RIM1? phosphorylation at serine-413 by protein
kinase A is not required for presynaptic
long-term plasticity or learning
Pascal S. Kaeser*†, Hyung-Bae Kwon‡, Jacqueline Blundell§, Vivien Chevaleyre‡¶, Wade Morishita?, Robert C. Malenka?,
Craig M. Powell§**, Pablo E. Castillo‡, and Thomas C. Su ¨dhof*†,††‡‡§§
Departments of *Neuroscience,§Neurology,††Molecular Genetics, and **Psychiatry and‡‡Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center, Dallas, TX 75390;‡Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461;
and?Department of Psychiatry and Behavioral Sciences, Nancy Pritzker Laboratory, Stanford University School of Medicine, Palo Alto, CA 94304
Contributed by Thomas C. S¨ udhof, July 11, 2008 (sent for review June 6, 2008)
Activation of presynaptic cAMP-dependent protein kinase A (PKA)
triggers presynaptic long-term plasticity in synapses such as cere-
bellar parallel fiber and hippocampal mossy fiber synapses. RIM1?,
a large multidomain protein that forms a scaffold at the presyn-
aptic active zone, is essential for presynaptic long-term plasticity in
these synapses and is phosphorylated by PKA at serine-413. Pre-
vious studies suggested that phosphorylation of RIM1? at serine-
413 is required for presynaptic long-term potentiation in parallel
fiber synapses formed in vitro by cultured cerebellar neurons and
that this type of presynaptic long-term potentiation is mediated by
binding of 14-3-3 proteins to phosphorylated serine-413. To test
the role of serine-413 phosphorylation in vivo, we have now
produced knockin mice in which serine-413 is mutated to alanine.
Surprisingly, we find that in these mutant mice, three different
forms of presynaptic PKA-dependent long-term plasticity are nor-
mal. Furthermore, we observed that in contrast to RIM1? KO mice,
RIM1 knockin mice containing the serine-413 substitution exhibit
normal learning capabilities. The lack of an effect of the serine-413
mutation of RIM1? is not due to compensation by RIM2? because
mice carrying both the serine-413 substitution and a RIM2? dele-
tion still exhibited normal long-term presynaptic plasticity. Thus,
phosphorylation of serine-413 of RIM1? is not essential for PKA-
dependent long-term presynaptic plasticity in vivo, suggesting
that PKA operates by a different mechanism despite the depen-
dence of long-term presynaptic plasticity on RIM1?.
active zone ? neurotransmitter release ? Rab3 ? synaptic vesicle ?
mediate use-dependent changes in synaptic transmission and are
implicated in many forms of learning and memory (1). Different
subsets of synapses exhibit distinct forms of long-term plasticity.
The more widely studied postsynaptic form of LTP is induced by
an NMDA receptor-dependent mechanism and involves the
regulation of AMPA-type glutamate receptors (2, 3). However,
excitatory synapses, such as those formed by hippocampal mossy
fibers (4, 5), cerebellar parallel fibers (6, 7), and the corticos-
triatal (8) and corticothalamic connections (9), as well as inhib-
itory synapses that contain presynaptic cannabinoid receptors
(10, 11), express presynaptic forms of long-term plasticity. A
common feature of these types of plasticity is that induction of
long-term plasticity depends on protein kinase A (PKA) acti-
vation, and expression is due to changes in the amount of
neurotransmitter release evoked by an action potential (12, 13).
In these synapses, calcium entry triggers the calcium/calmodu-
lin-sensitive adenylyl cyclase to synthesize cAMP, which in turn
activates presynaptic PKA. PKA activation is required for pre-
synaptic long-term plasticity (reviewed in ref. 14).
Two presynaptic proteins seem to be essential for most if not
all forms of PKA-dependent long-term plasticity: the low-
ynaptic long-term potentiation (LTP) and long-term depres-
sion (LTD) are central processes in the nervous system that
molecular-weight GTP-binding protein Rab3A, which is local-
ized to synaptic vesicles, and its effector protein RIM1?, which
is localized to active zones (15, 16). In contrast, other proteins,
such as synapsins and rabphilins—both of which are PKA
substrates—are not required for presynaptic, PKA-dependent,
long-term plasticity (17, 18). These findings indicated that a
presynaptic pathway that involves binding of Rab3A on synaptic
vesicles to RIM1? in the presynaptic active zone mediates
presynaptic plasticity but did not reveal how PKA regulates their
function in presynaptic long-term plasticity. The discovery that
RIM1? is a substrate for PKA at two sites (serine-413 and
serine-1548), and that in cultured cerebellar neurons, serine-413
but not serine-1548 was essential for long-term presynaptic
serine-413 (19). Consistent with this notion, phosphorylation of
serine-413 in RIM1? was shown to induce binding of 14-3-3
proteins, and this binding was found to regulate neurotransmit-
ter release (20).
Because of the central importance of presynaptic long-term
plasticity in brain function, we have now tested the relevance of
serine-413 phosphorylation of RIM1?, and 14-3-3 binding to
RIM1? in vivo. We show that phosphorylated serine-413 is the
only tight binding site for 14-3-3 proteins in RIM1? and that
14-3-3 binding is dependent on phosphorylation of serine-413.
More importantly, we show that in mice, mutating serine-413 to
alanine abolishes RIM1? phosphorylation and 14-3-3 protein
binding in vitro but does not detectably alter presynaptic long-
term plasticity. Our data thus suggest that, although the Rab3A/
RIM1? complex is essential for presynaptic long-term plasticity,
PKA regulates this type of plasticity by a mechanism that is
distinct from its phosphorylation of RIM1?.
Phosphoserine-413 Represents the Only Tight 14-3-3 Binding Site in
RIM1?. To identify specific interaction partners for RIM1?
phosphorylated on serine-413, we performed affinity chroma-
Author contributions: P.S.K., R.C.M., C.M.P., P.E.C., and T.C.S. designed research; P.S.K.,
H.-B.K., J.B., V.C., W.M., and C.M.P. performed research; P.S.K. contributed new reagents/
analytic tools; P.S.K., H.-B.K., J.B., V.C., W.M., R.C.M., C.M.P., P.E.C., and T.C.S. analyzed
data; and P.S.K., R.C.M., C.M.P., P.E.C., and T.C.S. wrote the paper.
The authors declare no conflict of interest.
†Present address: Department of Molecular and Cellular Physiology and Neuroscience
Institute, Stanford University School of Medicine, Palo Alto, CA 94304-5543.
¶Present address: Department of Neuroscience, Columbia University, New York, NY 10032.
§§To whom correspondence should be sent at the present address: Department of Molec-
ular and Cellular Physiology and Neuroscience Institute, Stanford University School of
Medicine, Palo Alto, CA 94304-5543. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 23, 2008 ?
vol. 105 ?
no. 38 www.pnas.org?cgi?doi?10.1073?pnas.0806679105
We purified RIM1-GST fusion proteins containing residues
351–596 of RIM1?, incubated them with PKA in the presence or
absence of ATP, and coupled them to amino-link chromatog-
raphy columns. We then bound solubilized rat brain proteins
containing phosphatase inhibitors to the column and analyzed
bound proteins by SDS/PAGE and Coomassie staining (Fig. 1 A
and B). One band of ?30 kDa specifically bound to phosphor-
ylated but not to nonphosphorylated RIM1-GST fusion protein
and was analyzed by mass spectroscopy and immunoblotting. We
found that this band consisted of multiple isoforms of 14-3-3
proteins (14-3-3?, 14-3-3?, and 14-3-3?; Fig. 1B). We confirmed
this finding with GST-pulldown experiments using GST-RIM1?
fusion proteins that contain residues 351–429 and 351–596 of
RIM1? [supporting information (SI) Fig. S1], and our data are
consistent with previously reported binding of phosphorylated
RIM1? to 14-3-3 proteins (20).
To test whether the affinity chromatography results reflect a
also bind to 14-3-3 proteins, we performed additional GST-
pulldown experiments using phosphorylated and nonphospho-
rylated GST-RIM1? fusion proteins. We first examined 14-3-3
binding to GST-fusion proteins containing residues 351–429 of
RIM1?, either with a wild-type sequence or with mutations that
substitute serine-413 for alanine, aspartate, or glutamate. These
mutations were used to test not only the phosphorylation
dependence of 14-3-3 protein binding with the alanine substi-
tution, but also the specificity of 14-3-3 protein binding with the
aspartate and glutamate substitutions that are thought to mimic
phosphorylated serines and threonines for some phosphoserine
and -threonine residues (21) but are known to be inactive for
(serine to alanine, serine to aspartate, and serine to glutamate)
inactivated binding of 14-3-3 proteins to RIM1? (Fig. 1C).
We next tested whether RIM1? contains other tight binding
sites for 14-3-3 proteins. This test was prompted by the fact that
14-3-3 proteins usually occur as dimers and function to induce
conformational changes, to mask protein domains, or to scaffold
proteins (23), and was further motivated by a previous study
indicating that RIM1? binds 14-3-3 proteins in a phosphoryla-
tion-independent manner between residues 241 and 287 (24).
For this purpose, we used GST-fusion proteins covering the
entire RIM1? sequence (Fig. 1A). However, we observed no
other 14-3-3 binding sites in RIM1? using GST-pulldown ex-
periments with or without phosphorylation by PKA (Fig. 1D).
Thus, RIM1? likely contains a single tight binding site for 14-3-3
that is generated by PKA phosphorylation of serine-413, al-
though the presence of additional low-affinity binding sites
cannot be excluded.
Generation and Characterization of Knockin Mice Containing a Serine-
413 to Alanine Substitution in RIM1?. To test the physiological role
of the phosphorylation of RIM1? at serine-413, we used ho-
mologous recombination to generate knockin (KI) mice in which
the RIM1 gene contains a serine-413 to alanine (S413A) sub-
stitution (Fig. 2A). In addition to the S413A point mutation,
silent BglI and SphI restriction sites were introduced into the
ORF for genotyping the S413A mutant locus. Homologous
recombination experiments were performed in embryonic stem
cells using selection with a neomycin resistance cassette (Fig.
2A) and were confirmed in the stem cells and in the mice we
generated from them by Southern blotting, PCR, and DNA
sequencing (Fig. 2 B–D). The neomycin resistance cassette was
removed from the mice by flp recombination, resulting in a
mouse line that contained only the serine-413 point mutation in
exon 6, a single frt recombination site in intron 6, and loxP sites
flanking exon 6. This mouse line (referred to as RIM1 S413A-KI
mice) was used for all experiments.
RIM1 S413A-KI mice were viable and fertile and exhibited no
impairment of survival or loss of weight when compared with
littermate wild-type control mice (Fig. 3 A and B). Immuno-
blotting showed that RIM1? expression was similar in littermate
wild-type and RIM1 S413A-KI mice (Fig. 3C). Importantly, a
previously characterized phospho-specific antibody directed
against phosphorylated serine-413 confirmed a complete ab-
sence of phosphoserine-413 RIM1? in RIM1 S413A-KI mice
(arrow, Fig. 3C). There was a cross-reactive band (marked with
and asterisk in Fig. 3C) at a slightly lower molecular weight that
was present in both the KI and the wild-type control mice. This
cross-reactive band is not RIM1? or RIM2? because it is still
present in RIM1? or RIM2? KO mice (data not shown).
Immunoblotting analyses of other brain proteins failed to detect
any major changes in the RIM1 S413A-KI mice (Fig. 3D).
Normal Presynaptic Long-Term Plasticity in RIM1 S413A-KI Mice. We
next tested whether the mutational block of RIM1? phosphor-
ylation in RIM1 S413A-KI mice impairs presynaptic long-term
plasticity using electrophysiological recordings in acute slices
from littermate wild-type control and RIM1 S413A-KI mice.
Surprisingly, no significant impairment in any form of presyn-
aptic plasticity was detected (Fig. 4 and Table S1). Specifically,
measurements of LTP at the cerebellar granule cell to Purkinje
cell synapse after repetitive stimulation of parallel fibers failed
to detect a decrease in LTP in the RIM1 S413A-KI mice (Fig.
overview of RIM1? and its protein domains. GST-fusion proteins that were
used for the biochemical characterization are indicated. SSA, SSB, SSC, splice
(B) Coomassie blue staining of the eluates of the affinity column that con-
tained phosphorylated GST-RIM1 351–596 and nonphosphorylated control
RIM1?. The arrow indicates the phospho-specific band that was submitted to
351–429 to 14-3-3? in GST-pulldown experiments from rat brain homoge-
nates. (D) In vitro binding of PKA phosphorylated and nonphosphorylated
GST-RIM1 fusion proteins to 14-3-3? in GST pulldown experiments from brain
homogenates. S/A, S413A-point mutant RIM1? fragment; wt, wild-type RIM1
fragment; *, cross-reactive band that sometimes appears in fresh brain ho-
mogenates with the 14-3-3? antibody.
RIM1? contains a single tight 14-3-3 binding site. (A) Schematic
Kaeser et al.
September 23, 2008 ?
vol. 105 ?
no. 38 ?
4A). Similarly, LTP in hippocampal mossy fiber synapses was
unchanged (Fig. 4B), as was endocannabinoid-dependent LTD
at inhibitory synapses in the CA1 area of the hippocampus
(I-LTD; Fig. 4C). Thus, different from rescue experiments in
cultured neurons (19), presynaptic long-term plasticity in acute
brain slices does not require phosphorylation of serine-413 in
RIM1?, even though it does require RIM1? itself (11, 16).
The lack of a plasticity phenotype in the RIM1 S413A-KI mice
this germline mutation. Although unlikely given that the RIM1?
KO also represents a germline mutation and does produce a
defect in plasticity (11, 16), we tested this possibility by analyzing
mice that carry both the RIM1 S413A-KI mutation and a
deletion of RIM2? (25). RIM2? is the only other RIM isoform
other than RIM1? that binds to Rab3; thus, any compensation
would likely have to operate through this isoform. However, we
found that mossy fiber LTP in these double-mutant mice was
also unchanged (Fig. 4D). Furthermore, parallel fiber LTP was
expressed in these double-mutant mice (double-mutant RIM1
S413A-KI/RIM2? KO mice: 138 ? 8%, 5 slices/3 mice; data not
shown). These experiments exclude the possibility that RIM2?
compensates for the loss of the serine-413 phosphorylation site
in RIM1 S413A-KI mice.
In addition to impaired presynaptic long-term plasticity,
RIM1? KO mice exhibit a major defect in short-term plasticity
at Schaffer collateral to CA1 pyramidal cell synapses (26). We
thus also measured two forms of short-term plasticity at these
synapses in RIM1 S413A-KI mice, but again we found no
difference (Fig. 5 and Table S1). Paired-pulse ratios in response
to 2 stimuli applied with a 40-ms interval were unchanged (Fig.
5A), as were responses to a train of 25 stimuli at 14 Hz (Fig. 5B).
RIM1 S413A-KI Mice Exhibit Normal Behaviors. As a further test of
the effect of the RIM1 S413A-KI mutation on brain function, we
monitored fundamental behavioral parameters in the RIM1
S413A-KI mice. Behavior is a sensitive test for overall brain
function, and RIM1? KO mice exhibit major impairments in
multiple behavioral parameters (27).
Using age-matched male littermate pairs that were either
wild-type or homozygous mutant for the S413A KI, we found no
significant behavioral differences (Fig. 6). Specifically, total
activity in a novel mouse cage (mixed ANOVA, P ? 0.4) was
similar in S413A KI and wild-type littermate control mice (Fig.
6A; see SI Materials and Fig. S2 for the analysis of locomotor
habituation and total distance traveled in an open field). Anx-
iety-like behavior (Fig. 6B) as measured by latency to enter the
H E R R H A D V A L
H E R R H S D V A L
ki/ki +/ki H2O
flp recombination site
cre recombination site
S413A-KI mice showing (from top to bottom) the wild-type (WT) RIM1 allele,
the S413A KI targeting construct, the RIM1 mutant allele after homologous
recombination, and the flp-excised KI allele. 5, 6, and 7, exons 5, 6 and 7; DT,
diphtherotoxin-expressing cassette; NR, neomycin resistance cassette; *,
S413A point mutation in exon 6; N, NcoI restriction sites that were used for
Southern blotting. (B) Southern blot from heterozygous embryonic stem cells
a 5? outside probe were used. The wild-type band is 8.0 kilobases (kb), the KI
band 3.8 kb. (C) PCR genotyping of the RIM1 S413A-KI mice including het-
3? loxP/frt site after flp-recombination, and the S413A point mutation/BglI
restriction site after BglI digest of the amplified fragments. (D) Sequencing
and protein sequences are indicated for comparison.
Generation of RIM1 S413A-KI mice. (A) KI strategy for the RIM1
510 15 20 25
body weight (g)
+/+ +/ki ki/ki
ki/ki+/+ ki/ki+/+ ki/ki+/+
offsprings from heterozygous matings of RIM1 S413A-KI mice; the gray
shaded area indicates the expected Mendelian ratio. (B) Body weight of RIM1
S413A-KI and control littermate mice from postnatal day 6 to 60. (C) Western
blotting for RIM1? with antibodies against the N terminus (Q703), the central
at a slightly lower molecular weight), and loading controls. GDI, GDP disso-
ciation inhibitor; VCP, valosin-containing protein). (D) Western blotting of
brain homogenates for several presynaptic and other proteins appears nor-
mal. Rph, rabphilin; RIM-BP2, RIM-binding protein 2; NSF, N-ethylmaleimide-
sensitive factor; Syn, synapsin; Syt 1, synaptotagmin-1.
www.pnas.org?cgi?doi?10.1073?pnas.0806679105 Kaeser et al.
light side of a dark/light box (P ? 0.5), time spent in the open
arms of an elevated plus maze (P ? 0.7), time spent in the center
also unchanged in the RIM1 S413A-KI mice. Two forms of
learning and memory that were severely affected in the RIM1?
KO mice showed no impairment in the RIM1 S413A-KI mice.
Twenty-four hours after training in context- and cue-dependent
fear conditioning, both wild-type and RIM1 S413A-KI mice
displayed similar cue (P ? 0.8) and contextual (P ? 0.2) fear
conditioning (Fig. 6D). During training in the Morris water
maze, latency to reach a submerged platform was similar in
control mice. Furthermore, RIM1 S413A-KI and control mice
displayed a significant preference for the target vs. all other
quadrants on day 12 after removal of the platform in the probe
trial (Fig. 6F; wildtype and RIM1 S413A-KI ? P ? 0.05).
Distance traveled, swim speed, and thigmotaxis were also nor-
mal in the RIM1 S413A-KI mice (data not shown). Taken
together, the RIM1 S413A-KI mice performed normally in these
behavioral tests, and they did not display any of the severe
deficits in emotional and spatial learning and memory that were
observed in the RIM1? KO mice (27).
RIM1? is an active zone protein that is essential for presynaptic
short- and long-term plasticity (11, 16, 26). Experiments with
cultured cerebellar neurons suggested that phosphorylation of
serine-413 of RIM1? by PKA acts as a phospho-switch that
controls synaptic strength in a use-dependent manner by mod-
ulating the presynaptic release machinery (19). To determine
whether a similar mechanism occurs in vivo during long-term
synaptic plasticity, we set out to test whether phosphorylation of
-100 10 203040
mossy fiber LTP
parallel fiber LTP
1 ki/ki, 2α +/+
1 ki/ki, 2α -/-
mossy fiber LTP
hippocampus are normal in RIM1 S413A-KI mice. (A) LTP at parallel fiber to
Purkinje cell synapses in response to a single tetanus (200 stimuli at 10 Hz,
CA3 pyramidal cell synapses evoked by a tetanus (125 pulses at 25 Hz) deliv-
were removed for clarity. fEPSP, field excitatory postsynaptic potential. (C)
Endocannabinoid-mediated long-term depression at hippocampal inhibitory
synapses (I-LTD) in wild-type and mutant mice was induced by theta-burst
stimulation (vertical arrow) consisting of a series of 10 bursts of 5 stimuli
(100-Hz burst, 200-ms interburst interval), which was repeated 4 times (5 s
apart). IPSC, inhibitory postsynaptic current. (D) Mossy fiber LTP in RIM1
S413A-KI/RIM2? KO double-mutant and RIM1 S413A-KI control mice. In A–D,
Cerebellar parallel fiber LTP, mossy fiber LTP, and I-LTD in the
fEPSP number in the train
Schaffer collateral to CA1 pyramidal neuron synapses. (A) Summary graph
(left) and averaged sample traces (right) of paired pulse facilitation (PPF)
measured at 40-ms interstimulus interval in RIM1 S413A-KI and control mice.
(B) Synaptic responses [field excitatory postsynaptic potentials (fEPSPs)] elic-
ited by a 14-Hz train of 25 stimuli in RIM1 S413A-KI and control mice. Values
are normalized to the first synaptic response of the stimulation train.
Short-term synaptic plasticity in RIM1 S413A-KI mice in excitatory
time (5 min bins)
# beam breaks
latency to enter light (s)
time in open arm (s)
time in center (s)
latency to platform (s)
% time in quadrant
target opposite rightleft
behavior, motor coordination, and spatial and emotional learning and mem-
ory. (A) Measurements of the spontaneous motor activity of RIM1 S413A-KI
and control mice were determined as the number of beam breaks per minute
behaviors probed as latency to enter the light side of a dark/light box, as the
time spent in the open arms of an elevated plus maze, and as the time spent
in the center of an open field. (C) Measurements of motor coordination
monitored as the time mice stay on an accelerating rotarod as a function of
trial number. (D) Freezing behavior to both context- and cue-dependent fear
conditioning 24 h after training. (E) Morris water maze analysis of spatial
learning during the initial 11 days of training as measured by latency to reach
time spent in each quadrant is indicated.
Kaeser et al.
September 23, 2008 ?
vol. 105 ?
no. 38 ?
RIM1? at serine-413 is required for presynaptic long-term
plasticity and learning and memory. To achieve this goal, we first
searched for specific binding proteins that interact with serine-
413 in a phospho-specific manner and then used KI mice to test
whether phosphorylation of serine-413 is essential for presyn-
aptic long-term plasticity or learning and memory. In brief, our
results demonstrate that although serine-413-phosphorylated
RIM1? indeed binds specifically to 14-3-3 proteins, phosphor-
ylation of serine-413 and binding of 14-3-3 proteins are not
essential for presynaptic plasticity or for learning and memory.
We found that multiple isoforms of 14-3-3 bind in vitro to
RIM1? when serine-413 is phosphorylated by PKA but that
there are no other tight 14-3-3 binding sites in RIM1? (Fig. 1).
We then generated KI mice in which serine-413 is substituted for
alanine and phosphorylation at serine-413 is abolished (Fig. 2).
We observed that homozygous KI mice are viable and fertile and
exhibit no obvious deficits in brain composition, survival, and
body weight (Fig. 3). Acute slice recordings revealed that
multiple forms of presynaptic long-term and short-term plastic-
ity are normal in the KI mice (Figs. 4 and 5). The KI mice also
performed normally in a panel of behavioral tasks (Fig. 6) that
are severely impaired in RIM1? KO mice (27). Taken together,
we confirm by a targeted genetic mutation that the phosphor-
ylation of RIM1? at serine-413 is not essential for presynaptic
long-term plasticity or learning. Because the S413A mutation
completely abolished 14-3-3 binding in vitro, our data suggest
that 14-3-3 binding to RIM1? is also not required for presynaptic
Our data raise three questions. First, what experimental
differences between this and previous studies might account for
the fact that such different conclusions were reached? Second,
is it possible that unspecified compensations in the genetic
manipulations, as opposed to the rescue experiments using the
cultured cerebellar neurons, might have led to the lack of a
phenotype? Third, what phosphorylation sites in the presynaptic
terminal could alternatively be involved in PKA-dependent
presynaptic plasticity given that RIM1? phosphorylation is
clearly not essential?
With regard to the experimental differences, it is important to
note that previous studies used LTP measurements in cultured
neurons in synapses that developed in vitro (7, 19). Here, we used
acute brain slices for characterizing three distinct and well-
defined synapses that exhibit presynaptic long-term plasticity. In
all 3 synapses, the S413A mutation in RIM1? did not impair
plasticity. The functional properties of synapses in cultures are
likely to be different from synapses in acute slices. Moreover,
previous studies delivered RIM1? by transfection, driven by a
CMV promoter, which might have led to overexpression and/or
partial mistargeting of RIM1? or other proteins. In contrast, in
the mouse line that we generated, S413A mutant RIM1? was
expressed as a KI mutation from the RIM1? promoter at levels
that were similar to those of wild-type RIM1?. If RIM1? had
been mistargeted, we should have observed a deficit similar to
that of RIM1? KO mice. Because long- and short-term plasticity
were normal in our experiments, mutant RIM1? was correctly
incorporated into the release machinery. Finally, the LTP
measurements in cultured cerebellar neurons were performed in
constitutive RIM1? KO neurons that developed in the absence
of RIM1?, which was only reconstituted a few days before the
recordings. In strong contrast, our experiments were performed
on neurons that contained all functional domains of RIM1
except for the serine-413 phosphorylation site throughout
Although compensation is often invoked when unexpected
KO phenotypes are encountered, two observations suggest that
compensation cannot explain the lack of a plasticity phenotype
in the S413A-KI mice. First, compensation would likely have to
occur through RIM2? because RIM2? is the only other RIM
isoform that binds to Rab3A. However, the RIM2? deletion on
top of the S413A KI did not change the lack of a phenotype in
RIM1 S413A-KI mice. Second, if the constitutive mutation of
serine-413 in RIM1? did elicit compensation, it would be
difficult to understand why the constitutive RIM1? KO would
not elicit the same compensation but still exhibited a major
What other phosphorylation sites could act as PKA-triggered
phorylation site of RIM1? is conserved in RIM2?, RIM1? (a
novel RIM1 isoform; P.S.K. and T.C.S., unpublished observa-
tion), and RIM2?, but only the ?-isoforms also bind Rab3, which
is required for mossy fiber LTP in addition to RIM1?. RIM2?
KO mice have no major defects in hippocampal synaptic trans-
mission (25), and our experiments in the RIM1 S413A-KI/
RIM2? KO mice show that RIM2? does not compensate for the
loss of phosphoserine-413 in RIM1?. We also formally excluded
the involvement of RIM1? because in the mutant RIM1
S413A-KI mice, RIM1? also lacks this PKA site (P.S.K and
T.C.S., unpublished observation). On the basis of the biochem-
ical data, it seems unlikely that RIM2? participates in presyn-
aptic LTP because it does not bind to Rab3, and RIM2?
expression levels are generally low in the dentate gyrus. Fur-
thermore, RIM2? did not compensate for the loss of LTP in
RIM1? KO mice (19). RIM1? has an additional PKA phos-
phorylation site at serine-1548, but according to in vitro data it
is unlikely that this site participates in LTP because mutant
RIM1? that lacked this serine residue still rescued in vitro LTP
(19). Besides, and in contrast to serine-413, serine-1548 phos-
phorylation is not prominent in mossy fiber terminals (19).
Overall, it is thus unlikely that RIM1? or RIM2? are direct
targets of PKA in triggering presynaptic LTP. This conclusion is
also in line with the observation that forskolin-dependent po-
acts as an activator of adenylyl cyclase and raises the level of total
Our findings suggest that there are PKA targets other than
RIM1? in the presynaptic terminal participating in long-term
plasticity. Synapsins have an N-terminal PKA phosphorylation
site that regulates binding to synaptic vesicles (28), but synapsins
are also not involved in presynaptic LTP (17). Similarly, rabphi-
lin, a Rab3 binding protein (29), is phosphorylated by PKA, but
presynaptic LTP is not affected in rabphilin-deficient mice (18).
Interestingly, synaptotagmin-12 is a presynaptic PKA target that
scales spontaneous release in response to activation of the
cAMP-PKA pathway in vitro (30). Whether it might play a role
in presynaptic long-term plasticity or whether other unknown
presynaptic PKA targets are involved remains to be elucidated.
Materials and Methods
Plasmid Construction, Preparation of GST-Fusion Proteins, and Affinity Chro-
matography. All expression plasmids were constructed in pGEX-KG and puri-
fied according to standard methods (31), plasmids were previously reported
description of affinity chromatography can be found in the SI Text. In brief,
the column (Pierce). Fresh rat brain extracts were incubated with the affinity
column overnight at 4°C in the presence of phosphatase inhibitors. Upon
multiple elutions at increasing salt concentrations, proteins were visualized
with Coomassie and silver staining. The phospho-specific binding partner was
identified by mass spectrometry. GST-pulldown assays were performed from
downscaled accordingly. Brain homogenates were prepared as described in
the SI Text.
procedures (34, 35) and as described in the SI Text. By targeting exon 6 of the
www.pnas.org?cgi?doi?10.1073?pnas.0806679105Kaeser et al.
RIM1 gene we replaced the serine-413 residue with alanine and introduced
BglI and SphI restriction sites for genotyping. The mouse line was submitted
to the Jackson Laboratories and is freely available to the community.
Electrophysiology. Synaptic transmission at parallel fiber to Purkinje cell syn-
apses, mossy fiber to CA3 pyramidal cell synapses, inhibitory synapses on CA1
pyramidal cells, and at excitatory Schaffer collateral to CA1 pyramidal cell
synapses were recorded in acute slice preparations of 3–6-week old RIM1
S413A mice and littermate control mice according to methods that were
previously described (11, 16, 26). Detailed methods and numeric values for
were performed in parallel in the laboratories of P.E.C and of R.C.M, and the
experimenters were blind to the results of the other laboratory. All other
electrophysiological recordings were performed in the laboratory of P.E.C.
Behavior. All behavioral experiments were carried out on RIM1 S413A-KI mice
and littermate male wild-type controls at age 3–6 months (n ? 12 pairs; age
difference among pairs ?5 weeks). Experimenters were blinded to the geno-
type and to all electrophysiological analyses during behavioral testing. The
conditioning, and Morris water maze. Mice were moved within the animal
facility to the testing room and allowed to habituate to the new location for
?1 h before testing. All behaviors were performed as previously described
(27). A detailed methodological description can be found in the SI Text.
to standard methods (34). Most antibodies were previously described (19, 32)
or are commercially available (14-3-3? from Transduction Laboratories, 14-
from Dr. N. Brose. All data are shown as mean ? SEM. Statistical significance
was determined by the ?2test (mouse survival), two-way ANOVA (electro-
physiological recordings), or the Student’s t test (two-tailed distribution,
paired, all other data) where no specific test is stated. All animal experiments
were conducted according to the institutional guidelines. Mouse weight
studies were performed on male littermate pairs, and the mice were weighed
every 2 days from postnatal day 6 through 14 and then on postnatal days 18,
24, and 60.
ACKNOWLEDGMENTS. We thank E. Borowicz, J. Mitchell, I. Kornblum, and L.
Fan for excellent technical assistance; Dr. Robert Hammer for blastocyst
injections of embryonic stem cells; and members of the Sudhof laboratory for
comments and advice. This work was supported by grants from the National
Disorders and Stroke to T.C.S. and R.C.M., DA17392 to P.E.C., MH065975 to
C.M.P.), by a Swiss National Science Foundation Postdoctoral Fellowship (to
P.S.K.), a Hirschl/Weill-Caulier Career Scientist Award (to P.E.C.), by the Blue
Gator Foundation (to C.M.P.), and National Alliance for Research on Schizo-
phrenia and Depression Young Investigator Awards (to P.S.K. and C.M.P.).
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no. 38 ?