;
Ullrich et al., 1994; Li et al., 1995). Only the
C
domains of synaptotagmins IV, VI, and VIII were unable to
bind phospholipids in a Ca
-regulated manner,
suggesting that they may not represent Ca
binding
domains. Ca
binding causes a conformational change in
synaptotagmin I and its first C
domain (Davletov and
Südhof, 1994). Crystallization of the first C
domain of synaptotagmin I confirmed that it forms a novel
Ca
binding module which is almost entirely composed
of
sheets and in which Ca
is coordinated
between apical loops (Sutton et al., 1995).
These
biochemical studies demonstrated that synaptotagmin is a major
Ca-binding protein of synaptic vesicles, suggesting
that it serves as a Ca
sensor in the synaptic vesicle
cycle. The best characterized Ca
action in the nerve
terminal is the triggering of neurotransmitter release, although
Ca
probably plays multiple additional roles in
regulating synaptic vesicle traffic. A detailed analysis of the
kinetics of neurotransmitter release in cultured hippocampal neurons
revealed that at least two components can be distinguished in
Ca
-dependent release: a major fast component that has
a low Ca
affinity and cannot be triggered by
Sr
, and a more minor slow component that is sensitive
to Sr
and has a higher apparent Ca
affinity (Goda and Stevens, 1994). To test the possible function
of synaptotagmin I as a Ca
sensor, mice homozygous
for a synaptotagmin I mutation were analyzed (Geppert et al.,
1994). In these mice, a selective impairment of the fast component of
Ca
-dependent neurotransmitter release but not of the
slow component was observed, suggesting that synaptotagmin I is the
major Ca
sensor for the fast component of release in
the synapses that were analyzed in the mutant.
Although the studies
on synaptotagmin I-mutant mice demonstrated an essential role for
synaptotagmin I in fast Ca-triggered release, the
apparent Ca
affinity of synaptotagmin I as measured
by phospholipid binding (EC
= 3-6
µM) was much higher than that of the putative exocytotic
Ca
sensor (>200 µM; Llinas et
al.(1992) and Heidelberger et al. (1994)). This apparent
paradox was resolved upon the discovery that synaptotagmins and their
first C
domains have multiple independent
Ca
-activated properties (Li et al., 1995).
Phospholipid binding triggered at Ca
concentrations
that are similar for most synaptotagmins, and syntaxin binding at
Ca
concentrations that differ between synaptotagmins.
Phospholipid binding is observed in the low micromolar range of
Ca
for all C
domains that exhibit
Ca
-dependent phospholipid binding. In contrast,
syntaxin binding requires high concentrations of Ca
for synaptotagmins I and II (>200 µM) but low
Ca
concentrations for synaptotagmins III and VII
(<10 µM). These studies provided a biochemical
correlate for the events during the last stage of synaptic vesicle
exocytosis and showed that a single C
domain may contain
multiple Ca
binding sites that have not yet been
defined structurally.
Multiple synaptotagmins are co-localized in
brain. The synaptotagmin I mutation gave a strong phenotype in spite of
the presence of other synaptotagmins; in fact, synaptotagmins I and III
are colocalized in the hippocampal neurons that are affected in the
knockout (Ullrich et al., 1994). This suggests that different
synaptotagmin isoforms perform distinct functions. The Ca binding properties of most synaptotagmins suggest that they also
function as Ca
sensors similar to synaptotagmin I.
The fact that synaptotagmin III has a higher Ca
affinity than synaptotagmins I in the syntaxin interaction and
that the slow component of neurotransmitter release is unimpaired in
the synaptotagmin I mutants raises the possibility that synaptotagmin
III could function as a Ca
sensor for the slow
component of release.
The defining property of the slow component of
release is its distinct Sr sensitivity compared to
the fast component (Goda and Stevens, 1994). We have now investigated
the possibility that the slow component of neurotransmitter release may
be mediated by synaptotagmin III by studying the relative
Sr
sensitivities of different synaptotagmins. Our
results demonstrate that although all synaptotagmins exhibit similar
Ca
affinities for phospholipid binding, they exhibit
dramatically different affinities for Sr
. Direct
comparison of the phospholipid binding properties of recombinant first
C
domains of synaptotagmin I and of the cytoplasmic domains
of purified brain synaptotagmin I demonstrated that they have very
similar properties, suggesting that the properties of the first C
domain reflect the properties of native synaptotagmin. Together
these studies show that results with the recombinant first C
domains accurately reflect the activities of the whole protein
and that synaptotagmin III has the requisite cation specificity of the
sensor for the slow component.
Figure 1:
Phospholipid
binding to the first C domains of synaptotagmins I, II,
III, V, and VII as a function of Ca
(top
panel) or Sr
(bottom panel).
Recombinant GST-synaptotagmin fusion proteins containing the first
C
domains of synaptotagmin I, II, III, V, and VII were
immobilized on glutathione agarose beads and incubated with
radiolabeled phospholipids at the concentrations of divalent cations
indicated. Beads were washed, and binding was determined by
scintillation counting and normalized to the range of 0% to 100% to
make the results with different recombinant proteins
comparable.
Figure 2:
Immobilization of purified full-length
synaptotagmin I (lane A) or of the cytoplasmic domains of
synaptotagmin I produced by proteolytic cleavage (lane B) on
Protein A-Sepharose via a synaptotagmin I-specific monoclonal antibody
(Cl41.1). The figure shows a Coomassie Blue-stained SDS gel of the
material used for divalent cation-dependent phospholipid binding
measurements to brain synaptotagmin I (Fig. 3). Intact
synaptotagmin I migrates on the gel as fuzzy monomeric and dimeric
bands because it is glycosylated and multimerizes via a sequence close
to the transmembrane region (Perin et al., 1991b).
Glycosylated sequences and the multimerization domain are not present
in the proteolytic cytoplasmic fragment immobilized by the antibody (lane B) which consists of the two C domains and
the C terminus. Filled arrows indicated positions of the
different forms of synaptotagmin I. Open arrows point to the
heavy and light chain of the monoclonal antibody used for the
immobilization. Numbers on the left indicate positions of
molecular weight markers.
Figure 3:
Phospholipid binding to the immobilized
cytoplasmic domains of synaptotagmin I from bovine brain as a function
of Ca (left panel), Sr
(middle panel), and Ba
(right
panel). In each panel, filled circles demonstrate phospholipid binding obtained in the presence of
synaptotagmin I, and open circles background binding obtained
in the absence of immobilizing antibody to control for nonspecific
cation-dependent phospholipid precipitation. Numbers in the left
upper corner of each panel give the binding constants calculated
for the experiment shown, half-maximal binding (EC
), and
apparent Hill coefficients (n). Bars indicate the
standard errors of the mean from triplicate determinations. The
experiment was repeated three times with similar
results.
Phospholipid binding to the immobilized
cytoplasmic fragment of native synaptotagmin I was measured as a
function of the Ca, Sr
, and
Ba
concentrations (Fig. 3). Native
synaptotagmin I containing both C
domains had an apparent
affinity for Ca
-dependent phospholipid binding that
was virtually identical with that observed for the GST-C
domain fusion protein (EC
= 5.4
µM), and its Hill coefficient was also very similar. Thus,
the Ca
/phospholipid binding properties of the single
first C
domain of synaptotagmin I accurately reflect the
properties of the cytoplasmic domain of the native protein. Both
Sr
and Ba
were found to mediate
phospholipid binding to native synaptotagmin I but required much higher
divalent cation concentrations than Ca
(Fig. 3). The Sr
concentration
dependence observed was similar to that for the recombinant first
C
domain ( Fig. 1and Fig. 3). Furthermore, in
studies measuring the Ca
dependence of syntaxin
binding of native synaptotagmin I, a Ca
dependence
was found that was indistinguishable from that of the first C
domain (data not shown; Li et al., 1995). Together these
data suggest that the cation-activated phospholipid binding properties
of synaptotagmin can be completely accounted for by the activities of
its first C
domain.
Figure 4:
Ability of Ca,
Sr
, and Ba
to trigger syntaxin
binding to the first C
domains of synaptotagmins. Purified
GST-synaptotagmin fusion proteins containing the first C
domains of the indicated synaptotagmins were immobilized on
glutathione agarose beads and incubated with solubilized brain
homogenates in the presence of the indicated divalent cations as
described under ``Experimental Procedures.'' Beads were
washed in the same buffers, and syntaxin bound was determined by
immunoblotting.
Figure 5:
Concentration dependence of
Sr-activated syntaxin binding to the first C
domains of synaptotagmins I and III. Binding measurements to the
immobilized GST-synaptotagmin fusion proteins were carried out in the
presence of the indicated Sr
concentrations as
described in Fig. 4. Syntaxin binding was measured by SDS-PAGE
and immunoblotting for syntaxin I.
Previous studies demonstrated that synaptotagmin I has the
Ca binding characteristics of the Ca
sensor for fast Ca
-triggered synaptic vesicle
exocytosis (Brose et al., 1992; Davletov and
Südhof, 1993, 1994; Chapman and Jahn, 1994; Li et al., 1995), and that, in its absence, fast
Ca
-dependent release in hippocampal synapses is
impaired (Geppert et al., 1994). These studies suggested that
synaptotagmin I serves as the major Ca
sensor in the
very last step of synaptic vesicle exocytosis in the hippocampus but
posed puzzling new questions. First, why are other synaptotagmins that
are colocalized with synaptotagmin I, especially synaptotagmin III, not
redundant with synaptotagmin I? Second, what is the nature of the slow
component of Ca
-dependent release that is unchanged
in the synaptotagmin I mutants? Third, how does synaptotagmin I act in
release? Although we are unable to answer these questions definitively
at present, results from the current study support a model whereby in
hippocampal synapses synaptotagmins I and III serve as distinct
Ca
sensors for the fast and slow components,
respectively, of Ca
-dependent neurotransmitter
release.
The major defining characteristics of the slow component of
neurotransmitter release is that it is triggered by Sr instead of Ca
whereas the fast component is not
(Goda and Stevens, 1994). In the current study we provide evidence that
synaptotagmins exhibit distinct Sr
binding
activities. We show that the first C
domains of all
Ca
-dependent synaptotagmins are activated to bind
phospholipids at similar Ca
concentrations but that
they differ markedly in their response to Sr
.
Synaptotagmins I and II required high Sr
concentrations for binding, whereas synaptotagmin III bound
phospholipids at relatively low Sr
concentrations.
More decisively, synaptotagmin III was the only synaptotagmin tested in
which Sr
can substitute for Ca
in
activating syntaxin binding, with a concentration dependence that is
similar to that of phospholipid binding.
Together with the previous
demonstration that the apparent Ca affinity of
synaptotagmin III is much higher than that of synaptotagmin I as
measured by syntaxin binding (Li et al., 1995), these data
show that synaptotagmin III has the requisite Ca
binding characteristics and specificity of the Ca
sensor for the slow component of neurotransmitter release.
Furthermore, the new data add to the complexity of C
domains by demonstrating that in spite of the similarity in the
Ca
dependence of phospholipid binding between
different C
domains, these differ dramatically in their
Sr
-activated properties. In addition, our results
indicate that the currently known properties of synaptotagmins are
insufficient to explain the functions of these proteins as
Ca
sensors in release. Besides the puzzle of why a
synapse should have separable fast and slow release components of
Ca
-dependent release, the fact remains that
synaptotagmin III cannot substitute for synaptotagmin I, even though
synaptotagmin III requires lower Ca
concentrations
than I. Thus, the syntaxin interaction of synaptotagmins in itself is
not sufficient to explain their in vivo functions. Clearly,
synaptotagmins I and III must not only have distinct functions in the
nerve terminal but also different Ca
-dependent
targets that have not yet been identified.
Almost all
Ca-dependent activities of synaptotagmins were
determined using recombinant proteins. In these studies, only the first
C
domain shows Ca
-dependent activities,
and, at least for the currently known Ca
-activated
targets of synaptotagmins, phospholipids, and syntaxins, the second
C
domain is not regulated by Ca
. Using
recombinant proteins, it was suggested that the second C
domain binds phospholipids independent of Ca
and that it acts synergistically with the first C
domain (Damer and Creutz, 1994). To gain a better understanding
of how the activities of recombinant single or double C
domains relate to those of native synaptotagmins and to determine
the behavior of the native protein, we purified synaptotagmin I from
brain and measured its Ca
-, Sr
-,
and Ba
-dependent phospholipid binding activities.
These experiments showed that the native protein and the isolated
recombinant first C
domain have very similar
Ca
-dependent properties, indicating that the
properties of the first C
domain fully account for the
known Ca
-dependent properties of native
synaptotagmin.
Native synaptotagmin showed little
Ca-independent phospholipid binding properties or
synergistic effect of two versus one C
domain.
Thus, in the native protein, the second C
domain does not
appear to contribute to Ca
-dependent or
Ca
-independent phospholipid binding. However, the
second C
domains of synaptotagmins bind AP-2 (Zhang et
al., 1994; Li et al., 1995) and polyinositol phosphates
(Fukuda et al., 1994) in a manner that does not require
Ca
, although this does not necessarily mean that the
second C
domain is not a Ca
binding
domain. Future studies will have to determine whether the second
C
domains of synaptotagmins are Ca
binding domains for unknown targets, and whether they
functionally cooperate with the first C
domains or if the
two C
domains serve completely separate functions (for
example in exo- and endocytosis). Regardless of these limitations in
our understanding, synaptotagmins have already now emerged as a focus
in Ca
-regulated membrane traffic with complex
Ca
-dependent properties that differ between isoforms.