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¹ Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA
² CNRS UMR 8078, Université Paris Sud, Hôpital Marie Lannelongue, 92350 Le Plessis-Robinson, France

	Abstract

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In many non-excitable cells, the predominant mode of agonist-activated Ca²⁺ entry switches from the arachidonic acid-regulated Ca²⁺ (ARC) channels at low agonist concentrations, to store-operated channels at high concentrations. Underlying this process is the inhibition of the ARC channels by a calcineurin-mediated dephosphorylation, which inhibits the ability of arachidonic acid to activate the channels. Following such a dephosphorylation, we found that restoration of the sensitivity of the ARC channels to arachidonic acid, as well as to low concentrations of carbachol, was specifically dependent on protein kinase A (PKA) activity. Inhibition of protein kinase C, protein kinase G or calmodulin-activated kinase had no effect. This action of PKA was unaffected by prolonged intracellular dialysis, whilst disruption of the binding of PKA to A-kinase anchoring proteins (AKAPs) inhibited currents through ARC channels, and blocked the PKA-dependent effects. AKAP79, a protein which scaffolds both PKA and calcineurin, was shown to be present in the cells. These data illustrate the significance of PKA-dependent phosphorylation and calcineurin-dependent dephosphorylation in the overall regulation of ARC channel activity, and indicate the key role of an AKAP, possibly AKAP79, in the spatial organization these processes.

(Received 9 May 2005; accepted after revision 24 June 2005; first published online 30 June 2005)
Corresponding author T. J. Shuttleworth: Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA. Email: trevor_shuttleworth{at}urmc.rochester.edu

	Introduction

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An enhanced rate of Ca²⁺ entry plays a critical role in agonist-activated cytosolic calcium concentration ([Ca²⁺]_i) signals in a variety of cells. In non-excitable cells, this enhanced entry of Ca²⁺ was, for many years, thought to occur exclusively via the so-called capacitative or store-operated mechanism first proposed by Putney (1986, 1990). In this, the entry of Ca²⁺ is activated entirely as a result of, and subsequent to, a depletion of the endoplasmic reticulum Ca²⁺ stores. During agonist action, this would normally occur as a result of the generation of inositol 1,4,5-trisphosphate (IP₃), but essentially anything that induces a depletion of the stores would activate this form of entry. The channels responsible are generically described as store-operated channels (SOCs) and come in various forms (Parekh & Penner, 1997). The original, and still most extensively characterized, is the highly Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ (CRAC) conductance and its close relatives, first described in T lymphocytes and mast cells (Hoth & Penner, 1992; 1993,; Zweifach & Lewis, 1993). Other, often less selective, SOCs are found in other cell types (Parekh & Putney, 2005). However, with a few possible exceptions, and despite intensive study, the molecular identity of these channels remains unknown. Moreover, although a putative regulator of the CRAC channels has recently been identified (Roos et al. 2005), the precise mechanism linking the depletion of the stores to the activation of these channels is also unclear.

In many non-excitable cells, stimulation with increasing concentrations of appropriate agonists is associated with a change from oscillatory to sustained elevated ‘plateau-type’ [Ca²⁺]_i signals. The role of SOCs in modulating the amplitude of the sustained [Ca²⁺]_i signals, when intracellular stores are profoundly depleted, is clear. However, oscillatory [Ca²⁺]_i signals are typically associated with only a partial, and very brief depletion of the stores (Park et al. 2000), raising questions as to whether these channels could function effectively during such signals (Shuttleworth, 1999). As a result, in more recent years, various non-capacitative modes of Ca²⁺ entry have been described, whose activity may be more relevant during such oscillatory [Ca²⁺]_i signals. Of these, perhaps the most widely distributed is an entry pathway whose activation is exclusively dependent on the generation of arachidonic acid (Shuttleworth, 1996; Munaron et al. 1997; Shuttleworth & Thompson, 1998; Broad et al. 1999). In certain of these cases, the channels responsible (arachidonate-regulated calcium (ARC) channels), have been extensively characterized and shown to be highly Ca²⁺-selective conductances whose biophysical features, although superficially similar to the CRAC channels, reveal that they represent an entirely distinct conductance (Mignen & Shuttleworth, 2000, 2001; Mignen et al. 2003a). As such, their presence in a variety of different cell types has been demonstrated, along with their specific activation at low agonist concentrations. Current evidence from several cell types, including both cell lines and acutely isolated cells, indicates that it is these ARC channels that provide the predominant mode of Ca²⁺ entry at such agonist concentrations (Mignen et al. 2001, 2005). Only at high agonist concentrations, when a prolonged and profound depletion of the stores occurs, are the co-existing SOCs activated. Moreover, the activation of the SOCs as agonist concentrations are increased coincides with an inhibition of the ARC channels (Mignen et al. 2001, 2005). Thus, the change from an oscillatory to a sustained [Ca²⁺]_i signal as agonist concentrations are increased, coincides with a switch in the predominant mode of agonist-activated Ca²⁺ entry from the ARC channels at low concentrations, to the SOC channels at high concentrations – a phenomenon we have described as the ‘reciprocal regulation of Ca²⁺ entry’ (Mignen et al. 2001). The key action underlying this reciprocal regulation is the inhibition of ARC channels by the sustained elevated [Ca²⁺]_i signals induced as a result of SOC channel activation. Biochemical investigations have demonstrated that this inhibition is mediated by the Ca²⁺–calmodulin-dependent phosphatase calcineurin (Mignen et al. 2003b). Interestingly, we found that this calcineurin-dependent dephosphorylation not only inhibits previously activated ARC channels, but also renders the channels insensitive to subsequently applied arachidonic acid. This suggests that phosphorylation is required for the effective stimulation of the ARC channels by arachidonic acid. The purpose of the following study was to examine the nature of this phosphorylation, and its effects on the activity of the ARC channels.

	Methods

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Cell preparation

HEK293 cells stably transfected with the human m3 muscarinic receptor (m3-HEK cells) were used throughout. These cells were cultured in an incubator in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and antibiotics with 5% CO₂ at 37°C, as previously described (Shuttleworth & Thompson, 1998).

Whole-cell patch-clamp experiments

Patch-clamp recordings of macroscopic whole-cell currents were performed using an Axopatch-200B patch-clamp amplifier (Axon Instruments) as previously described (Mignen & Shuttleworth, 2000; Mignen et al. 2003b). Internal (pipette) solutions contained (mM): caesium acetate 140, NaCl 10, MgCl₂ 1.22, EGTA 5, CaCl₂ 1.89, MgATP 0.5 and Hepes 10 (pH 7.2), unless otherwise indicated. Calculated free [Ca²⁺] was 100 nM. The extracellular (bath) solution contained (mM): NaCl 140, MgCl₂ 1.2, CaCl₂ 10, CsCl 5, D-glucose 10 and Hepes 10 (pH 7.4). Whole-cell currents were recorded during 250-ms hyperpolarizing voltage pulses to –80 mV delivered every 2 s from a holding potential of 0 mV. Current–voltage (I–V) relationships were recorded using 150 ms voltage ramps from –100 to +30 mV. Data were sampled at 20 kHz during the voltage steps and at 5.5 kHz during the voltage ramps and digitally filtered off-line at 1 kHz. Initial I–V relationships obtained before activation of ARC currents, were averaged and used for leak subtraction of subsequent current recordings. Changes in the external (bath) solution were by perfusion of the patch-clamp chamber (approximately 1.5 ml min^–1). All experiments were carried out at room temperature (20–22°C). Data are presented as means ± S. E. M.

Cytosolic calcium measurements

Changes in [Ca²⁺]_i in single individual cells were determined following loading with the fluorescent probe indo-1. Approximately 24 h after plating onto glass coverslips, cells were loaded by incubation with 4 µM acetoxymethylester form of indo-1 (indo-1 AM; Molecular Probes) for 12 min, washed in saline, and then incubated for a further 30 min at 37°C to allow for complete hydrolysis of the acetoxymethylester. The coverslips formed the base of a microscope perfusion chamber and were continually perfused with Hepes-buffered saline containing (mM): NaCl 132.5, KCl 4.8, CaCl₂ 1.3, MgSO₄ 1.2, KH₂PO₄ 1.2, glucose 6, NaHepes 9.05 and Hepes free acid 5.95 (pH 7.4). Measurements were performed using an inverted epifluorescence microscope (Nikon TE 200) with a 40 x oil immersion objective lens (numerical aperture, 1.3). Cells were excited at 345 nm using a monochromator (TILL Polychrome IV), and fluorescence measurements of single individual cells, isolated by a cell framing adapter, were obtained simultaneously at emitted wavelengths of 405 nm and 485 nm using photon-counting photomultipliers (IonOptix). Background subtraction and calculation of the resulting 405 nm/485 nm fluorescence ratios were performed on-line using the IonOptix software. All experiments were performed at room temperature.

Protein kinase A activity measurements

Membrane preparations were obtained by homogenization of washed cells in homogenization medium containing (mM): EDTA 1, DTT 1, leupeptin 0.1 and Tris-HCl 25 (pH 7.2) followed by three centrifugations at 14 000 g at 4°C for 10 min, and the supernatant was discarded each time. The final pellet was resuspended in homogenization medium containing 0.01% Triton X-100 and 2 mg ml^–1 bovine serum albumin (BSA). This was assayed for protein kinase A (PKA) activity using a fluorescence-based kemptide phosphorylation assay kit (PepTag, Promega) following the manufacturers instructions, as modified by Karege et al. (2001). This involved incubation of the homogenates in a reaction buffer containing (mM): ATP 10, MgCl₂ 10 and Tris-HCl 20 (pH 7.4), PKA catalytic subunit and fluorescently labelled kemptide. The reaction was terminated by heating at 95°C for 10 min, followed by separation of the phosphorylated from the non-phosphorylated kemptide by electrophoresis on a 0.8% agarose gel. Phosphorylated and non-phosphorylated fluorescent substrate bands were visualized under UV illumination, excised and quantified by spectrofluorimetry. PKA-independent phosphorylation was determined by addition of protein kinase A inhibitory peptide (PKI) (5 µM) to the reaction 10 min prior to adding the kemptide, whilst maximal PKA activity was determined by inclusion of cAMP (1 µM) in the reaction buffer.

Detection of AKPA79 in the m3-HEK cells

Detection of the presence of AKAP79, a protein which scaffolds both PKA and calcineurin, involved both RT-PCR and immunoprecipitation followed by Western blotting. For RT-PCR, total cellular mRNA was extracted from cells using Qiagen RNeasy according to the manufacturer's instructions. A one-step RT-PCR reaction was performed using Superscript One-Step RT-PCR with Platinum Taq (Invitrogen) on 2 µg template RNA using primers designed to amplify the region between nucleotides 42 and 677 of human AKAP79. The absence of genomic DNA was verified by omitting the RT/Platinum Taq mix and substituting 2 units of Platinum Taq DNA polymerase (Invitrogen). For immunoprecipitation and Western blotting, lysates from the m3-HEK cells were prepared by Dounce homogenization on ice in buffer A (140 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 0.1% Triton X-100, 2 nM okadaic acid, protease inhibitor cocktail and 50 mM Tris-HCl; pH 7.4). The homogenates were centrifuged at 1000 g at 4°C for 5 min and the supernatant transferred to a fresh tube and centrifuged for 30 min at 16 000 g at 4°C. The resulting pellet was rehomogenized in buffer B (500 mM NaCl, 1.5% Triton X-100, 2 nM okadaic acid, protease inhibitor cocktail and 5 mM Tris-HCl; pH 7.4), precleared using Pansorbin cells (Calbiochem) for 30 min at 4°C, and centrifuged at 16 000 g for 2 min at 4°C. The appropriate antibody for immunoprecipitation (AKAP79 or calcineurin, BD Biosciences Clontech) was added to the supernatant and incubated overnight at 4°C. Protein A beads were added to the sample for a further 2 h, after which the immunocomplexes were pelleted, washed, extracted with gel-loading buffer, and boiled for 5 min. Proteins were separated on a 10% SDS-PAGE gel, transferred on to nitrocellulose, and Western blots performed using the appropriate antibody (AKAP79 and calcineurin, BD Biosciences Clontech; RII-subunit of PKA, Biomol). A goat anti-mouse secondary antibody (Bio-Rad) was used. Detection was by chemiluminescence using ‘Western Lightning’ (Pierce).

Statistical analysis

Data are reported as means ± S.E.M. When appropriate, means were compared by unpaired Student's t test. P < 0.05 was considered significant.

	Results

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ARC channels are constitutively phosphorylated

As noted above, the calcineurin-dependent dephosphorylation prevents the activation of ARC channels by arachidonic acid. Consequently, the fact that arachidonic acid is able to activate the ARC channels in otherwise resting cells, suggests that the relevant molecule (possibly the channels themselves) must be, at least partially, in a constitutively phosphorylated state. To identify which kinase(s) might be responsible for this resting phosphorylation, we began by examining various common kinase inhibitors for their effects on the currents through ARC channels activated by exogenous addition of arachidonic acid (8 µM). To maximize any effects of these kinase inhibitors in these initial experiments, the normal amount of ATP (0.5 mM) was omitted from the pipette solution. As can be seen, preincubation with the cell-permeable PKA inhibitory peptide, myristolated PKI (myr-PKI, Biomol, 20 µM for 10–30 min), significantly decreased the macroscopic whole-cell currents through the ARC channels (Fig. 1). Inward currents measured at –80 mV declined from a value of 0.56 ± 0.01 pA pF^–1 (n = 20) in control cells to 0.38 ± 0.03 pA pF^–1 (n = 12) in myr-PKI-treated cells. In contrast, inhibition of protein kinase C (PKC) with calphostin C (0.5 µM for 30 min), protein kinase G with KT5823 (1 µM for 30–60 min) or calmodulin (CaM)-kinase with KN93 (10 µM for 30–60 min) had no effect (mean inward currents at –80 mV were 0.52 ± 0.02 pA pF^–1, n = 10, 0.57 ± 0.06 pA pF^–1, n = 3 or 0.52 ± 0.02 pA pF^–1, n = 6, respectively). Although these data suggest that PKA may be responsible for the resting level of phosphorylation necessary for ARC channel activation, addition of forskolin (10 µM) induced only a relatively small, but statistically significant, increase in inward ARC currents measured at –80 mV to 0.68 ± 0.03 pA pF^–1 (n = 10) (Fig. 1). However, this is entirely consistent with our suggestion that the ARC channels, or a regulator of the channels, are in a phosphorylated state under resting conditions.

View larger version (18K): [in this window] [in a new window]   Figure 1.  Inhibition of PKA-dependent phosphorylation reduces ARC channel activity The effect of forskolin (10 µM) and incubation in the presence of the PKA inhibitor myr-PKI (20 µM for 10–20 min) on the I–V relationship (A) and the mean inward currents measured at –80 mV (B) from cells activated by addition of 8 µM arachidonic acid. For the I–V curves: control cells, n = 5; myr-PKI-treated cells, n = 4 forskolin-treated cells, n = 6; for mean inward currents: control cells, n = 20; myr-PKI-treated cells, n = 12; forskolin-treated cells, n = 10. ***P < 0.001.

Because of this apparently relatively high level of constitutive phosphorylation in the resting state, we argued that it would first be necessary to drive the cells into a dephosphorylated state in order to examine the effects of phosphorylation. As already discussed, we have previously shown that elevated levels of cytosolic Ca²⁺ effectively inhibit the ability of arachidonic acid to activate the ARC channels in a process dependent on the Ca²⁺–calmodulin-dependent phosphatase calcineurin. To induce such a dephosphorylation in intact cells we decided to utilize the endogenous purinergic P2Y receptors of HEK293 cells that are known to be coupled to phospholipase C and the generation of IP₃ (Schachter et al. 1997). We argued that activation of these receptors with a high concentration of ATP would generate a sustained increase in [Ca²⁺]_i and that this, in turn, would result in the activation of calcineurin and the induction of the dephosphorylation-dependent inactivation of the ARC channels, as we have previously demonstrated (Mignen et al. 2003b). Consistent with this, addition of ATP (100 µM) induced a large and prolonged increase in [Ca²⁺]_i in the m3-HEK cells (Fig. 2A). This was shown to be a specific P2Y-dependent effect as preincubation with a combination of the P2Y antagonists PPADS (100 µM) and suramin (50 µM) completely abolished the ATP-induced increase in [Ca²⁺]_i. However, a similar increase in [Ca²⁺]_i induced by the subsequent addition of a high concentration of the muscarinic agonist carbachol (10 µM) was unaffected (Fig. 2A). Subsequent measurement of macroscopic ARC currents in cells that had similarly been exposed to ATP (100 µM for 5 min) revealed that these currents were markedly reduced (Fig. 2B). Mean inward currents measured at –80 mV declined from 0.50 ± 0.02 pA pF^–1 (n = 7) in control cells, to 0.21 ± 0.02 pA pF^–1 (n = 13) in cells pretreated with external ATP. This effect on the currents was abolished in the presence of PPADS and suramin (mean inward current at –80 mV, 0.56 ± 0.05 pA pF^–1, n = 5) confirming that the response involved P2Y receptors (Fig. 2C). Moreover, the effect of external ATP on the measured currents was similarly abolished by preincubation with the calcineurin inhibitor cyclosporin A (1 µM for 15–45 min; mean inward current, 0.45 ± 0.02 pA pF^–1, n = 12; Fig. 2C). This confirms that the observed ATP-dependent inhibition of the ARC channel currents involved the activation of the phosphatase calcineurin, as predicted.

View larger version (14K): [in this window] [in a new window]   Figure 2.  Activation of endogenous P2Y receptors with external ATP inhibits the currents through ARC channels A, representative trace showing that addition of 100 µM ATP (added at the black arrow) induces an increase in [Ca2+]i as measured by intracellularly loaded indo-1. This increase is blocked by the P2Y receptor antagonists PPADS (100 µM) and suramin (50 µM) (grey trace). However, addition of these antagonists does not affect the [Ca2+]i signal induced by addition of the muscarinic agonist carbachol (10 µM) (added at grey arrow). B, the arachidonic acid-activated (8 µM) I–V curves from control cells, and from cells exposed to 100 µM ATP for 5 min (n = 5 in both cases). C, the effects of exposure to ATP (100 µM) on the mean inward arachidonic acid-activated currents measured at –80 mV in control cells, and in cells pretreated with PPADS plus suramin (100 µM and 50 µM, respectively) or cyclosporin A (1 µM for 15–45 min). control cells, n = 7; cells exposed to ATP, n = 13; cells treated with PPADS and suramin, n = 5; cyclosporin-treated cells, n = 12. ***P < 0.001 versus control; NS, not significantly different versus control.

By using these ATP-pretreated (i.e. ‘dephosphorylated’) cells with their reduced ARC channel activity, we were able to examine the effects of phosphorylation on the currents through the ARC channels. In such cells, preincubation with myr-PKI (20 µM) had no significant effect on the macroscopic ARC channel currents (mean inward currents at –80 mV: myr-PKI-treated cells 0.22 ± 0.03 pA pF^–1, n = 4; control, 0.23 ± 0.04 pA pF^–1, n = 6; Fig. 3A and B). This is consistent with the prediction that the treatment with external ATP had induced a calcineurin-mediated dephosphorylated state in the cells leading to the inhibition of the ARC channels. However, addition of forskolin (10 µM) to these ATP-pretreated cells induced a greater than 2.5-fold increase in the inward currents activated by arachidonic acid to reach a value of 0.58 ± 0.04 pA pF^–1 (n = 6) as measured at –80 mV (Fig. 3A and B). Moreover, this effect of forskolin was completely blocked by preincubation with myr-PKI (mean inward current at –80 mV, 0.27 ± 0.02 pA pF^–1, n = 4), confirming that the forskolin-induced increase in current involves a PKA-dependent step. Additional experiments demonstrated that these effects were not unique to the ARC channels when activated by exogenous arachidonic acid as essentially the same responses were seen in cells in which the channels were activated by a low concentration of the muscarinic agonist carbachol (0.5 µM). Once again, pretreatment with ATP (100 µM) markedly reduced the carbachol-activated inward currents as measured at –80 mV from 0.40 ± 0.06 pA pF^–1 (n = 6) to 0.18 ± 0.01 pA pF^–1 (n = 7), whilst addition of forskolin (10 µM) restored the currents to their normal value (0.39 ± 0.05 pA pF^–1, n = 5) (Fig. 3C).

View larger version (15K): [in this window] [in a new window]   Figure 3.  PKA-dependent phosphorylation modulates ARC channel activity following ATP-pretreatment The effect of forskolin (10 µM) and incubation in the presence of the PKA inhibitor myr-PKI (20 µM for 10–20 min) on the I–V curves (A) and the mean inward currents measured at –80 mV (B) from ATP-pretreated cells activated by addition of 8 µM arachidonic acid. For the I–V curves: ATP-treated control cells, n = 7; myr-PKI-treated cells, n = 5; forskolin-treated cells, n = 5; myr-PKI cell treated with forskolin, n = 4. For the mean inward currents: control cells, n = 6; myr-PKI-treated cells both alone and with forskolin, n = 4; forskolin-treated cells, n = 8. C, the effect of ATP-pretreatment (100 µM for 5 min) and the subsequent addition of forskolin (10 µM) on the mean inward currents measured at –80 mV in cells activated by addition of a low concentration (0.5 µM) of carbachol. Control, n = 3; ATP-pretreated cells, n = 7; forskolin-treated cells, n = 5.

Spontaneous phosphorylation and recovery of ARC channel activity

We next examined the ability of this resting PKA activity to recover ARC channel activity following pretreatment with external ATP. To determine this, cells were patch clamped at different time points after pre-exposure to ATP (100 µM) and the activity of the ARC channels determined by measurement of the macroscopic current at –80 mV induced by addition of 8 µM arachidonic acid. The data obtained show that the channel activity fully recovered within 30 min (Fig. 4A). Moreover, this spontaneous recovery of ARC channel activity is markedly inhibited by incubation with myr-PKI, demonstrating that it results from a PKA-dependent process (Fig. 4A). Significantly, in cells in which whole-cell patch-clamp conditions were initiated within the first few minutes after ATP exposure, the rate of the recovery of ARC channel activity during the subsequent maintenance of whole-cell patch-clamp conditions was no slower than that seen in intact cells. A representative trace is shown in Fig. 4B, which illustrates the initial activation of a current equal to approximately –0.25 pA pF^–1 on addition of 8 µM arachidonic acid in a cell that was patch clamped 6 min after exposure to external ATP (100 µM). After a short stabilization period, this current progressively increases over the succeeding 18 min to reach a stable value of approximately –0.50 pA pF^–1. Despite this doubling of the arachidonic acid-activated current, the shape of the I–V relationship remains constant (Fig. 4C). The mean data obtained from this type of experiment are shown in Fig. 4D. Arachidonic acid-activated currents in cells patch clamped 10 min after the beginning of ATP exposure were equal to –0.29 ± 0.02 pA pF^–1 (n = 4). During continued whole-cell patch-clamp conditions, this current progressively increased to reach a stable value of –0.55 ± 0.03 pA pF^–1 over the subsequent 13.75 ± 2.66 min (equivalent to 23.75 ± 2.66 min from the initiation of ATP exposure). These data clearly demonstrate that the rate of the spontaneous PKA-dependent recovery of arachidonic acid-activated current is not inhibited by prolonged intracellular dialysis during maintained whole-cell patch-clamp conditions.

View larger version (18K): [in this window] [in a new window]   Figure 4.  Following dephosphorylation, a spontaneous PKA-dependent rephosphorylation reactivates ARC channels A, cells were exposed to ATP (100 µM) and then subsequently whole-cell patch clamped at the times indicated and the arachidonic acid-activated currents at –80 mV determined (•). The normal mean arachidonic acid-activated currents measured in control cells (, n = 7) and after 5 min of ATP exposure (100 µM; , n = 13) are shown. The mean arachidonic acid-activated current in ATP-treated cells that were continuously incubated in the presence of myr-PKI (20 µM) for the time indicated before whole-cell patch clamping is shown (, n = 9). B, representative trace of an experiment showing the progressive development of the inward arachidonic acid-activated current in a cell maintained under whole-cell patch-clamp conditions. The cell was treated with 100 µM ATP and 6 min later whole-cell patch clamp was initiated. Addition of arachidonic acid (8 µM) at time zero induced a rapid development of the inward current measured at –80 mV (a). This current then gradually increases to reach a stable value after an additional approximately 18 min (b). C, individual I–V curves for the cell shown in (B) taken at points a and b. The data are plotted as a five-point moving average for clarity. D, mean data from experiments similar to that shown in B. Cells (n = 4) were exposed to ATP (100 µM) at time zero. Ten min later whole-cell patch-clamp conditions were initiated, and the arachidonic acid-activated inward currents determined (first filled circle). Whole-cell patch-clamp conditions were maintained until the arachidonic acid-activated currents stabilized, and the mean value of that current and the time at which it was achieved are indicated (second filled circle). The data for intact cells (see A above) are included for comparison.

Our finding that prolonged intracellular dialysis does not interfere with the spontaneous PKA-dependent recovery of ARC channel activity suggests that the phosphorylation process does not involve any freely diffusible moiety, and that activation of the PKA probably occurs close to its site of action. One way in which this is known to be achieved in many cells is by the anchoring of PKA close to its downstream targets via members of the A-kinase anchoring protein (AKAP) family of proteins (Rubin, 1994; Colledge & Scott, 1999; Wong & Scott, 2004). To examine whether such an AKAP might be involved in the phosphorylation-dependent reactivation of ARC channel activity we utilized the Ht31 peptide (Promega), a synthetic peptide that disrupts the binding of PKA to AKAPs thereby disrupting the AKAP-dependent scaffolding of PKA to its targets (Rubin, 1994; Rosenmund et al. 1994; Colledge & Scott, 1999). Pre-treatment of cells with the cell-permeable stearated version of Ht31 (st-Ht31, 40 µM for 10–20 min) significantly reduced the magnitude of the inward ARC currents stimulated by exogenous arachidonic acid (8 µM) and measured at –80 mV, from a mean value of 0.50 ± 0.02 pA pF^–1 (n = 7) to 0.34 ± 0.03 pA pF^–1 (n = 8) (Fig. 5A). Incubation with a control peptide (st-Ht31P) in which a single isoleucine at position 10 in the peptide is substituted with a proline, was without effect on the ARC channel currents (mean inward current at –80 mV, 0.53 ± 0.01 pA pF^–1, n = 6). Similarly, inclusion of Ht31 (30 µM) in the pipette also inhibited the arachidonic acid-induced ARC channel currents (mean inward current at –80 mV, 0.28 ± 0.03 pA pF^–1, n = 5) (Fig. 5B and C). Following incorporation of Ht31 into the cell via the patch pipette, exposure to ATP (100 µM) produced a further modest reduction in the arachidonic acid-activated inward currents to a value of 0.20 ± 0.02 pA pF^–1 (n = 10). Of more importance, such inclusion of Ht31 in the cell markedly inhibited the subsequent forskolin-mediated recovery of normal ARC channel activity with inward currents at –80 mV only reaching a value of 0.31 ± 0.01 pA pF^–1 (n = 6) (Fig. 5B and C), compared to a value of 0.58 ± 0.04 pA pF^–1 (n = 6) in the absence of Ht31 (see Fig. 3B). Moreover, pretreatment with st-Ht31 (40 µM) also blocked the spontaneous recovery of ARC currents following exposure to external ATP. Thus, in experiments similar to those illustrated in Fig. 4C, inward currents at –80 mV measured 40 min after initial exposure to ATP reached a value of only 0.31 ± 0.02 pA pF^–1 (n = 5) in the st-Ht31-treated cells, compared to a value of 0.51 ± 0.01 pA pF^–1 (n = 6) in control cells. As such, the st-Ht31-treated cells behaved very similarly to those treated with myr-PKI (mean inward current at 40 min after ATP exposure, 0.28 ± 0.02 pA pF^–1, n = 9, see Fig. 4C). We conclude that disrupting the binding of PKA to an AKAP by Ht31 both inhibits the ability of arachidonic acid to effectively activate resting ARC channel activity, and blocks the PKA-dependent recovery of activity following a calcineurin-mediated dephosphorylation.

View larger version (13K): [in this window] [in a new window]   Figure 5.  The effects of phosphorylation on ARC channel activity involve an AKAP A, mean inward arachidonic acid-activated currents measured at –80 mV in control cells (n = 7), in cells treated with st-Ht31 (n = 8) and in cells treated with the control peptide st-Ht31P (n = 6). ***P < 0.001 versus control. Mean I–V curves (B) and mean inward currents measured at –80 mV (C) for the arachidonic acid-activated current in cells patch clamped with a pipette solution containing Ht31 (30 µM), in such cells subsequently exposed to ATP (100 µM for 5 min) and following addition of forskolin (10 µM). For the I–V curves: Ht31 cells, n = 4; ATP-treated cells, n = 6; addition of forskolin, n = 3. For the mean inward currents: Ht31 cells, n = 5; ATP-treated cells, n = 10; addition of forskolin, n = 6. *P < 0.05 versus Ht31 control; NS, not significant versus Ht31 control.

View larger version (56K): [in this window] [in a new window]   Figure 6.  AKAP79 is present in the m3-HEK cells A, results from a one-step RT-PCR reaction performed on total RNA from cells using primers specific for AKAP79. The predicted RT-PCR product was 635 base pairs. The negative control was the same reaction carried out in the absence of reverse transcriptase. B, representative Western blot showing the immunoreaction of a crude cell membrane preparation immunoprecipitated with an antibody to AKAP79 and blotted with the same antibody. For comparison, a whole-cell lysate blotted with the AKAP79 antibody is shown. The immunoreaction of a crude cell membrane preparation immunoprecipitated with an antibody to calcineurin and then blotted with an AKAP79 antibody (C) or with an antibody to the RIIß subunit of PKA (D).

	Discussion

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The data obtained have shown that the activation of the ARC channels is specifically dependent on a PKA-mediated phosphorylation. We could find no evidence for the involvement of PKC, protein kinase G, or CaM-kinase in this phosphorylation. Activation of PKA reverses the inhibitory effect of the calcineurin-dependent dephosphorylation on the ARC channels, resulting in a restoration of the ability of both arachidonic acid and low concentrations of the muscarinic agonist carbachol to activate the channels. Consistent with the notion that the phosphorylation status in resting cells must be relatively high, we found that such cells displayed a PKA activity that was approximately 30% of the maximal activity. The basis for this level of activity is not entirely clear, but it should be remembered that we were using a cultured cell line and the presence of, for example, various growth factors in the serum in which the cells are maintained, may have resulted in some activation of adenylyl cyclases. Clearly, the balance between the opposing effects of phosphorylation versus dephosphorylation on the channel activity are weighted strongly towards phosphorylation, as evidenced by the limited effect of forskolin on ARC channel currents in the resting cells. However, it is quite possible that this balance may be set at a different level in different cell types, or in the same cells under different conditions.

Whatever the underlying cause of this resting phosphorylation, our data have demonstrated that following induction of a calcineurin-dependent dephosphorylation, the relatively high resting PKA activity results in the spontaneous rephosphorylation and restoration of ARC channel activity. Significantly, this rephosphorylation is unaffected by prolonged dialysis of the intracellular environment during whole-cell patch clamp, suggesting some form of structural association or coupling between the PKA and its target. Our data indicate that the basis for this is the involvement of an AKAP in the PKA-dependent phosphorylation effects on ARC channel activity. AKAPs are a diverse group of scaffolding proteins that share the property of possessing an amphipathic helix domain that binds the type II regulatory subunit (RII) dimer of the PKA holoenzyme, together with specialized anchoring domains that scaffold the AKAP–PKA complex to specific intracellular sites close to their substrates (Rubin, 1994; Colledge & Scott, 1999; Wong & Scott, 2004). As such, they are a well-established means of attaining high specificity in phosphorylation events in the face of the broad substrate specificity of PKA. In the data presented here, the Ht31 peptide, which specifically disrupts the interactions between AKAPs and PKA, inhibited the ability of ARC channels to be activated by arachidonic acid, and blocked the PKA-dependent restoration of ARC channel activity following a calcineurin-induced dephosphorylation.

It is interesting that both the effects of PKA-dependent phosphorylation reported in this study, and the calcineurin-dependent dephosphorylation effects on ARC channel activity previously reported (Mignen et al. 2003b), are resistant to conditions of whole-cell dialysis. Because of this, we suggest that the relevant AKAP involved may be AKAP79. AKAP79 is unique in that it scaffolds both PKA and calcineurin, along with PKC, and is targeted to membranes via an interaction with acidic phospholipids, particularly phosphatidylinositol 4,5-bisphosphate (PIP2) (Dell'Acqua et al. 1998). Compared to neuronal tissues, where AKAP79 is abundant (Carr et al. 1992; Klauck et al. 1996), expression of endogenous AKAP79 is relatively low in the m3-HEK cells used here. In neuronal tissues, AKAP79 plays a role in the regulation of a variety of synaptic proteins including postsynaptic excitatory neurotransmitter receptors. In particular, AKAP79 has been shown to be critical in the regulation of the phosphorylation–dephosphorylation balance that determines the overall activity of AMPA-type glutamate receptors. In much the same way as we have illustrated in this study, Ht31-induced disruption of PKA binding to AKAP79 interferes with the phosphorylation of the AMPA channel and favours the down-regulation of channel activity by calcineurin (Tavalin et al. 2002). AKAP79 has also been associated with the regulation of other ion channels including the inwardly rectifying potassium channel Kir2.1 (Dart & Leyland, 2001), and both the skeletal and cardiac forms of L-type Ca²⁺ channels (Johnson et al. 1994, 1997; Gao et al. 1997).

Although we have shown that phosphorylation profoundly influences the activity of the ARC channels, exactly how this change in activity is achieved is unclear. Importantly, we do not know whether it is the channels themselves that are being phosphorylated, or some distinct regulator of channel activity. Resolving this question is problematic as the molecular identity of the ARC channels is unknown. Either way, we can be certain that the effect of phosphorylation is not to actually open the ARC channels – only arachidonic acid does this. Instead, our data indicate that either phosphorylation is required for the channels to be opened by arachidonic acid or, once opened by arachidonic acid, phosphorylation results in a markedly higher overall activity of the channels, presumably via increases in open probability and/or single channel conductance. The latter possibility is, perhaps, supported by the fact that a residual inward current of approximately 0.2 pA pF^–1 at –80 mV is consistently seen even after all the treatments to inhibit phosphorylation (e.g. PKI or Ht31). Interestingly, a similar level of residual activity was observed after maximal activation of calcineurin by elevated cytosolic [Ca²⁺] in our previous report (Mignen et al. 2003b). Such a residual activity may reflect the intrinsic channel activity in the completely dephosphorylated state. However, it also remains possible that none of these treatments is capable of achieving a complete dephosphorylation. In any event, the effects of the opposing actions of PKA-dependent phosphorylation and calcineurin-dependent dephosphorylation are probably best described as exerting a modulating effect on overall ARC channel activity, rather than regulating the channel activity per se.

This clear role of phosphorylation and dephosphorylation in the modulation of the ARC channels stands in contrast to the situation with store-operated pathways for Ca²⁺ entry. Although several studies have indicated an involvement of various protein kinases in the apparent regulation of Ca²⁺ entry via such pathways, relatively few of these report direct measurement of the conductances involved. Because of this, it is uncertain whether the observed effects involve contributions from other components of the overall [Ca²⁺]_i homeostasis system including membrane potential, phospholipase C activity, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and plasma membrane calcium (PMCA) pumps and mitochondrial function, rather than any direct effect on channel activity per se. Using a variety of approaches including kinase inhibitors, blockade of ATP hydrolysis and reductions in internal Mg²⁺ concentrations, Bodding (2001) could find no evidence for any role of protein kinases in the activation of CRAC currents in RBL cells. However, Parekh & Penner (1995) have shown that, whilst cAMP and cGMP were without affect, stimulation of PKC enhanced the inactivation of CRAC currents in these cells. More recently, Shi et al. (2004) have demonstrated a complex [Ca²⁺]_i-dependent regulation of the TRPC6 and TRPC7 channels that appear to underlie the agonist-activated entry of Ca²⁺ in portal vein myocytes. These authors found that activation of TRPC6 by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), required a ‘priming’ calmodulin-dependent phosphorylation that appeared to involve CaM-kinase. In contrast, inhibition of calmodulin increased TRPC7 activity. At high levels of [Ca²⁺]_i, both channels were inhibited via a process that was dependent on PKC activity. It is interesting, at least for TRPC6, that the overall regulation appears to show many parallels to that reported here for the ARC channels. In both cases, activation by the relevant endogenous messenger (diacylglycerol for TRPC6 and arachidonic acid for ARC) requires a prior phosphorylation by the appropriate kinase (CaM-kinase for TRPC6 and protein kinase A for ARC). At high levels of [Ca²⁺]_i, channel activity is inhibited (by a PKC-dependent phosphorylation for TRPC6 and by a calcineurin-dependent dephosphorylation for ARC).

Clearly, the data presented here illustrate that the overall activity of ARC channels is subject to a tight regulation that reflects a balance between the modulatory influences of PKA-dependent phosphorylation and calcineurin-dependent dephosphorylation. Moreover, the specificity of this modulation is greatly enhanced by the involvement of an AKAP, possibly AKAP79. Given the demonstrated role of the ARC channels in modulating agonist-activated oscillatory [Ca²⁺]_i signals in various non-excitable cell types, factors that alter the existing balance between these two opposing influences will profoundly affect the ability of the ARC channels to conduct Ca²⁺ ions into the cell following activation by appropriate agonists, with obvious consequences for the overall [Ca²⁺]_i signals produced.

	References

Top Abstract Introduction Methods Results Discussion References

 
Bodding M (2001). Activation of store-operated Ca²⁺ entry in RBL cells without the contribution of protein kinases. Naunyn Schmiedebergs Arch Pharmacol 363, 633–638.[CrossRef][Medline]

Broad LM, Cannon TR & Taylor CW (1999). A non-capacitative pathway activated by arachidonic acid is the major Ca²⁺ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin. J Physiol 517, 121–134.[Abstract/Free Full Text]

Carr DW, Stofko-Hahn RE, Fraser ID, Cone RD & Scott JD (1992). Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. J Biol Chem 267, 16816–16823.[Abstract/Free Full Text]

Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM & Scott JD (1995). Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267, 108–111.[Abstract/Free Full Text]

Colledge M & Scott JD (1999). AKAPs: from structure to function. Trends Cell Biol 9, 216–221.[CrossRef][Medline]

Dart C & Leyland ML (2001). Targeting of an A kinase anchoring protein, AKAP79, to an inwardly rectifying potassium channel, Kir2.1. J Biol Chem 276, 20499–20505.[Abstract/Free Full Text]

Dell'Acqua ML, Faux MC, Thorburn J, Thorburn A & Scott JD (1998). Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate. EMBO J 17, 2246–2260.[CrossRef][Medline]

Gao T, Yatani A, Dell'Acqua ML, Sako H, Green SA, Dascal N, Scott JD & Hosey MM (1997). cAMP-dependent regulation of cardiac L-type Ca²⁺ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19, 185–196.[CrossRef][Medline]

Hoth M & Penner R (1992). Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355, 353–356.[CrossRef][Medline]

Hoth M & Penner R (1993). Calcium release-activated calcium current in rat mast cells. J Physiol 465, 359–386.[Abstract/Free Full Text]

Johnson BD, Brousal JP, Peterson BZ, Gallombardo PA, Hockerman GH, Lai Y, Scheuer T & Catterall WA (1997). Modulation of the cloned skeletal muscle L-type Ca²⁺ channel by anchored cAMP-dependent protein kinase. J Neurosci 17, 1243–1255.[Abstract/Free Full Text]

Johnson BD, Scheuer T & Catterall WA (1994). Voltage-dependent potentiation of L-type Ca²⁺ channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 91, 11492–11496.[Abstract/Free Full Text]

Karege F, Schwald M, Lambercy C, Murama JJ, Cisse M & Malafosse A (2001). A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex. Brain Res 903, 86–93.[CrossRef][Medline]

Klauck TM, Faux MC, Labudda K, Langeberg LK, Jaken S & Scott JD (1996). Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271, 1589–1592.[Abstract]

Mignen O & Shuttleworth TJ (2000). I(ARC), a novel arachidonate-regulated, noncapacitative Ca²⁺ entry channel. J Biol Chem 275, 9114–9119.[Abstract/Free Full Text]

Mignen O & Shuttleworth TJ (2001). Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca²⁺ channels in HEK293 cells. Comparison with endogenous store-operated channels. J Biol Chem 276, 21365–21374.[Abstract/Free Full Text]

Mignen O, Thompson JL & Shuttleworth TJ (2001). Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca²⁺ entry pathways. J Biol Chem 276, 35676–35683.[Abstract/Free Full Text]

Mignen O, Thompson JL & Shuttleworth TJ (2003a). Ca²⁺ selectivity and fatty acid specificity of the noncapacitative, arachidonate-regulated Ca²⁺ (ARC) channels. J Biol Chem 278, 10174–10181.[Abstract/Free Full Text]

Mignen O, Thompson JL & Shuttleworth TJ (2003b). Calcineurin directs the reciprocal regulation of calcium entry pathways in nonexcitable cells. J Biol Chem 278, 40088–40096.[Abstract/Free Full Text]

Mignen O, Thompson JL, Yule DI & Shuttleworth TJ (2005). Agonist activation of arachidonate-regulated Ca²⁺-selective (ARC) channels in parotid and pancreatic acinar cells. J Physiol 564, 791–801.[Abstract/Free Full Text]

Munaron L, Antoniotti S, Distasi C & Lovisolo D (1997). Arachidonic acid mediates calcium influx induced by basic fibroblast growth factor in Balb-c 3T3 fibroblasts. Cell Calcium 22, 179–188.[CrossRef][Medline]

Parekh AB & Penner R (1995). Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells. Proc Natl Acad Sci U S A 92, 7907–7911.[Abstract/Free Full Text]

Parekh AB & Penner R (1997). Store depletion and calcium influx. Physiol Rev 77, 901–930.[Abstract/Free Full Text]

Parekh AB & Putney JW Jr (2005). Store operated calcium channels. Physiol Rev 85, 757–810.[Abstract/Free Full Text]

Park MK, Petersen OH & Tepikin AV (2000). The endoplasmic reticulum as one continuous Ca²⁺ pool: visualization of rapid Ca²⁺ movements and equilibration. EMBO J 19, 5729–5739.[CrossRef][Medline]

Putney JW Jr (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1–12.[Medline]

Putney JW Jr (1990). Capacitative calcium entry revisited. Cell Calcium 11, 611–624.[CrossRef][Medline]

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S et al. (2005). STIM1, an essential and conserved component of store-operated Ca²⁺ channel function. J Cell Biol 169, 435–445.[Abstract/Free Full Text]

Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD & Westbrook GL (1994). Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 368, 853–856.[CrossRef][Medline]

Rubin CS (1994). A kinase anchor proteins and the intracellular targeting of signals carried by cyclic AMP. Biochim Biophys Acta 1224, 467–479.[Medline]

Schachter JB, Sromek SM, Nicholas RA & Harden TK (1997). HEK293 human embryonic kidney cells endogenously express the P2Y1 and P2Y2 receptors. Neuropharmacology 36, 1181–1187.[CrossRef][Medline]

Shi J, Mori E, Mori Y, Mori M, Li J, Ito Y & Inouue R (2004). Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells. J Physiol 561, 415–432.[Abstract/Free Full Text]

Shuttleworth TJ (1996). Arachidonic acid activates the noncapacitative entry of Ca²⁺ during [Ca²⁺]_i oscillations. J Biol Chem 271, 21720–21725.[Abstract/Free Full Text]

Shuttleworth TJ (1999). What drives calcium entry during [Ca²⁺]_i oscillations? – challenging the capacitative model. Cell Calcium 25, 237–246.[CrossRef][Medline]

Shuttleworth TJ & Thompson JL (1998). Muscarinic receptor activation of arachidonate-mediated Ca²⁺ entry in HEK293 cells is independent of phospholipase C. J Biol Chem 273, 32636–32643.[Abstract/Free Full Text]

Tavalin SJ, Colledge M, Hell JW, Langeberg LK, Huganir RL & Scott JD (2002). Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long-term depression. J Neurosci 22, 3044–3051.[Abstract/Free Full Text]

Wong W & Scott JD (2004). AKAP signalling complexes: focal points in space and time. Nat Rev Mol Cell Biol 5, 959–970.[CrossRef][Medline]

Zweifach A & Lewis RS (1993). Mitogen-regulated Ca²⁺ current of T lymphocytes is activated by depletion of intracellular Ca²⁺ stores. Proc Natl Acad Sci U S A 90, 6295–6299.[Abstract/Free Full Text]

	Acknowledgements

 
We thank Pauline Leakey for excellent technical assistance, and Dr David I. Yule for advice and helpful comments. This work was supported by a grant from the National Institutes of Health (GM040457) to T.J.S. O.M. was supported in part by funds from the Alfred and Eleanor Wedd Endowment.

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