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* The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden;
Institut für Medizinische Biochemie und Molekularbiologie, Universitätskrankenhaus Eppendorf, Universität Hamburg, 20246 Hamburg, Germany; and
Institut für Pharmakologie und Toxikologie, Albert-Ludwig Universität Freiburg, 79104 Freiburg, Germany
1Correspondence: The Rolf Luft Center for Diabetes Research L3, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: yang.shaonian{at}enk.ks.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adenylyl cyclase • calcium channel • hippocampus • inositol polyphosphate • protein kinase A
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Much less is known
about the signal transduction pathways activated by these latter
inositol polyphosphates compared with the well-characterized second
messenger effect of inositol 1,4,5-trisphosphate in mobilizing
intracellularly bound Ca2+ (1
, 2)
.
InsP6 is the most abundant of inositol
polyphosphates, including inositol 1,4,5-trisphosphate, in cells
(1
, 2)
. In accordance, several specific
InsP6 binding proteins have been revealed in cell
organelles (2
, 4)
, which include synaptotagmin, the plasma
membrane clathrin assembly proteins 2 and 3, Golgi coatomer, Arrestin,
and endosomal p100 (2
, 4)
. The possibility that
InsP6 acts as a general intracellular signaling
molecule in native excitable cells, including neurons, is suggested
from recent findings (5
6
7
8)
. InsP6
levels transiently change in several cell types after stimulation
(5
, 8)
. Microinjection of InsP6 into
Xenopus oocytes, expressing the substance P receptor,
significantly diminishes the desensitization of substance P
receptor-mediated, Ca2+-dependent
Cl- current responses (6)
.
InsP6 has been shown to enhance insulin
exocytosis from permeabilized HIT T15 cells through activation of
protein kinase C (7)
. In vitro biochemical analysis shows
that InsP6 significantly inhibits the activity of
purified catalytic subunits of serine/threonine protein phosphatases
types 1, 2A, and 3 as well as corresponding holoenzymes in cell
extracts (8)
. Furthermore, intracellular application of
InsP6 dramatically potentiates
Ca2+ channel activity in insulin-secreting cell
lines (8)
. However, the role of
InsP6 in intracellular signaling in neurons is
not known.
Voltage-gated Ca2+ channels of various kinds play
important and specific roles in neuronal signaling (9)
. In
the present study, we wanted to clarify whether
InsP6 modulates Ca2+
channel activity in the neuron and, if so, whether this effect is
associated with a certain Ca2+ channel subtype
that may have a specific functional implication. We selected cultured
pyramidal neurons from rat hippocampus as a model since specific
high-affinity InsP6 binding sites are most
abundantly distributed in the pyramidal cell layer of the hippocampus
compared with other inositol polyphosphate binding sites and other
areas of the brain (10)
. Levels of
InsP6 in the neuron have been shown to
dramatically increase after depolarization with high
K+ (5)
.
It has been shown that almost all cloned 
1
subunits (Ca2+ conducting subunits), including
1A, 1B,
1C, 1D,
1E, 1G,
1H, and 1I subunits,
of voltage-gated Ca2+ channels are present in the
hippocampal neuron (9
, 11
12
13)
. The low voltage-gated
T-type Ca2+ channel distributes predominantly in
dendrites and is a crucial component in electrophysiological rhythm
generators (14
, 15)
. The 1E
subunit of the R-type Ca2+ channel displays a
different subcellular distribution and is observed mainly in the soma
and proximal dendrites (9)
, although activation of this
channel has been shown to evoke a slight release of neurotransmitters
(16)
. The 1A subunit of the high
voltage-gated P/Q-type channel is predominantly visualized in
presynaptic terminals (9)
. Accordingly, the most
appreciable role of the P/Q-type channel is to trigger neurotransmitter
release in the active zone of presynaptic terminals (17
, 18)
. The high voltage-gated N-type Ca2+
channel plays the same role at the neurotransmitter release sites as
the P/Q-type channel does (17
, 18)
. However, the
1B subunit of this channel is mainly localized
in dendrites and nerve terminals (9)
. Labeling of the
1B subunit in the soma is low
(9)
.
The 1C and 1D
subunits of high voltage-gated L-type Ca2+
channels exist mainly in the soma and proximal dendrites
(11)
. The relatively high density of L-type
Ca2+ channels in the cell body suggests the
involvement of this channel in cellular signaling from the plasma
membrane to the nuclei—for example,
Ca2+-dependent gene expression (19)
.
Indeed, increases in postsynaptic Ca2+ levels in
the hippocampal neuron through the L-type Ca2+
channel stimulate gene expression and potentiate synaptic transmission
(19
, 20)
. It is well established that protein kinase A
(PKA) -induced phosphorylation of the L-type Ca2+
channel increases its activity (12
, 21)
. Increases in the
activity and density of L-type Ca2+ channels in
the hippocampal neuron during aging have been suggested to underlie the
vulnerability of neurons to age-associated neurodegenerative conditions
(22)
. The hippocampus is an important structure in
relation to synaptic plasticity, e.g., learning and memory (23
, 24)
. The synaptic plasticity is dramatically modulated by
changes in N-, P/Q-, and L-type Ca2+ channel
activity (20
, 25)
. We now demonstrate that
InsP6 specifically enhances the activity of
L-type Ca2+ channels in hippocampal neurons by
activation of the adenylyl cyclase (AC)-PKA cascade. The
InsP6-enhanced Ca2+ influx
through L-type Ca2+ channels may initiate several
intracellular events involved in molecular mechanisms of higher
functions of the brain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 27)
.
Hippocampal cell culture
Eighteen-day-old pregnant Sprague-Dawley rats (B&K Universal AB,
Stockholm, Sweden) were killed by CO2 inhalation.
Fetuses (E18) were removed and kept in glass Petri dishes on ice. This
was rapidly followed by removal of the brain, which was placed in
ice-cold Ca2+/Mg2+-free
Hank’s balanced solution (pH 7.3). The brain was hemisected and
dissection of hippocampi was performed under a stereomicroscope. The
hippocampi were incubated in 0.1% trypsin (Life Technologies Ltd.,
Paisley, UK), diluted in
Ca2+/Mg2+-free Hank’s
balanced solution at 37°C for 15 min and rinsed twice with
Ca2+/Mg2+-free Hank’s
balanced solution. The hippocampi were subsequently triturated through
a Pasteur pipette into single cells in Dulbecco’s modified Eagle
medium (DMEM)/nutrient mix F12 (Life Technologies). Corning Petri
dishes were coated with poly-L-lysine hydrobromide (MW 30,000–70,000,
Sigma, St. Louis, MO). The cells were plated in Corning Petri dishes
containing DMEM/nutrient mix F12 and incubated at 37°C in 5%
CO2 for 11–16 days.
Electrophysiological recordings
Whole-cell currents were recorded in isolated pyramidal-type
cells exhibiting a triangular soma with distinct processes after 11–16
days in culture. Pipettes were pulled from borosilicate glass
capillaries (Hilgenberg, Malsfeld, Germany) on a horizontal
programmable puller (DMZ Universal Puller, Zeitz-Instrumente, Augsburg,
Germany). Typical electrode resistance was 3–5 M
. Electrodes were
filled with a standard internal solution containing (in mM) 150
N-methyl-D-glucamine, 10 ethylene glycol-bis(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1
MgCl2, 2 CaCl2, 5
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES),
and 3 Mg-ATP (pH 7.2). The bath solution contained (in mM) 140
tetraethylammonium chloride, 1 MgCl2, 5, or 10
CaCl2, 10 HEPES, and 10 glucose (pH 7.4). After
obtaining a seal, the holding potential was set at -80 mV during the
course of an experiment. The first set of depolarizing voltage pulses
(100 ms) between -70 and 40 mV was applied in 10 mV increments at 0.5
Hz. This depolarization protocol was used to evaluate current–voltage
relationships in cells filled with the standard internal solution alone
or together with 20 µM myo-inositol hexakis[dihydrogen
phosphate] (InsP6) (Sigma), 20 and 100 µM
myo-inositol 1,3,4,5,6-pentakisphosphate
(InsP5) (Sigma), respectively. This approach was
also applied to assess current–voltage relationships in cells
preincubated with the specific AC inhibitor 2',5'-dideoxyadenosine
(2',5'-dd-Ado, Calbiochem, La Jolla, CA) and PKA inhibitor
N-[2-((p-bromo
cinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, Calbiochem) for
30 min with and without further application of
InsP6. The second set of depolarizing voltage
pulses (100 ms, 0.05 Hz) from a holding potential of -80 mV to a test
potential of 0 mV was used to evoke maximum peak
Ca2+ currents. This protocol was used to examine
the possible differences in effects of nimodipine (Research
Biochemicals International, Natick, MA) and
8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate
(8-CTP-cAMP, Calbiochem) on maximum peak Ca2+
currents between control cells and cells filled with 20 µM
InsP6. The last set of depolarizing voltage
pulses (100 ms, 0.05 Hz) to -40 mV from a set of holding potentials
from -110 to -60 mV was used for optimal recordings of low
voltage-gated Ca2+ currents (28)
.
Whole-cell currents were recorded with an Axopatch 200 amplifier (Axon
Instruments, Foster City, CA) and filtered at 1 kHz. All recordings
were registered at room temperature (
22°C) when a stable amplitude
of the whole-cell Ca2+ currents was reached,
5–10 min after breaking the patch. The amplitude of whole-cell
Ca2+ currents was normalized by the capacitance
of cells. Acquisition and analysis of data were done using the software
program pCLAMP (Axon Instruments).
Preparation of hippocampal samples
Thirteen-day-old Sprague-Dawley rats (B&K Universal AB) were
killed by decapitation. Their brains were rapidly removed from the
skull and immediately hemisected on ice. Hippocampi were dissected and
chopped into small pieces. Hippocampi for AC, phosphodiesterase (PDE),
and PKA activity assays were homogenized with a
motor-driven TeflonTM-glass homogenizer (20 strokes) in 300
µl of ice-cold homogenization buffer containing 50 mM Tris-HCl, 1 mM
EGTA, 10% sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
antipain, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml
pepstatin, pH 7.4. The homogenate was centrifuged at 1000 g
for 10 min. Supernatant 1 was collected for PDE activity assay or again
centrifuged at 15,000 g for 10 min to obtain supernatant 2
as cytosol preparations for PKA activity assays and pellet as membrane
preparations for the plasma membrane-associated enzyme AC activity
assay (29
, 30)
.
Assay of adenylyl cyclase activity
AC activity of hippocampal membrane preparations was quantified
by measuring the rate of conversion of ATP to cAMP. The reaction
mixture contained 25 mM Tris-HCl (pH 7.4), 60 µM EGTA, 1 mM
MgCl2, 10 mM isobutyl-methyl-xanthine, 5 mM
phosphocreatine, creatine phosphokinase 125 U/ml, 0.1 mM GTP, 0.1 mM
ATP, and 1 mM cAMP, 15 µCi/ml 32P-ATP, and
10 µM forskolin. AC activity assay of hippocampal membrane
preparations (16 µg protein) was performed at 30°C in 100 µl
reaction mixture in the presence or absence of 0.002–20 µM
InsP6. For activation of PKA in the hippocampal
cytosol (see below), exogenous cAMP and
32P-ATP were omitted in the reaction mixture.
After 30 min, the reaction was terminated by addition of 1 ml stopping
solution consisting of 50 mM Tris-HCl (pH 7.5), 2.6 mM ATP, 4.3 mM
cAMP, 10 mM CaCl2, and 0.5% lauryl sulfate.
3H-cAMP (20,000 cpm) was included to monitor
cAMP recovery from the samples. Sequential chromatography over AG50-X4
(200–400 mesh, hydrogen form; Bio-Rad, Hercules, CA) and alumina
(neutral, WN-3, Sigma) columns was used to separate ATP from cAMP
(29)
.
Assay of cAMP phosphodiesterase activity
PDE activity of hippocampal homogenates was determined by the
rate of hydrolysis of cAMP. Hippocampal homogenates (32 µg of protein
for high-Km PDE activity assay or 50 µg of protein
for low-Km PDE activity assay) were incubated at
34°C for 10 min in 400 µl of the reaction mixture containing 40 mM
Tris-HCl (pH 8.0), 1.25 mM 2-mercaptoethanol, 10 mM
MgCl2, 0.075% bovine serum albumin, 1 (for
high-Km PDE activity assay) or 200 µM cAMP (for
low-Km PDE activity assay), and 130,000 cpm
3H-cAMP in the presence or absence of 0.002–20
µM InsP6. The reaction was stopped by addition
of 400 µl stopping solution containing 40 mM Tris-HCl (pH 7.4) and 10
mM EDTA. Samples were boiled for 2 min and kept on ice. The second
incubation was performed in the presence of excessive 5'-nucleotidase,
crotalus atrox (Sigma) at 34°C for 10 min and terminated by addition
of 2 ml ice-cold ethanol; 2 ml of stirred AG1-X8 resin slurry (Bio-Rad)
was added and allowed to equilibrate for 15 min at 4°C. The resin was
then spun down. The supernatant was collected and counted
(30)
.
Assay of protein kinase A activity
The PepTag® nonradioactive PKA assay kit
(Promega, Madison, MI) was used to analyze PKA activity in hippocampal
cytosol preparations. In brief, 2 µg Kemptide labeled with
fluorescence (a highly selective substrate for PKA) (31)
and the hippocampal cytosol (12.5 µg protein) mixed with AC reaction
product in hippocampal membrane preparations (see above) in the
presence or absence of 0.02–20 µM InsP6 were
incubated for 30 min at 30°C. PKA reaction buffer contained 20 mM
Tris-HCl, 10 mM MgCl2, and 1 mM ATP, pH 7.4. The
reaction was stopped by putting the samples in boiling water for 10
min; 0.8% agarose gel electrophoresis was used to separate the
phosphorylated from the nonphosphorylated Kemptide in terms of
difference in its net charge (the phosphorylated version: -1, the
nonphosphorylated version: +1). Phosphorylated and nonphosphorylated
substrate fluorescence were quantified by densitometry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 27)
. As shown in Fig. 1A
, the basal levels of InsP6 in several
regions tested were similar. However, electrically evoked convulsive
seizure (ECS) stimulated accumulation of InsP6 in
the hippocampus more potently than in other regions. Narcosis treatment
induced a decrease in InsP6 levels in brain areas
tested, especially in the cortex (P<0.05) (Fig. 1)
.
According to the documented extracellular space (20%) in the brain
(32)
and assuming an even intracellular distribution, the
intracellular concentrations of InsP6 were
estimated to be 13 µM in control hippocampi and 22 µM in hippocampi
subjected to ECS.
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Intracellular InsP6 potentiates high voltage-gated
Ca2+ currents
To evaluate whether intracellular InsP6
modulates voltage-gated Ca2+ currents in cultured
hippocampal neurons, we first examined the effect of
InsP6 on the current–voltage relationship of
depolarization-activated Ca2+ currents. 20 µM
of InsP6 was chosen on the basis of results from
the InsP6 mass assay. Inclusion of
InsP6 in the recording pipette enhanced
whole-cell Ca2+ currents generated by a set of
depolarizing voltage pulses (100 ms) between -70 and 40 mV in 10 mV
increments from a holding potential of -80 mV (Fig. 2Ai
, Aii
). Compiled data illustrate that the
InsP6-treated cells (n=40) exhibited
larger Ca2+ currents during depolarizations in
the range from -20 to 10 mV from a holding potential of -80 mV
compared with control cells (n=39) (Fig. 2B
).
|
To test the specificity of InsP6 in the
modulation of voltage-activated Ca2+ currents, we
next applied InsP5 into cells. Replacement of
InsP6 with an equimolar concentration of
InsP5 (20 µM, n=42) did not affect
voltage-gated Ca2+ currents compared with
controls (n=41) (Fig. 2Ci
, Cii
, D
).
Increasing the concentration of InsP5 to 100 µM
significantly enhanced high voltage-gated Ca2+
currents in the cells (n=42) (Fig. 2Ci
, Ciii
,
D
). However, 100 µM InsP5 was still
less potent than 20 µM InsP6 (Fig. 2B
, D
).
Although the above protocol did not reveal effects of
InsP6 during smaller voltage steps (from -70 to
-30 mV), modulatory effects of intracellular
InsP6 on low voltage-gated
Ca2+ currents could not be excluded. Optimal
recordings of low voltage-gated Ca2+ currents
require higher concentrations of extracellular
Ca2+ and more negative holding potentials, since
these currents are small and partially inactivated at -80 mV in
cultured hippocampal neurons (28)
. We therefore
investigated the possible effect of intracellular
InsP6 on low voltage-gated
Ca2+ currents in cells bathed in a solution
containing 10 mM Ca2+ and depolarized to -40 mV
from a set of holding potentials from -110 to -60 mV. Figure 2Ei
, Eii
shows typical low voltage-activated
Ca2+ current traces after depolarizing pulses to
-40 mV from holding potentials more negative than -70 mV. These agree
with previous findings in hippocampal neurons (28)
. The
representative voltage-activated Ca2+ current
traces display no difference between a control cell (Fig. 2Ei
) and a cell filled with 20 µM
InsP6 (Fig. 2Eii
). Compiled data
illustrate that intracellular application of 20 µM
InsP6 (n=37) did not affect the peak
and steady-state Ca2+ current density compared
with controls (n=34) (Fig. 2F
).
L-type Ca2+ channel blocker abolishes the potentiation
of high voltage-gated Ca2+ currents by
InsP6
To examine whether a specific subtype of the voltage-gated
Ca2+ channel is modulated by
InsP6 in the hippocampal neuron, which is
equipped with all the types of voltage-gated Ca2+
channels described, including L, N, P/Q, R, and T types (9
, 11
, 13
, 21
, 28)
, we exposed the InsP6-treated
and control cells to 6 µM nimodipine, a blocker of the L-type
Ca2+ channel. Figure 3A
shows typical whole-cell Ca2+ current
traces and changes in Ca2+ current density from
an InsP6-treated and a control cell before and
during the exposure to nimodipine. The voltage-gated
Ca2+ current density measured during
depolarization from a holding potential of -80 mV to 0 mV was
significantly larger (P<0.05) in
InsP6-treated cells (n=41) than in
control cells (n=39) before exposure to nimodipine (Fig. 3
A, B
). In contrast, there was no significant
difference in the voltage-gated Ca2+ current
density between InsP6-treated and control cells
during exposure to nimodipine (Fig. 3A
, B
). The
net percentage decrease in high voltage-gated
Ca2+ currents produced by nimodipine was
significantly larger (P<0.01) in cells filled with
InsP6 (26±1.5%) than
non-InsP6-treated cells (17±1.4%) (Fig. 3C
). Under our experimental conditions, 20% of the
overall Ca2+ currents were blocked by nimodipine,
in agreement with previous findings (33)
. The degree of
inhibition of this specific L-type Ca2+ channel
blocker varies between cells and depends on the holding potential
(28)
. In fact, a few hippocampal neurons are insensitive
to the dihydropyridine Ca2+ channel blocker, as
reported here and in other studies (28)
. However, we
included all the neurons tested in order to properly compare the
difference between control and InsP6-treated
cells. In contrast, the study (28)
demonstrating a higher
percentage of L-type Ca2+ currents compared with
the present work excluded neurons insensitive to the dihydropyridine
Ca2+ channel blocker.
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InsP6 enhances high voltage-gated Ca2+
currents by stimulating adenylyl cyclase-PKA cascade
The activity of the L-type Ca2+ channel has
been demonstrated to be potentiated by the activation of PKA in
hippocampal neurons (21)
. To investigate whether
InsP6 also modulated PKA activity as a mechanism
to enhance L-type voltage-gated Ca2+ currents, we
examined the direct effect of InsP6 on PKA
activity. InsP6 at concentrations from 0.02 to
200 µM did not influence the activity of purified PKA catalytic
subunits or corresponding holoenzymes in hippocampal cytosol
preparations (data not shown). Although there were no direct effects of
InsP6 on PKA activity, possible indirect effects
of InsP6 on PKA activity cannot be excluded
through AC and cAMP PDE, which determine the intracellular level of
cAMP, an endogenous PKA activator. Therefore, we assessed effects of
InsP6 on the activity of AC in hippocampal
membrane preparations and PDE in hippocampal homogenates. The
dose–activity curve of AC and PDE was used to determine the half peak
doses of these enzymes. As shown in Fig. 4A
, C
, E
, the half peak doses of AC,
high-Km, and low-Km PDEs were 16,
32, and 50 µg of proteins, respectively; 2 and 20 µM of
InsP6 significantly increased the activity of AC
in hippocampal membrane preparations containing 16 µg of protein
(P<0.05) (Fig. 4B
). However,
InsP6 at concentrations from 0.002 to 20 µM did
not influence the activity of high-Km and
low-Km PDEs in hippocampal homogenates containing 32
and 50 µg of proteins, respectively (P>0.05) (Fig. 4D
, F
). These data suggested that
InsP6 might indirectly affect PKA activity
through AC pathway. We first incubated hippocampal membrane
preparations containing membrane-associated AC with different
concentrations of InsP6 under favorable
conditions for AC. Subsequently the cAMP produced by AC in hippocampal
membrane preparations was used to activate PKA holoenzyme in
hippocampal cytosol preparations. This protocol revealed that PKA
activity in hippocampal cytosol preparations was dose-dependently
related to the amount of hippocampal cytosolic proteins with a half
peak dose of around 12.5 µg protein (Fig. 5A
). InsP6 at 20 µM significantly
enhanced the activity of PKA, activated by cAMP produced by AC in
hippocampal membrane preparations (P<0.05) (Fig. 5B
).
|
|
To assess subsequent physiological consequences of the indirect
stimulation of PKA by InsP6, we used the
membrane-permeable cAMP analog 8-CTP-cAMP (1 mM), a PKA activator, to
examine whether intracellular application of
InsP6 could counteract the effect of PKA
activator on the L-type Ca2+ channel activity
(21)
. Figure 5C
shows that a cell pretreated
with InsP6 exhibited larger
Ca2+ currents but a less pronounced
8-CTP-cAMP-induced increase in Ca2+ channel
activity than a control cell. Compiled data show a significant increase
(P<0.05) in voltage-gated Ca2+
current density in InsP6-filled cells
(n=40) compared with control cells (n=41) (the
control group vs. the InsP6-treated group:
-28.9±1.2 pA/pF vs. -35.5±2.5 pA/pF) before treatment with
8-CTP-cAMP (Fig. 5D
). The voltage-gated
Ca2+ current density observed in cells filled
with InsP6 (-49.2±3.5 pA/pF) was no longer
different from that in control cells (-47.0±1.9 pA/pF) during
treatment with 8-CTP-cAMP (Fig. 5D
). Taken together, Fig. 5E
illustrates larger net increases in voltage-gated
Ca2+ currents by 8-CTP-cAMP in control cells
(66.2±5.5%) than in cells filled with InsP6
(40.5±3.4%).
The stimulatory effect of 8-CTP-cAMP on Ca2+
currents was blocked by nimodipine, confirming that this cAMP analog
acted on L-type Ca2+ channels. A typical
recording of voltage-gated Ca2+ currents before
and during these treatments is illustrated in Fig. 5F
.
19.5 ± 2.3% of voltage-gated Ca2+ currents
were blocked by nimodipine (n=5) (Fig. 5G
). The
increase in voltage-gated Ca2+ currents by
treatment with 8-CTP-cAMP was much less in cells exposed to nimodipine
(10.1±4.1%) than in cells not exposed (66.2±5.5%) (Fig. 5E
, G
).
The above results indicate that intracellular
InsP6 specifically enhanced L-type
Ca2+ channel activity by raising cAMP levels.
However, the involvement of PKA was uncertain since the possible
cAMP-dependent but PKA-independent modulation of
Ca2+ channel activity could not be ruled out. To
ascertain the involvement of PKA in the action of
InsP6, preincubation of cells with the specific
AC and PKA inhibitors 2',5'-dd-Ado and H-89 was used in combination
with intracellular application of InsP6. The
incubation with 100 µM 2',5'-dd-Ado completely blocked activation of
AC by 25 µM forskolin. In contrast, this treatment did not affect the
stimulatory effect of 8-CTP-cAMP on PKA, which was abolished by the
preincubation with 1 µM H-89 (Fig. 6A
).Intracellular application of InsP6 significantly
increased whole-cell Ca2+ currents (Fig. 6Bi
, B ii
, C
). This stimulatory effect was
abolished by pretreatment with either 2',5'-dd-Ado or H-89 (Fig. 6Biii
, Biv
, Bv
, Bvi
, C
).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 34
, 35)
. Hippocampal pyramidal neurons possess multiple types of
voltage-gated Ca2+ channels (9
, 11
, 13
, 21
, 28)
. These channels play distinct roles in the regulation of
neuronal structure and function of the hippocampus corresponding to
their subcellular locations and electrophysiological properties
(9)
. The 1C and
1D subunits, the Ca2+
conducting subunits of the L-type Ca2+ channel,
are localized in cell bodies and proximal dendrites of hippocampal
pyramidal neurons (11)
. Consistent with this localization,
the L-type Ca2+ channel plays a key role in the
activation of the transcription factor CREB (cAMP response element
binding protein) by Ca2+-dependent
phosphorylation in the nucleus of hippocampal pyramidal neurons
(34
, 35)
. It has been shown that opening of the L-type
Ca2+ channel by either synaptic stimulation or
direct depolarization induces translocation of calmodulin to the
nucleus, activation of calmodulin-dependent kinase, and elevation in
cAMP levels (35)
. This leads to activation of PKA and
thereby CREB phosphorylation (35)
. Therefore, the L-type
Ca2+ channel has been considered a critical
element in excitation–transcription coupling in hippocampal pyramidal
neurons (9)
.
Depolarization with high K+ dramatically
increases InsP6 levels in cerebellar granule
cells (5)
. The amplitude and duration of this
depolarization-induced change in InsP6 levels are
comparable to that in inositol 1,4,5-trisphosphate levels
(5)
. Our experiments not only confirmed that
InsP6 levels were significantly elevated in
activated brain neurons, but also uncovered that inhibition of neuronal
activity by narcosis lowered InsP6 levels in
central neurons. These data suggest that InsP6
may act as an intracellular signaling molecule in neurons in terms of
activity-dependent changes of InsP6 mass. The
hippocampus was most sensitive to electrical challenge with regard to
percentage accumulation of InsP6. In addition,
intracellular application of InsP6 significantly
potentiates L-type Ca2+ channel activity in
insulin-secreting cells (8)
. To explore the possible
signal function of InsP6, we evaluated the effect
of InsP6 on voltage-gated
Ca2+ channels, particularly L-type
Ca2+ channel activity in cultured hippocampal
pyramidal neurons.
The present study established a fundamental role for intracellular
InsP6 signaling in neurons, i.e., the
voltage-dependent enhancement of L-type Ca2+
channel activity. We observed that this enhancement by
InsP6 took place at depolarizations to potentials
from -30 to 20 mV. However, InsP6 had no effect
on voltage-gated Ca2+ channel activity when
neurons were depolarized to potentials more negative than -30 mV and
more positive than 20 mV. Low voltage-gated Ca2+
channel currents recorded under optimal conditions were unaltered by
intracellular application of InsP6. Furthermore,
a higher concentration of InsP5 (100 µM)
produced a smaller effect on voltage-gated Ca2+
channel activity than 20 µM InsP6. These data
indicate that InsP6 is relatively specific in the
modulation of voltage-gated Ca2+ channel activity
and also rule out nonspecific effects of inositol polyphosphates. It
should be noted that there is no chelating effect on
Ca2+ by 20 µM InsP6,
dissolved in a solution containing 10 mM EGTA (7)
.
Intracellular InsP6 selectively potentiated high
voltage-gated Ca2+ channel activity at membrane
potentials in the range of the action potential, which is the basis of
the signal-carrying ability of neurons.
The InsP6-enhanced Ca2+
current was effectively abolished by the L-type
Ca2+ channel blocker nimodipine. This
indicates that InsP6 selectively modulated the
L-type Ca2+ channel, although all the types of
voltage-gated Ca2+ channels described exist in
the hippocampal neuron (9
, 11
, 13
, 21
, 28)
. The neuron is
one of the most specialized cells in an organism in terms of its
morphology and function. The typical neuron has three characteristic
microscopic structures—dendrites, soma, and axon—for receiving,
processing and transmitting signals. The subcellular localization of
the L-type Ca2+ channel in the soma of
hippocampal pyramidal neurons (11)
underlies the key role
of the L-type Ca2+ channel in controlling
Ca2+-dependent gene expression (19
, 34
, 35)
. This suggests that intracellular
InsP6 is probably involved in the modulation of
gene expression and development by selectively modulating L-type
Ca2+ channel activity, the extent of which
remains to be explored.
Activation of PKA is known to phosphorylate the L-type
Ca2+ channel (12)
and thus to
significantly potentiate its activity in hippocampal neurons
(21)
. Therefore, the possible involvement of PKA in the
stimulatory effects of InsP6 was examined. The
results show that InsP6 did not alter the
activity of either purified PKA catalytic subunits or PKA holoenzymes
in the hippocampal cytosol. This argues against a direct effect of
InsP6 on PKA. However, it is still unclear
whether the PKA pathway plays a role in the regulation of the L-type
Ca2+ channel by InsP6,
since InsP6 may indirectly affect PKA through
modulation of the machinery producing or degrading intracellular cAMP,
an endogenous PKA activator. We found that InsP6
significantly enhanced AC activity in hippocampal membrane preparations
without influencing PDE. Physiological consequences of the
InsP6 effect on AC were examined in both in vitro
and in vivo experiments. In the presence of
InsP6, more cAMP was produced by AC in the
hippocampal membrane preparation, resulting in a more effective
activation of PKA in the hippocampal cytosol that in the absence of
InsP6. Furthermore, the effect of 8-CPT-cAMP, a
membrane-permeable cAMP analog, on L-type Ca2+
channel activity was counteracted by pretreatment with
InsP6. PKA and AC inhibitors completely blocked
the stimulatory effect of InsP6 on voltage-gated
Ca2+ currents. Other serine/threonine protein
kinases, including protein kinases C and G as well as
Ca2+/calmodulin-dependent kinase, are unlikely to
be involved in the mechanism by which InsP6
enhanced voltage-gated Ca2+ currents, since they
either inhibit or do not affect high voltage-gated
Ca2+ channel activity in hippocampal neurons
(36
37
38)
. In our hands, activation of PKC did not
influence L-type Ca2+ channel activity in the
hippocampal neuron (data not shown). Hence, the effect of
InsP6 on L-type Ca2+
channel activity was most likely due to increases in phosphorylation
state at PKA phosphorylation sites of this channel by
InsP6-mediated activation of the AC-PKA cascade
and inhibition of serine/threonine protein phosphatases, which was
described in our previous work (5
6
7
8)
. It is unlikely that
InsP6 directly interacts with the subunits of
voltage-gated Ca2+ channel proteins, since
screening and characterization of InsP6 binding
proteins in the plasma membrane of neurons clearly show that these
proteins do not match any subunit of the voltage-gated
Ca2+ channel in terms of their molecular masses
and functions (39
40
41)
.
L-type Ca2+ currents accounted for 20% of the
overall Ca2+ currents obtained in hippocampal
neurons under our experimental conditions. It is obvious that other
types of Ca2+ channels contribute to the major
fraction of Ca2+ currents in these neurons
(28
, 37)
. These Ca2+ channels have
been shown to be phosphorylated (11)
. The
voltage-dependent and selective enhancement of L-type
Ca2+ channel activity by
InsP6 may be explained by the fact that a
conformational change of the PKA phosphorylation site sequence in the
L-type Ca2+ channel is voltage dependent. The
conformation of this phosphorylation site sequence in a certain
depolarization range is well recognized by PKA and serine/threonine
phosphatases (42
43
44)
. It has been shown that treatment
with okadaic acid counteracts voltage-dependent potentiation of the
L-type Ca2+ channel due to its phosphorylation at
PKA phosphorylation sites (42)
. Okadaic acid has also been
demonstrated to preferentially facilitate the phosphorylation of the
L-type Ca2+ channel compared with other types of
Ca2+ channels—for example, the N-type
Ca2+ channel (45)
.
A model for the effects of InsP6 on L-type
Ca2+ channel activity in the hippocampal neuron
is given in Fig. 7
. The balance between serine/threonine protein phosphatase activity and
PKA activity determines the phosphorylation state at PKA
phosphorylation sites of the L-type Ca2+ channel
(Fig. 7A
). Obviously, decreases in serine/threonine protein
phosphatase activity and/or increases in PKA activity elevate the
phosphorylation state and thereby enhance L-type
Ca2+ channel activity. It has been demonstrated
that a rise in the phosphorylation state at PKA phosphorylation sites
increases channel open probability and enhances the availability of the
L-type Ca2+ channel in hippocampal pyramidal
neurons (21)
. InsP6 inhibited the
activity of serine/threonine protein phosphatases, attenuating the
dephosphorylation at PKA phosphorylation sites of the L-type
Ca2+ channel. It simultaneously stimulated the
AC-PKA cascade, facilitating the phosphorylation at PKA phosphorylation
sites of the L-type Ca2+ channel. As a result,
intracellular InsP6 up-regulated the
phosphorylation state of the L-type Ca2+ channel
and thereby increased the ability of the L-type
Ca2+ channel to conduct
Ca2+ currents (Fig. 7B
). Taken
together, our findings provide a novel signaling pathway for
InsP6 in the hippocampal neuron.
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ACKNOWLEDGMENTS |
|---|
Received for publication December 5, 2000.
Revision received April 4, 2001.
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REFERENCES |
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