Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Yao, Wei-Dong and Chun-Fang Wu. Distinct Roles of CaMKII and PKA in Regulation of Firing Patterns and K+ Currents in Drosophila Neurons. J. Neurophysiol. 85: 1384-1394, 2001. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the cAMP-dependent protein kinase A (PKA) cascades have been implicated in neural mechanisms underlying learning and memory as supported by mutational analyses of the two enzymes in Drosophila. While there is mounting evidence for their roles in synaptic plasticity, less attention has been directed toward their regulation of neuronal membrane excitability and spike information coding. Here we report genetic and pharmacological analyses of the roles of PKA and CaMKII in the firing patterns and underlying K+ currents in cultured Drosophila central neurons. Genetic perturbation of the catalytic subunit of PKA (DC0) did not alter the action potential duration but disrupted the frequency coding of spike-train responses to constant current injection in a subpopulation of neurons. In contrast, selective inhibition of CaMKII by the expression of an inhibitory peptide in ala transformants prolonged the spike duration but did not affect the spike frequency coding. Enhanced membrane excitability, indicated by spontaneous bursts of spikes, was observed in CaMKII-inhibited but not in PKA-diminished neurons. In wild-type neurons, the spike train firing patterns were highly reproducible under consistent stimulus conditions. However, disruption of either of these kinase pathways led to variable firing patterns in response to identical current stimuli delivered at a low frequency. Such variability in spike duration and frequency coding may impose problems for precision in signal processing in these protein kinase learning mutants. Pharmacological analyses of mutations that affect specific K+ channel subunits demonstrated distinct effects of PKA and CaMKII in modulation of the kinetics and amplitude of different K+ currents. The results suggest that PKA modulates Shaker A-type currents, whereas CaMKII modulates Shal-A type currents plus delayed rectifier Shab currents. Thus differential regulation of K+ channels may influence the signal handling capability of neurons. This study provides support for the notion that, in addition to synaptic mechanisms, modulations in spike activity patterns may represent an important mechanism for learning and memory that should be explored more fully.
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INTRODUCTION |
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The
Ca2+/calmodulin-dependent protein kinase II
(CaMKII) and cAMP-dependent protein kinase A (PKA) cascades play
critical roles in learning and memory behaviors in both vertebrates
(Byrne and Kandel 1996; Chen and Tonegawa
1997
) and invertebrates (Davis 1996
;
Tully et al. 1996
). CaMKII and PKA are necessary for
spatial memory performance in mice (Huang et al. 1995
;
Mayford et al. 1996
; Silva et al.
1992a
,b
). In Drosophila, inhibition of CaMKII by
genetic transformation alters both nonassociative and associative conditioning (Griffith et al. 1993
) and disruptions of
cAMP pathways by mutations that alter the dunce (a
phosphodiesterase gene), rutabaga (an adenylyl cyclase
gene), or PKA gene impair learning and memory in a variety of
behavioral paradigms (Aceves-Pina and Quinn 1979
;
Drain et al. 1991
; Dudai et al. 1976
;
Duerr and Quinn 1982
; Goodwin et al.
1997
; Heisenberg et al. 1985
; Li
et al. 1995
; Quinn et al. 1974
; Siegel
and Hall 1979
; Spatz et al. 1974
;
Wustmann et al. 1996
).
The cellular basis of CaMKII- and PKA-mediated behavioral plasticity
has been intensively studied. The major focus has been modifications in
synaptic strength and nerve terminal sprouting (Bailey and
Kandel 1993; Bliss and Collingridge 1993
),
especially in the context of hippocampal long-term potentiation in
mammals (Huang et al. 1995
; Malenka et al.
1989
; Malinow et al. 1989
). In
Drosophila, inhibition of CaMKII and mutations of
dnc and rut genes alter synaptic transmission and
nerve terminal arborization at larval neuromuscular junctions
(Wang et al. 1994
; Zhong and Wu 1991
;
Zhong et al. 1992
).
Modulation of intrinsic membrane properties of neurons can profoundly
change network dynamics and performance (Getting 1989; Harris-Warrick and Marder 1991
) and may represent
another important cellular mechanism for behavioral plasticity, one
that has received less attention in learning and memory studies. To
more fully understand the cellular basis of learning and memory, it
will be important to determine the roles of PKA and CaMKII in
regulation of intrinsic membrane properties in neurons. Although
dnc and rut mutations have been shown to affect
central neuron firing patterns (Zhao and Wu 1997
), it is
not known to what degree this effect is mediated by PKA, a downstream
component of the cAMP pathway. It is also not known how the electrical
activities of central neurons are regulated by CaMKII or how this
regulation compares with modulation by PKA. Some useful insights into
the cellular operations that subserve learning and memory processes may
be gained from such studies given the apparently similar deficiencies
in synaptic and behavioral plasticity caused by gene knockouts of these
two kinases in mammalian systems (Huang et al. 1995
;
Mayford et al. 1996
; Silva et al.
1992a
,b
).
Inhibition of CaMKII causes limbic epilepsy in mice (Butler et
al. 1995) and enhanced nerve firing in Drosophila
(Griffith et al. 1994
), suggesting a possible role
for CaMKII in the regulation of ion channels. In
Drosophila, voltage-dependent K+
currents are altered by dnc and rut
(Delgado et al. 1998
; Zhao and Wu 1997
;
Zhong and Wu 1993
), mutations that affect cAMP
metabolism (Byers et al. 1981
; Levin et al.
1992
; Livingston et al. 1984
), raising the
possibility that K+ channels may be downstream
targets of PKA regulation. Correspondingly, central neurons of these
memory mutants show aberrant spike frequency coding (Zhao and Wu
1997
). Furthermore K+ channel mutants
display abnormal habituation in a defined neural circuit, similar to
altered habituation found in dnc and rut flies (Engel and Wu 1996
, 1998
). These findings suggest a
close link among K+ channel activity, neuronal
spike patterns, and behavioral plasticity.
The Drosophila "giant" neuron culture system has
facilitated patch-clamp characterization of neuronal spike activity and
the underlying ionic currents (Renger et al. 1999;
Saito and Wu 1991
; Wu et al. 1990
;
Yao and Wu 1999a
,b
; Zhao et al. 1995
).
Two transgenic fly strains, ala1 and ala2, carry
a heat-shock inducible mini-gene for a specific peptide inhibitor of
CaMKII (Griffith et al. 1993
). DC0X4 is an EMS-induced
mutation that inactivates a catalytic subunit of PKA, causing
dramatically reduced PKA activity (Li et al. 1995
). These Drosophila strains, together with several mutants and
transformants of K+ channel subunits, allowed us
to study how PKA and CaMKII regulate neuronal firing patterns and how
such effects may be mediated by modulations of identified
K+ channels.
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METHODS |
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Fly stocks
Canton S (CS) was the wild-type control strain used in all
experiments. ShM and
eag1 were both from the collection of Dr.
S. Benzer at Cal Tech. ShM is a null
allele of Sh (Zhao et al. 1995).
Shab1 was generated in Dr. S. Singh's Lab at SUNY Buffalo. Three lines of ShD transformants were
generated by inserting a P-element vector containing a ShD cDNA fused
to a hsp-70 promoter into the first, second, or third chromosome in the
ShM genetic background. No basal
expression of ShD channels is observed at room temperature (Zhao
et al. 1995
).
DC0X4, a cold-sensitive allele
of the DC0 gene that encodes the major catalytic subunit
(RI) of PKA, is lethal at 18°C but viable at 25°C (Li et al.
1995). The transgenic CaMKII inhibitor strains ala1
and ala2 were generated with P-element insertions into the first and second chromosome, respectively (Griffith et al.
1993
). Expression of the inhibitory peptide is under control of
the heat shock promoter hsp-70. The ala transformants show a
low basal level of expression of the inhibitory peptide even without
heat-shock treatment (Griffith et al. 1993
). All fly
stocks were maintained on standard Drosophila media at
20-22°C except for DC0X4,
which was kept at 25°C. All strains used were homozygous for the
corresponding mutations.
Cell culture and electrophysiology
The procedure for culturing Drosophila "giant"
neurons has been described previously (Saito and Wu
1991; Wu et al. 1990
). Briefly, embryos at the
gastrulation stage were gently dissociated in modified Schneider medium
(GIBCO, Grand Island, NJ) containing 200 ng/ml Insulin (Sigma, St.
Louis, MO), 20% fetal bovine serum (FBS), 50 µg/ml streptomycin, and
50 U/ml penicillin. Cells were washed in the above medium and
resuspended in medium containing 2 µg/mg Cytochalasin B (Sigma), and
then plated on glass coverslips. Cultures were maintained in humidified
chambers at room temperature (20-25°C) for 2-5 days prior to recordings.
Standard whole cell patch-clamp recording has been described previously
(Saito and Wu 1991). Recording bath solutions
(Jan and Jan 1976
) contained (in mM) 128 NaCl, 2 KCl, 4 MgCl2, 1.8 CaCl2, and 35.5 sucrose, buffered with 5 HEPES at pH 7.1. Patch pipettes were filled
with solution containing (in mM) 144 KCl, 1 MgCl2, 0.5 CaCl2, and 5 EGTA, buffered with 10 HEPES, pH 7.1. K+ currents
were isolated by adding TTX (0.2 µM) and Cd2+
(0.2 mM) to the bath solution. PKA inhibitor Rp-cAMPS and CaMKII inhibitors KN-62 and KN-93 were from Calbiochem (La Jolla, CA). Recordings were obtained at room temperature from isolated neurons with
a patch clamp amplifier (Axopatch 1B, Axon Instruments, Foster City,
CA). Data acquisition and analysis were carried out using pCLAMP and
Axograph software (Axon Instruments).
Heat-shock protocol
Induction of transgene expression in ShD, ala1, and
ala2 cultures was accomplished by placing cell cultures in a
38.5°C incubator for 45 min. The heat-shock temperature and duration
were established in previous studies to be sufficient for induction of
either peptide (Griffith et al. 1993; Wang et al.
1994
; Zhao et al. 1995
). Detectable expression
of the CaMKII inhibitory peptide in ala lines, but not ShD
channels, was significantly induced by the cellular stress imposed by
the current cell culture procedures. ala phenotypes with or
without heat shock did not differ, and thus results from heat-shock-treated and non-heat-shocked neurons were pooled in all experiments.
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RESULTS |
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Alterations of excitability and firing patterns in ala and DC0 neurons
Whole cell current-clamp experiments were performed on the soma of
isolated giant neurons to minimize complications introduced by synaptic
connectivity. On injection of depolarizing currents, three types of
voltage responses were observed in both wild-type and mutant neurons:
all-or-none, graded, and nonregenerative (Saito and Wu
1991). Among these, the all-or-none type action potentials have
been best characterized (Zhao and Wu 1997
) and will be
emphasized here. In wild-type cells with all-or-none action potentials,
firing patterns can be functionally classified into adaptive, tonic, and delayed categories based on their instantaneous spike frequency and
first spike latency (Fig. 1), following
the criteria described previously (Zhao and Wu 1997
).
Specifically, the delay to first spike, and the intervals between the
first two spikes (ffirst) and
the last two spikes (flast) in
the spike train were used to distinguish three different firing
patterns. "Delayed" showed a delayed onset (>100 ms) of the first
spike. "Adaptive" displayed decreasing firing frequency within a
spike train and normally conformed to the criteria of latency <100 ms
and
flast/ffirst < 0.7. "Tonic" showed firing patterns with latency <100 ms but flast/ffirst > 0.7, hence the firing frequency in these neurons was relatively
constant.
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It should be noted that such categorization was based on spike trains
during the first run of a current-clamp protocol immediately after a
whole cell configuration was established. This stimulation protocol
consisted of a series of 400-ms current steps ranging from 30 to 300 pA. The spike train in response to a current pulse at the strength of
40 pA above threshold was taken for categorizing the firing patterns.
Firing patterns classified this way represented a condition closer to
the "native" excitability states of the neuron.
Because both CaMKII and PKA cascades are implicated in mechanisms
subserving learning and memory, we sought to discern how the two
systems modulate distinct aspects of neuronal firing patterns. ala1 and ala2 neurons, in which CaMKII activity
is inhibited by a specific inhibitory peptide, typically displayed
prolonged action potential waveforms (Figs. 1 and
2) and abnormal spontaneous activity (Fig. 3) with the following
characteristics. First, the spike duration, which was determined as the
width at the inflection point during an action potential take-off in a
spike train, was substantially prolonged in ala neurons
(Fig. 2, Table 1). Action potential
prolongation was observed in spike trains of all categories in current
injection experiments (Table 1). Second, during sustained step current
injection, all-or-none spikes in the tonic and adaptive categories in
ala neurons exhibited a progressive decrease in spike size
(along with an increase in spike duration over the period of
stimulation), in contrast to the well-maintained spike shape in
wild-type cells (Fig. 1). This might reflect an inefficient repolarization mechanism. Third, a subpopulation of ala
neurons fired highly irregular spike pattern in response to current
injection that could not be classified into the three categories (Fig.
1). Fourth, long-lasting spontaneous bursting activity with sporadic occurrence and varying frequency was observed in 25-30% of
ala neurons compared with only 5% in wild type (Fig. 3),
although the resting membrane potentials were similar (data not shown). The action potentials within spontaneous spike trains were also prolonged (data not shown). This phenotype suggests that CaMKII plays a
significant role in maintaining the quiescent state of neurons.
Persistent rhythmic firing lasting for minutes (data not shown) (cf.
Yao and Wu 1999a) has been observed in a small population of cultured neurons. Such cells in wild-type cultures presumably correspond to endogenous pace-making cells in the normal CNS
(Yao and Wu 1999a
). The percentage of these putative
pace-making cells in ala (3-5%) was similar to that
of wild-type culture (2%; Fig. 3). The incidence of adaptive, tonic,
and delayed firing as well as the spike amplitude and the mean spike
frequency in response to suprathreshold stimulation were also
summarized in Table 1.
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Both abnormal spontaneous activity and aberrant spike frequency coding
have been demonstrated in neurons with dnc and
rut mutations (Zhao and Wu 1997), both of
which disrupt cAMP metabolism and cause learning and memory
deficiencies (Aceves-Pina et al. 1983
; Byers et
al. 1981
; Dudai et al. 1976
; Livingstone
et al. 1984
). Although the majority of the cAMP effects in
eukaryotes may be mediated through activation of PKA, intracellular
cAMP has also been shown to interact directly with ion channels in some
cell types (Delgado et al. 1991
; Dhallan et al.
1990
). Thus to establish the functional roles of PKA in
mediating the phenotypes of the memory mutants dnc and
rut, it is necessary to employ mutants such as
DC0X4 in which direct perturbation of the
kinase activity occurs.
We found that very few DC0X4 neurons
displayed spontaneous sporadic bursting activities with a percentage
not statistically different from that of wild type (Fig. 3). This
indicates that the increased spontaneous activity in dnc and
rut neurons (Zhao and Wu 1997) may be
mediated by mechanisms other than PKA modulation. The resting membrane
potential, distribution of firing patterns, and action potential
duration are not affected in DC0X4 (Table
1 and data not shown). The firing frequency was significantly increased
in adaptive and tonic DC0X4 neurons. The
action potential amplitude was significantly decreased in adaptive but
slightly reduced in tonic neurons in
DC0X4. We noticed that the most remarkable
mutant phenotype of firing patterns in
DC0X4, however, is the erratic firing rate
during current injection (Figs. 1 and 4).
These findings show that abnormality in firing frequency coding can
occur without altered action potential duration (Figs. 1 and 2). With
respect to this particular phenotype, the DC0X4 results were in general consistent
with the dnc and rut study (Zhao and Wu
1997
), suggesting that defective frequency coding caused by
abnormal cAMP levels may be mediated by PKA.
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In contrast, frequency coding of all-or-none spike trains in ala neurons in response to current injections appeared normal, and the instantaneous firing rates in tonic and adaptive categories were indistinguishable between ala and wild type (Figs. 1 and 4). It is remarkable that the spike frequency coding remained intact despite the fact that the action potential duration in some ala neurons is significantly prolonged compared with wild-type neurons (Figs. 1, 2, and 4 and Table 1). These results indicate that perturbations of CaMKII and PKA activities exert influences on distinct neuronal firing parameters. CaMKII appears to play a major role in defining the width of individual action potentials and maintaining their waveform during sustained activity. PKA, on the other hand, is more important to the regularity of spike frequency coding.
Altered stability of spike frequency coding in ala
and DC0
neurons
Responses to individual current pulses can reveal erratic patterns of spike trains in mutant neurons, such as ala1, ala2, and DC0X4. However, a single-pulse paradigm cannot determine the stability of the spike patterns generated by a given neuron. Such endogenous stability of firing patterns is important to ensure a reliable and consistent input-output relationship during signal processing by the neuron. We examined this problem by using a revised paradigm involving repetitive stimulation at a low frequency. When neurons were stimulated by step current injections (80-120 pA) at 0.5 Hz or lower to minimize the interaction between successive pulses, wild-type neurons displayed highly reproducible firing patterns in response to these identical repeated stimuli (Fig. 5A). The firing pattern, in particular the number of spikes in each spike train, varied to a much greater extent in ala and DC0X4 neurons. To quantify the variation in the number of spikes evoked by a fixed current pulse for each genotype, the coefficient of variance (CV = SD/Mean) was determined for 20 repeated stimuli and presented in relation to the mean spike number for each cell (Fig. 5B). A markedly increased CV was seen in a large proportion of ala1, ala2, and DC0X4 neurons compared with wild type, indicating significant intrinsic instability in the mutant neurons. Although our frequency analysis of different firing patterns indicates that mutant neurons do not fire fewer action potentials in general (Table 1), the variability among responses to identical stimuli was most evident for neurons that generated fewer spikes (Fig. 5B). Thus both CaMKII and PKA play crucial roles in maintaining the stability of neuronal firing patterns, which may be important for the proper performance of neural circuits during information processing.
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Altered K+ currents in ala and DC0 neurons
Voltage-activated K+ currents regulate
membrane repolarization and hence excitability and firing patterns in
neurons (Hille 1992; Rudy 1988
). The
altered neuronal firing patterns and aberrant spontaneous
hyperexcitability observed in ala and
DC0X4 neurons suggested that CaMKII and
PKA affect K+ current mechanisms. In the giant
neuron culture system, various voltage-activated
K+ currents and the effects of mutations of
identified K+ channel subunits have been well
characterized (Yao and Wu 1999a
; Zhao et al.
1995
). Abnormality in kinetics and amplitude of different current components may be readily determined. Some of the striking mutant phenotypes observed in current clamp experiments might be
correlated to changes in K+ current properties
that could be revealed in voltage-clamp records.
Voltage-activated K+ currents were examined in
saline containing TTX and Cd2+ to eliminate
inward Na+ and Ca2+
currents as well as outward Ca2+-activated
K+ currents (Saito and Wu 1991).
Two types of K+ currents with different
inactivation kinetics can be seen in wild-type neurons (Fig.
6A) (cf. Delgado et al.
1998
). The decay of Type 1 currents could be fitted by a
two-exponential process with fast [
1 = 68.3 ± 4.1 (SE) ms] and slow (
2 = 1,133 ± 221 ms) components. Type 1 currents were encountered in
the majority of giant neurons (~60%, n = 59). Type 2 currents, found in the remaining cells (~40%), inactivated with a
single-exponential time course (
= 1,066 ± 485 ms). Both
4-aminopyridine (4-AP)- and TEA-sensitive components were found in the
two types of currents. In general, Type 1 currents contained a greater
4-AP-sensitive, fast-inactivating component, and Type 2 currents were
more sensitive to TEA (data not shown).
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We found similar fractions of neurons in wild-type and mutant cultures
expressing Type 1 versus Type 2 currents [wild type (WT): 73 vs. 27%;
ala1: 75 vs. 25%; ala2: 73 vs. 27%;
DC0X4: 62 vs. 38%, no significant
differences under 1-sample binomial tests]. In ala1 and
ala2 but not in DC0X4 neurons,
1 of the double-exponential approximation for
Type 1 currents was significantly reduced compared with WT neurons (Fig. 6). The specific effect of ala mutations on the fast
decay time constant is consistent with previous pharmacological studies that showed accelerated decay of K+ currents
after CaMKII inhibition (Peretz et al. 1999
; Yao
and Wu 1999b
). However, the slow inactivation time constant
(
2, not shown) in Type 1 currents and the
decay rate (
) of Type 2 currents did not significantly differ among
genotypes (Fig. 6). These results suggest that CaMKII may
differentially regulate certain channels with distinct kinetic
properties in Drosophila neurons.
To examine how the amplitudes of IA and IK in the two current types were affected in ala and DC0X4 neurons, we used a prepulse paradigm to separate the early, inactivating currents (IA) and the delayed, noninactivating currents (IK; Fig. 7A). We found that both ala and DC0X4 mutations affected neurons displaying Type 1 currents. In these cells, current density of IA was reduced significantly in both ala and DC0X4 neurons (Fig. 7B). When the current-voltage relations (I-V curves) are normalized for the three genotypes (Fig. 7C), it is apparent that the reductions in current amplitude were independent of membrane potentials. The current density of IK was also reduced in ala and DC0X4 neurons. However, there was an apparent voltage dependence of this effect with more severe reduction at lower membrane potentials (especially in ala neurons) (Fig. 7C), which is also reflected in the significantly shifted half-activation voltage in ala and DC0X4 (Fig. 7D). The amplitudes of IA and IK components of Type 2 currents were not significantly different between WT and ala neurons [WT: IA = 13.17 ± 1.29, IK = 19.85 ± 0.77 (n = 3); ala: IA = 13.59 ± 4.32, IK = 17.31 ± 2.39 (n = 3); P > 0.05, t-tests; consistent results were also obtained from nonseparated IA and IK (data not shown). All currents were recorded at +20 mV]. In contrast, the DC0X4 mutation did not affect IA but decreased IK components of Type 2 current significantly [DC0X4: IA = 19.02 ± 2.78, IK = 9.01 ± 2.09 (n = 5), P < 0.01]. Taken together, ala affects both the kinetics and amplitude of Type 1 currents, whereas DC0X4 selectively affects current amplitude but in both Type 1 and Type 2 currents. Such distinct effects on current density may contribute to the differences in the regulation of excitability and firing patterns by CaMKII and PKA. Further current- and voltage-clamp correlation analysis in the same cell may reveal how neurons of different firing patterns express varying portions of CaMKII- and PKA-sensitive K+ current components.
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Genetic dissection of CaMKII and PKA modulation on K+ currents
Voltage-gated K+ channel subunits in Drosophila neurons, encoded by the identified
genes Sh, Shal, Shab, and
Shaw, generate K+ currents with
distinct kinetics and voltage dependence when expressed in heterologous
systems (Butler et al. 1989
; Iverson and Rudy 1990
; Timpe et al. 1988
). Viable point mutations
of Sh (Wu and Ganetzky 1992
) and Shab (Singh and
Singh 1999
) provide powerful tools to dissect the downstream
effectors of PKA and CaMKII for modulating K+
currents. Highly selective pharmacological agents that inhibit CaMKII
and PKA are also available to enhance the genetic approach.
KN-62 and -93 are specific inhibitors of CaMKII (Sumi et al.
1991; Tokumitsu et al. 1990
) while Rp-cAMPS
(adenosine 3',5'-cyclic monophosphorothioate, Rp-Isomer; 50 µM)
selectively inhibits PKA activity. When applied to the bath solution,
CaMKII inhibitors KN-62 (10 µM) and KN-93 (1 µM) and PKA inhibitor
Rp-cAMPS (50 µM) all reduced both the peak and sustained
K+ currents in WT neurons (Fig.
8).
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Sh mutations alter A-type currents in muscle (Salkoff
and Wyman 1981; Wu and Haugland 1985
) and in
neurons (Baker and Salkoff 1990
). The null mutation
ShM deletes all Sh subunits in neurons and
could be used to test whether Sh subunits are a target for CaMKII or
PKA modulation. As shown in Figs. 9 and
10, the non-Sh currents that remain in ShM neurons, including both the A-type and
the sustained components, were significantly affected by KN-93 in both
amplitude and kinetics to a degree similar to those of WT neurons. In
contrast, the non-Sh currents were apparently resistant to modulation
by Rp-cAMPS (Figs. 9B and 10B).
|
|
To directly assay the modulation of an identified Sh subunit by
CaMKII kinase and PKA, we used ShD transgenic strains in which a copy
of ShD cDNA was inserted into the first, second, or third chromosome in
the ShM host background to express ShD
channels in neurons lacking endogenous Sh channels. The expressed ShD
current, induced by heat shock (see METHODS), exhibited
fast inactivation kinetics and extremely slow recovery from
inactivation (Zhao et al. 1995
). These distinct kinetic
properties allow the isolation of this current from the native
K+ currents by a twin pulse paradigm
(Renger et al. 1999
; Zhao et al. 1995
).
As shown in Figs. 9 and 10, ShD current was inhibited by Rp-cAMPS but
not by KN-93. Taken together, the results support the idea that the Sh
product is a target for PKA, but not for CaMKII, modulation.
The Shab gene has been proposed to be responsible for the
major slowly inactivating delayed rectifier K+
currents in Drosophila neurons (Tsunoda and Salkoff
1995b) and muscle cells (Singh and Singh 1999
).
We found that a point mutation, Shab1
(Singh and Singh 1999
), eliminated most of
the steady-state currents in giant neurons (Fig. 9). This mutation
allowed us to analyze further how CaMKII and PKA modulate the different
K+ currents in neurons. Figures 9 and 10 show
that in Shab-deficient neurons, the transient A current
(more prominent due to the removal of the delayed rectifier) was still
significantly modified by KN-93 and Rp-cAMPS. However, these two drugs
did not significantly affect the small sustained non-Shab components
remaining in Shab-deficient neurons.
The A current in Drosophila neurons is thought to be
primarily composed of Shal currents (Tsunoda and Salkoff
1995a) with a smaller contribution from Sh (Baker and
Salkoff 1991
). The sustained current consists of a number of
components including the delayed rectifiers Shab (Tsunoda and
Salkoff 1995a
) and the steady-state components of A-type Sh and
Shal currents. Currents conferred by Shaw subunits have also been
implicated as part of the sustained current (Tsunoda and Salkoff
1995a
). The suppression of A-type currents in
Shab1 neurons is consistent with the idea that
CaMKII and PKA modulate Shal and Sh channels, respectively (see
preceding text). In contrast, the lack of significant effects of CaMKII
inhibitors on the non-Shab sustained current in
Shab1 neurons suggests that modulation of
the Shab current may be responsible for part of the reduction in
sustained currents of WT neurons by those drugs.
In summary, a working scheme about PKA and CaMKII modulation of K+ channels can be proposed based on the genetic and pharmacological data: modulation of A currents is mediated by PKA on Sh channels and CaMKII on Shal channels (Fig. 11), whereas modulation of delayed rectifier K+ currents by CaMKII is mediated primarily by Shab. Future work on Shaw mutants, when they become available, should provide further evidence concerning the genetic basis of CaMKII and PKA modulation of K+ currents.
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DISCUSSION |
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In this study, we report that CaMKII and PKA modulate different aspects of neuronal firing properties, resulting at least in part from differential targeting of voltage-gated K+ channels by the two enzymes. Our results establish roles for the two Ca2+/CaM-activated pathways in neuronal spike shaping and firing patterning. These properties provide another regulatory mechanism, in addition to modulation of synaptic efficacy, which may contribute to behavioral modifications.
Differential regulation of neuronal function by PKA
and CaMKII
Neuronal firing patterns carry highly structured temporal
information. Firing rate and action potential shape are two major parameters that dictate the timing and amount of neurotransmitter release. Neuronal electrical activity can be highly plastic, being affected by both short- and long-term modulatory mechanisms
(Getting 1989; Marder et al. 1996
;
McCormick 1992
; Turrigiano et al. 1994
, 1995
). Such functional plasticity has important implications
not only for behavioral modification but also for activity-dependent refinement of proper neural circuits during development (Budnick et al. 1990
; Cline 1991
; Hubel and Wiesel
1970
). Given the critical roles of PKA and CaM kinase in
behavioral plasticity, it is not surprising that at the cellular level,
both enzymes regulate neuronal firing properties. Comparisons of how
the two signaling pathways modulate neuronal membrane properties can
provide insights into the cellular mechanisms underlying behavioral
modifications (Fig. 11).
The activity-dependent accumulation of Ca2+ could
activate both PKA and CaM kinase activity through a
Ca2+/calmodulin-dependent mechanism (Fig.
11B). Direct binding of Ca2+/CaM is
required for CaMKII activation while
Ca2+/CaM-dependent activation of adenylyl cyclase
stimulates cAMP synthesis, which in turn activates PKA.
DC0X4 neurons in which the PKA catalytic
subunit is defective showed aberrant frequency coding, unstable firing
patterns, enhanced firing frequency, and decreased spike amplitude, but
no changes in action potential width (Figs. 1, 2, and 4, Table 1).
These phenotypes are in general consistent with the defects found in dnc and rut neurons (Zhao and Wu
1997) which have a defective cAMP phosphodiesterase and
adenylyl cyclase, respectively. The exception is that dnc
and rut neurons display spontaneous activity not seen in
DC0X4, which raises the possibility of
regulation mediated by direct cAMP binding to ion channels
(Delgado et al. 1991
; Dhallan et al.
1990
; Nakamura and Gold 1987) or via
non-DC0-dependent PKA pathways. In comparison, neurons of
ala1 and ala2 transformants, which express a
CaMKII inhibitory peptide (Griffith et al. 1993
), exhibited unstable firing patterns in repeated stimulation trials, but
no fluctuations in spike frequency coding during single current injections. Furthermore the action potential waveform was prolonged in
ala neurons. Because CaMKII is highly abundant at the
synapse (Kennedy 1997
; Nairn et al.
1985
), its regulation of action potential duration could
influence the dynamics of Ca2+ influx and thus
the amount of transmitter release. Indeed, enhancement of synaptic
currents and irregularity of release patterns has been reported for the
larval neuromuscular junction of Drosophila ala
transformants (Wang et al. 1994
).
The preceding observations suggest that different second-messenger
systems may regulate distinct aspects of neuronal firing properties.
This can enhance the capacity of the nervous system to fulfill specific
functional requirements for a variety of behavioral tasks. Based on
previous observations, cAMP and CaM kinase pathways are known to exert
different effects on nerve terminal growth and synaptic transmission at
the larval neuromuscular junction (Wang et al. 1994;
Zhong and Wu 1991
; Zhong et al. 1992
),
which demonstrates that differential regulation of different aspects of
neuronal function by the two signaling cascades can occur within the
same cell. It will be interesting to further investigate firing patterns between these mutants in neurons associated with identified neural circuits underlying distinct behaviors.
Correlation of K+ current modulation with firing patterns
The firing properties of Drosophila "giant" neurons
are shaped by a plethora of ionic currents, including voltage-activated outward K+ currents, inward
Ca2+ and Na+ currents, and
Ca2+-activated outward K+
currents (Saito and Wu 1993). IA
has been shown in this preparation to modulate the onset, the duration,
and the frequency of action potentials whereas
IK primarily contributes to the
repolarization of the action potential, determining its duration
(Saito and Wu 1993
; Yao and Wu
1999a
; Zhao and Wu 1997
). The distinct
patterns of adaptive, tonic, and delayed firing reflect differences in expression of different K+ currents (Zhao
and Wu 1997
). Consistent with this notion, our preliminary
results suggest that Type 1 currents can support all three types of
firing patterns while Type 2 neurons only fire adaptive spikes
(unpublished observations).
Although a complete explanation of mutant firing patterns will require
knowledge of both inward and outward currents, some insights can be
obtained by an initial analysis of K+ currents.
For example, kinase inhibitors and K+ channel
blockers can be used to explore the contributions of pharmacologically
and kinetically distinct K+ current components.
Specifically, 4-AP has been shown to increase the firing frequency and
shorten the latency to the onset of spikes, which is particularly
striking for delayed type neurons, whereas TEA broadens the duration of
action potentials without changing the firing rate, which is most
obvious for adaptive type neurons (Zhao and Wu 1997).
Current- and voltage-clamp correlation studies from the same cells
demonstrated that K+ currents in adaptive cells
have a large TEA-sensitive component and delayed cells a large
4-AP-sensitive component (Zhao and Wu 1997
). In our
preliminary experiments we found that KN-93 prolonged action potential
duration especially in adaptive cells and Rp-cAMPS greatly reduced the
latency and turned adaptive cells into fast-spiking cells (Yao and Wu,
Peng and Wu, unpublished observations). These observations are
consistent with the hypothesis that PKA has a major effect on Sh
IA currents and CaM kinase affects
non-Sh currents including Shab IK.
However, the kinase inhibitors did not completely mimic the modulation
of either IA or
IK channel blockers (data not shown),
suggesting that kinase inhibitors may affect firing patterns in a more
complicated manner. For example, INa
and ICa are known to affect the
initiation and duration of action potentials and may be modulated by
protein kinases. A more complete understanding of the ionic basis of
firing patterns would require voltage-and current-clamp correlation
analysis in the same cells of each firing pattern and should include
not only K+ currents but also other ionic currents.
Distinct regulation of K+ channel subunits by different second-messenger systems
It has been established that a variety of voltage-gated
K+ channels in Drosophila neurons are
composed of a combination of pore-forming subunits, including Sh,
Shal, Shab, and Shaw, and auxiliary
subunits such as hyperkinetic
(Yao and Wu 1999a
). Based on the genetic and
pharmacological analyses presented above, a scheme of
K+ channel modulation by PKA and CaMKII can be
proposed (Fig. 11B).
Our data show that KN-93 affected
IA in WT neurons and non-Sh transient
currents in ShM mutant neurons to a
similar degree, suggesting that Shal, but not Sh, is modulated by
CaMKII. This was supported by the fact that ShD current is resistant to
KN-93 treatment. Since Sh RNA undergoes extensive
alternative splicing (Kamb et al. 1988; Pongs et
al. 1988
; Schwarz et al. 1988
), further tests of
whether other Sh products are modulated by CaMKII may be performed in
future genetic and pharmacological studies. On the other hand, our
results show that Rp-cAMPS modulated ShD currents in ShD transformant neurons but not the non-Sh currents in ShM
mutants, which is consistent with previous findings: the Sh polypeptide contains PKA phosphorylation sites (Schwarz et al. 1988
)
responsible for modulation of inactivation of Sh channels expressed in
Xenopus oocytes (Drain et al. 1994
), and in
Drosophila muscle, the transient Sh current is modified by
dnc mutations and by acute application of cAMP analogs
(Zhong and Wu 1993
). Taken together, these observations demonstrate that Sh channels are a target of PKA but probably not of CaMKII.
The sustained K+ current consists of multiple
components as discussed in RESULTS, but its major
component, the delayed rectifier current, is thought to be mediated by
the Shab subunit (Fig. 9) (cf. Singh and Singh 1999;
Tsunoda and Salkoff 1995b
). The fact that the sustained
currents in both WT and ShM neurons were
substantially suppressed, following KN-93 treatments, to a level
comparable to the steady-state currents in
Shab1 neurons (see preceding text) (cf.
Peretz et al. 1998
; Singh and Singh 1999
;
Yao and Wu 1999b
) strongly suggests that the delayed rectifier Shab channel mediates the majority of CaMKII effects on
sustained K+ currents.
The differential effects of CaMKII and PKA on distinct K+ channel subunits discussed in the preceding text may contribute to the action potential phenotypes observed in ala and DC0X4 neurons. While the modulation of Sh currents by PKA appears to be important for generating regular firing patterns for reliable frequency coding, modulation of non-Sh currents by CaMKII may contribute to the control of action potential repolarization and duration. Both PKA and CaMKII, however, may regulate the stability of firing patterns through possible effects on multiple channel types (see preceding text), which may be critical for reliability and precision in signal processing in the nervous system.
In conclusion, PKA and CaMKII target different K+
channel subunits to confer differential regulation of neuronal
excitability and spike patterning. The two second-messenger systems may
modulate separate parameters of neuronal function, including nerve
terminal outgrowth (Wang et al. 1994; Zhong et
al. 1992
), synaptic function and plasticity (Wang et al.
1994
; Zhong and Wu 1991
), and neuronal firing
patterns (Zhao and Wu 1997
; this study). These
parameters might be conditioned differentially during nervous system
activity and thus may underlie specific behavioral modifications by
different stimulus paradigms and in different model systems.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Jeff Engel for comments on the manuscript.
This work was supported by National Institutes of Health grants to C.-F. Wu.
Present address of W.-D. Yao: HHMI/Dept. of Cell Biology, Duke University Medical Center, Durham, NC 27710.
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FOOTNOTES |
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Address for reprint requests: C.-F. Wu (E-mail: cfwu{at}blue.weeg.uiowa.edu).
Received 11 April 2000; accepted in final form 19 December 2000.
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REFERENCES |
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