Inositol hexakisphosphate increases L-type Ca2ⴙ channel activity by stimulation of adenylyl cyclase SHAO-NIAN YANG,*,1 JIA YU,* GEORG W. MAYR,† FRED HOFMANN,‡ OLOF LARSSON,* AND PER-OLOF BERGGREN* *The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden; †Institut fu¨r Medizinische Biochemie und Molekularbiologie, Universita¨tskrankenhaus Eppendorf, Universita¨t Hamburg, 20246 Hamburg, Germany; and ‡Institut fu¨r Pharmakologie und Toxikologie, Albert-Ludwig Universita¨t Freiburg, 79104 Freiburg, Germany ABSTRACT Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2ⴙ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2ⴙ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2ⴙ channel activity.—Yang, S.-N., Yu, J., Mayr, G. W., Hofmann, F., Larsson, O., Berggren, P.-O. Inositol hexakisphosphate increases L-type Ca2ⴙ channel activity by stimulation of adenylyl cyclase. FASEB J. 15, 1753–1763 (2001)
Key Words: adenylyl cyclase 䡠 calcium channel 䡠 hippocampus 䡠 inositol polyphosphate 䡠 protein kinase A Stimulation of plasma membrane receptors with neurotransmitters, hormones, and growth factors evokes the hydrolysis of phosphatidylinositols and hence generation not only of inositol 1,4,5-trisphosphate, but also a series of other inositol polyphosphates such as inositol tetrakisphosphate, inositol pentakisphosphate (InsP5), and inositol hexakisphosphate (InsP6) (1–3). 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, includ0892-6638/01/0015-1753 © FASEB
ing 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– 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 highaffinity 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 1
Correspondence: The Rolf Luft Center for Diabetes Research L3, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail:
[email protected] 1753
(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, 11–13). 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/Qtype 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.
MATERIALS AND METHODS Mass assay of InsP6 The rat brain was stimulated with electrodes by applying 50 mA pulses at 200 Hz for 0.2 s, which invariably triggered an immediate epileptic seizure. The untreated rat brains were used as controls. The hippocampus, cerebellum, cortex, and striatum were quickly dissected, weighed precisely, and soni1754
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cated for 20 s in an ice-cold solution containing 0.5 M perchloric acid and 0.1 M acetic acid. The homogenate was kept on ice for 30 min and centrifuged at 10,000 g for 10 min; the resulting supernatant was collected. The pellet was resuspended, homogenized as above, and extracted for 15 min; 1 nmol of Ins(1, 2,3,4,5)P5 was added in the pooled extracts as an internal standard. This InsP5 isomer is present in low concentration in all animal cells and thus allowed us to monitor the recovery of inositol polyphosphates in samples subjected to several processing steps. The procedure to perform high-performance liquid chromatography with metal dye detection (HPLC-MDD) has been described elsewhere (26, 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, ZeitzInstrumente, 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(-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 M myo-inositol hexakis[dihydrogen phosphate] (InsP6) (Sigma), 20 and 100 M 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
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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 M 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 wholecell 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 Teflon™-glass homogenizer (20 strokes) in 300 l of ice-cold homogenization buffer containing 50 mM Tris-HCl, 1 mM EGTA, 10% sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml antipain, 5 g/ml aprotinin, 5 g/ml leupeptin, and 5 g/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).
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 M cAMP (for low-Km PDE activity assay), and 130,000 cpm 3H-cAMP in the presence or absence of 0.002–20 M InsP6. The reaction was stopped by addition of 400 l 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 g Kemptide labeled with fluorescence (a highly selective substrate for PKA) (31) and the hippocampal cytosol (12.5 g protein) mixed with AC reaction product in hippocampal membrane preparations (see above) in the presence or absence of 0.02–20 M 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.
RESULTS
Assay of adenylyl cyclase activity
InsP6 levels in brain regions
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 M EGTA, 1 mM MgCl2, 10 mM isobutyl-methylxanthine, 5 mM phosphocreatine, creatine phosphokinase 125 U/ml, 0.1 mM GTP, 0.1 mM ATP, and 1 mM cAMP, 15 Ci/ml ␣32P-ATP, and 10 M forskolin. AC activity assay of hippocampal membrane preparations (16 g protein) was performed at 30°C in 100 l reaction mixture in the presence or absence of 0.002–20 M InsP6. For activation of PKA in the hippocampal cytosol (see below), exogenous cAMP and ␣32PATP 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).
To define InsP6 concentrations in rat brain regions, we measured InsP6 levels by HPLC-MDD, which has been successfully used in mass assay of inositol polyphosphates (26, 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 M in control hippocampi and 22 M in hippocampi subjected to ECS.
Assay of cAMP phosphodiesterase activity
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 M of InsP6 was chosen on the basis of results from the InsP6 mass assay. Inclusion of
PDE activity of hippocampal homogenates was determined by the rate of hydrolysis of cAMP. Hippocampal homogenates (32 g of protein for high-Km PDE activity assay or 50 g of protein for low-Km PDE activity assay) were incubated at 34°C for 10 min in 400 l of the reaction mixture containing 40 INOSITOL HEXAKISPHOSPHATE ENHANCES Ca2⫹ CURRENTS
Intracellular InsP6 potentiates high voltage-gated Ca2ⴙ currents
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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, ii shows typical low voltageactivated 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 voltageactivated Ca2⫹ current traces display no difference between a control cell (Fig. 2Ei) and a cell filled with 20 M InsP6 (Fig. 2Eii). Compiled data illustrate that intracellular application of 20 M InsP6 (n⫽37) did not affect the peak and steady-state Ca2⫹ current density compared with controls (n⫽34) (Fig. 2F).
Figure 1. InsP6 levels in brain regions after electrically evoked convulsive seizure (ECS) and narcosis treatments. A) Similar basal levels of InsP6 were observed in the brain regions tested. ECS treatment significantly increased InsP6 levels in the regions tested. Narcosis treatment induced a significant decrease in the cortex and a trend toward a decrease in InsP6 levels in other regions tested. Data are presented as means ⫾ se, n ⫽ 6 –15. Statistical significance was evaluated by one-way ANOVA, followed by least significant difference (LSD) test. **P ⬍ 0.01 vs. controls, ⫹P ⬍ 0.05 vs. controls. B) Percentage changes in InsP6 levels show that ECS treatment accumulated more InsP6 in the hippocampus than in other regions. Narcosis treatment decreased InsP6 levels more potently in the cortex than in other regions tested.
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, ii). 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 M, n⫽42) did not affect voltage-gated Ca2⫹ currents compared with controls (n⫽41) (Fig. 2Ci, ii, D). Increasing the concentration of InsP5 to 100 M significantly enhanced high voltagegated Ca2⫹ currents in the cells (n⫽42) (Fig. 2Ci, iii, D). However, 100 M InsP5 was still less potent than 20 M InsP6 (Fig. 2B, D). 1756
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L-type Ca2ⴙ channel blocker abolishes the potentiation of high voltage-gated Ca2ⴙ currents by InsP6 To examine whether a specific subtype of the voltagegated 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 M 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 InsP6treated 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. 3A, 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 neu-
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Figure 2. Effects of intracellular application of InsP6 and InsP5 on voltage-gated Ca2⫹ currents. A) Sample whole-cell Ca2⫹ current traces, generated by a set of depolarizing voltage pulses (100 ms, 0.5 Hz) between ⫺70 and 40 mV in 10 mV increments from a holding potential of ⫺80 mV, from a control cell (i, capacitance: 28.2 pF) and a cell filled with 20 M InsP6 (ii, capacitance: 28.0 pF). B) Summary graph of current density–voltage relationships showing that InsP6-treated cells (filled circles, n⫽40) displayed larger Ca2⫹ currents than control cells (open circles, n⫽39) during depolarizations in the range ⫺20 to 10 mV from a holding potential of ⫺80 mV. Data are presented as means ⫾ se. Statistical significance was evaluated by unpaired Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01. C) Sample whole-cell Ca2⫹ current traces generated by the same depolarization protocol as above from a control cell (i, capacitance: 30.5 pF), a cell filled with 20 M InsP5 (ii, capacitance: 31.3 pF), and a cell filled with 100 M InsP5 (iii, capacitance: 30.0 pF). D) Summary graph of current density–voltage relationships showing that intracellular application of 20 M InsP5 (filled circles, n⫽42) had no significant effect on voltage-gated Ca2⫹ currents compared with controls (open circles, n⫽41). However, cells (n⫽42) filled with a higher concentration of InsP5 (100 M) (open triangles, n⫽42) exhibited larger Ca2⫹ currents when depolarized within the range ⫺30 to 0 mV from a holding potential of ⫺80 mV. Data are presented as means ⫾ se. Statistical significance was evaluated by one-way ANOVA, followed by LSD test. *P ⬍ 0.05 vs. controls. E) Sample whole-cell Ca2⫹ current traces, generated by a set of depolarizing voltage pulses (100 ms, 0.05 Hz) to ⫺40 mV from a set of holding potentials from ⫺110 to ⫺60 mV, from a control cell (i, capacitance: 30.9 pF) and a cell filled with 20 M InsP6 (ii, capacitance: 31.3 pF). F) Summary graph showing that intracellular application of 20 M InsP6 (n⫽37) did not influence the Ca2⫹ current density at holding potentials from ⫺110 to ⫺60 mV in both transient peak (circles) and steady-state inactivation (triangles) parts vs. controls (n⫽34). Data are presented as means ⫾ se. Statistical significance was evaluated by unpaired Student’s t test.
rons 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. InsP6 enhances high voltage-gated Ca2ⴙ 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 M 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 INOSITOL HEXAKISPHOSPHATE ENHANCES Ca2⫹ CURRENTS
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 g of proteins, respectively; 2 and 20 M of InsP6 significantly increased the activity of AC in hippocampal membrane preparations containing 16 g of protein (P⬍0.05) (Fig. 4B). However, InsP6 at concentrations from 0.002 to 20 M did not influence the activity of high-Km and low-Km PDEs in hippocampal homogenates containing 32 and 50 g 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 g protein (Fig. 5A). InsP6 at 20 M 1757
Figure 3. The L-type Ca2⫹ channel blocker nimodipine abolishes the enhanced high voltage-gated Ca2⫹ current by intracellular application of InsP6. A) Examples showing time courses of voltage-gated Ca2⫹ currents evoked by repetitive depolarizing voltage steps from ⫺80 mV to 0 mV (100 ms, 0.05 Hz) in a control cell (capacitance: 44.5 pF, open circles) and a cell filled with 20 M InsP6 (capacitance: 38.7 pF, filled circles) before and during exposure to nimodipine. Insets show individual Ca2⫹ current traces registered at the points sequentially numbered in the time course curves. Calibration bars are 400 pA and 10 ms. B) Summary graph shows that whole-cell Ca2⫹ current density in cells filled with 20 M InsP6 (filled column to the left, n⫽41) is significantly higher than that in control cells (open column to the left, n⫽39) before exposure to nimodipine. There was no significant difference in Ca2⫹ current density between cells filled with 20 M InsP6 (filled column to the right) and control cells (open column to the right) when the L-type Ca2⫹ channel was blocked with 6 M nimodipine. Data are presented as means ⫾ se. Statistical significance was evaluated by unpaired Student’s t test. *P ⬍ 0.05. C) The net percentage decrease in voltage-gated Ca2⫹ currents by nimodipine was smaller in control cells (open column, n⫽39) than in InsP6-treated cells (filled column, n⫽41). Data are presented as means ⫾ se. Statistical significance was evaluated by unpaired Student’s t test. **P ⬍ 0.01.
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 1758
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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 voltagegated 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 voltagegated 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 M 2⬘,5⬘-ddAdo completely blocked activation of AC by 25 M 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 M H-89 (Fig. 6A). Intracellular application of InsP6 significantly increased whole-cell Ca2⫹ currents (Fig. 6Bi, ii, C). This stimulatory effect was abolished by pretreatment with either 2⬘,5⬘-dd-Ado or H-89 (Fig. 6Biii–vi, C).
DISCUSSION Cultured pyramidal neurons of the hippocampus have been used as one of the best in vitro systems to examine the molecular and cellular basis of synaptic plasticity, which plays a central role in learning and memory. A series of molecular mechanisms involved in synaptic plasticity, including protein phosphorylation, gene expression, and neurotransmitter release, has been demonstrated to be regulated by voltage-gated Ca2⫹ chan-
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Figure 4. InsP6 increases adenylyl cyclase (AC) activity without influencing cAMP PDE in hippocampal preparations. A) Dose–activity relationships of AC in hippocampal membrane preparations. The amount of hippocampal membrane preparations is indicated by protein content. B) InsP6 at 2 to 20 M significantly increases AC activity in hippocampal membrane preparations (16 g protein). Data are presented as means ⫾ se, n ⫽ 5. Statistical significance was evaluated by one-way ANOVA, followed by LSD test. *P ⬍ 0.05. C) Dose–activity relationships of high-Km PDE in the hippocampal homogenate. The amount of the hippocampal homogenate is indicated by protein content. D) InsP6 at concentrations 0.002 to 20 M had no effect on high-Km PDE activity in the hippocampal homogenate (32 g protein). Data are presented as means ⫾ se, n ⫽ 5. Statistical significance was evaluated by one-way ANOVA, followed by LSD test. E) Dose–activity relationships of low-Km PDE in the hippocampal homogenate. The amount of the hippocampal homogenate is indicated by protein content. F) InsP6 at concentrations 0.002 to 20 M did not alter low-Km PDE activity in the hippocampal homogenate (50 g protein). Data are presented as means ⫾ se, n ⫽ 5. Statistical significance was evaluated by one-way ANOVA, followed by LSD test.
nels (9, 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 INOSITOL HEXAKISPHOSPHATE ENHANCES Ca2⫹ CURRENTS
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 M) produced a smaller effect on voltage-gated Ca2⫹ channel activity than 20 M InsP6. These data indicate that InsP6 is relatively specific in the modulation of voltagegated Ca2⫹ channel activity and also rule out nonspecific effects of inositol polyphosphates. It should be 1759
Figure 5. InsP6 increases protein kinase A (PKA) activity in the mixture of hippocampal membrane and cytosol preparations and counteracts the enhanced high voltage-gated Ca2⫹ currents induced by 8-CTP-cAMP. A) Dose– activity relationships of PKA in the mixture of hippocampal membrane and cytosol preparations in phosphorylation of the PKA-selective substrate Kemptide. Inset shows the phosphorylated and nonphosphorylated Kemptide fluorescence signals produced by different amounts of PKA in hippocampal cytosol preparations, which was activated by cAMP produced by AC in hippocampal membrane preparations. The amount of hippocampal cytosol preparations is indicated by protein content. B) InsP6 at 20 M significantly increased PKA activity in the mixture of hippocampal membrane and cytosol preparations in phosphorylation of the PKAselective substrate Kemptide compared with control. Inset showing an example of the phosphorylated and nonphosphorylated Kemptide fluorescence signals observed at different concentrations of InsP6 (N: no hippocampal preparation). Data are presented as means ⫾ se, n ⫽ 8. Statistical significance was evaluated by oneway ANOVA, followed by LSD test. *P ⬍ 0.05. C) Examples of time courses of voltage-gated Ca2⫹ currents evoked by repetitive depolarizing voltage steps from ⫺80 mV to 0 mV (100 ms, 0.05 Hz) in a control cell (capacitance: 26.5, open circles) and a cell filled with 20 M InsP6 (capacitance: 26.0 pF, filled circles), before and during the exposure to 8-CTP-cAMP. Insets show individual Ca2⫹ current traces registered at the sequential numbers of the points in the time course curves. Calibration bars are 400 pA and 10 ms. D) The basal value of Ca2⫹ current density measured during the depolarization from ⫺80 to 0 mV was significantly different between control cells (open column to the left, n⫽41) and InsP6-treated cells (filled column to the left, n⫽40). The difference in Ca2⫹ current density between control (open column to the right) and InsP6-treated cells (filled column to the right) was no longer present during treatment with 8-CTP-cAMP. Data are presented as means ⫾ se. Statistical significance was evaluated by unpaired Student’s t test. *P ⬍ 0.05. E) The net percentage increase in voltage-gated Ca2⫹ currents by 8-CTP-cAMP was much larger in control cells (open column, n⫽41) than in InsP6-treated cells (filled column, n⫽40). Data are presented as means ⫾ se. Statistical significance was evaluated by an unpaired Student’s t test. **P ⬍ 0.01. F) A time course of the percentage change in voltage-gated Ca2⫹ currents, normalized by the first three recordings evoked by repetitive depolarizing voltage steps from ⫺80 mV to 0 mV (100 ms, 0.05 Hz) in a cell exposed to nimodipine, followed by treatment with 8-CTP-cAMP. Insets show individual Ca2⫹ current traces registered at the sequential numbers of the points in the time course curves. Calibration bars are 400 pA and 10 ms. G) Summary graph showing that voltage-gated Ca2⫹ currents markedly decreased during the exposure to nimodipine (open column, n⫽5). Pretreatment with nimodipine (filled column) dramatically diminished the increase in voltage-gated Ca2⫹ currents by 8-CTP-cAMP compared with non-pretreatment (open column in panel E).
noted that there is no chelating effect on Ca2⫹ by 20 M 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, pro1760
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cessing 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
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Figure 6. Protein kinase A (PKA) and adenylyl cyclase (AC) inhibitors block effects of InsP6 on voltage-gated Ca2⫹ currents. A) Examples of time courses of voltage-gated Ca2⫹ currents evoked by repetitive depolarizing voltage steps from ⫺80 mV to 0 mV (100 ms, 0.05 Hz) in a cell preincubated with the PKA inhibitor H-89, followed by stimulation with 8-CTPcAMP (capacitance: 34.5 pF, open circles) or cells pretreated with the AC inhibitor 2⬘,5⬘-dd-Ado, followed by stimulation with forskolin (capacitance: 23.5 pF, filled triangles) and 8-CTP-cAMP (capacitance: 25.0 pF, filled circles), respectively. Insets show individual Ca2⫹ current traces registered at the sequential numbers of the points in the time course curves. The bar under the time course curves denotes the period of application of 8-CTP-cAMP and forskolin. Calibration bars are 400 pA and 10 ms. B) Sample whole-cell Ca2⫹ current traces, generated by a set of depolarizing voltage pulses (100 ms, 0.5 Hz) between ⫺70 and 40 mV in 10 mV increments from a holding potential of ⫺80 mV, from a control cell (i, capacitance: 27.4 pF), a cell filled with 20 M InsP6 (ii, capacitance: 29.3 pF), H-89-pretreated cells with (iv, capacitance: 27.2 pF) and without further application of 20 M InsP6 (iii, capacitance: 27.9 pF), and 2⬘,5⬘-dd-Ado-pretreated cells with (vi, capacitance: 27.5 pF) and without further application of 20 M InsP6 (v, capacitance: 30.5 pF), respectively. C) Summary graph of current density–voltage relationships showing that InsP6-treated cells (filled circles, n⫽37) exhibited larger Ca2⫹ currents than control cells (open circles, n⫽36) during depolarizations in the range ⫺20 to 10 mV from a holding potential of ⫺80 mV. Application of 20 M InsP6 in cells subjected to preincubation with H-89 INOSITOL HEXAKISPHOSPHATE ENHANCES Ca2⫹ CURRENTS
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 –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 ACPKA cascade and inhibition of serine/threonine protein phosphatases, which was described in our previous work (5– 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 – 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 selec-
(filled triangles, n⫽36) no longer affected voltage-gated Ca2⫹ currents compared with cells subjected to only H-89 preincubation (open triangles, n⫽36). Likewise, application of 20 M InsP6 in cells subjected to preincubation with 2⬘,5⬘-ddAdo (filled triangles, n⫽36) produced no effect on voltagegated Ca2⫹ currents compared with cells subjected to only 2⬘,5⬘-dd-Ado-pretreatment (open triangles, n⫽36). Data are presented as means ⫾ se. Statistical significance was evaluated by one-way ANOVA, followed by LSD test. *P ⬍ 0.05, **P ⬍ 0.01 vs. control, H-89 and 2⬘,5⬘-dd-Ado pretreatments with and without further application of InsP6. 1761
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.
Figure 7. Model describing how intracellular InsP6 potentiates L-type Ca2⫹ currents in hippocampal neurons. A) The ability of the L-type Ca2⫹ channel to conduct Ca2⫹ depends on the phosphorylation state of this channel at PKA phosphorylation sites, the extent to which is determined by the balance between PKA and serine/threonine protein phosphatase (PPase) activities. AC: adenylyl cyclase, P: phosphoryl group. B) Increase in PKA activity and/or decrease in serine/ threonine protein phosphatase activity potentiate the phosphorylation of the L-type Ca2⫹ channel and thus enhances channel activity. InsP6 directly inhibits serine/threonine protein phosphatases and indirectly stimulates PKA through the AC pathway, thereby elevating L-type Ca2⫹ channel activity through the potentiation of the phosphorylation state of this channel at PKA phosphorylation sites.
tive 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– 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 1762
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We thank Dr. Bjo¨rn Owe-Larsson for help in the establishment of hippocampal cell culture, Dr. Ernst Habermann for help in mass assay of InsP6, and Dr. Christopher J. Barker for discussions. This work was supported by grants from the Swedish Medical Research Council (72X-09890, 72X-09891, 72XS-12708, and 72X-00034), the Swedish Diabetes Association, the Nordic Insulin Foundation Committee, Fredrik and Ingrid Thurings Foundation, Funds of Karolinska Institutet, Berth von Kantzows Foundation, the Novo Nordisk Foundation, and the Swedish Society for Medical Research.
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