Arachidonate-regulated Ca2+-selective (ARC) channel activity is modulated by phosphorylation and involves an A-kinase anchoring protein - PubMed (original) (raw)

Arachidonate-regulated Ca2+-selective (ARC) channel activity is modulated by phosphorylation and involves an A-kinase anchoring protein

Olivier Mignen et al. J Physiol. 2005.

Abstract

In many non-excitable cells, the predominant mode of agonist-activated Ca(2+) entry switches from the arachidonic acid-regulated Ca(2+) (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.

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Figures

Figure 1

Figure 1. Inhibition of PKA-dependent phosphorylation reduces ARC channel activity

The effect of forskolin (10 μ

m

) and incubation in the presence of the PKA inhibitor myr-PKI (20 μ

m

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 μ

m

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.

Figure 2

Figure 2. Activation of endogenous P2Y receptors with external ATP inhibits the currents through ARC channels

A, representative trace showing that addition of 100 μ

m

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 μ

m

) and suramin (50 μ

m

) (grey trace). However, addition of these antagonists does not affect the [Ca2+]i signal induced by addition of the muscarinic agonist carbachol (10 μ

m

) (added at grey arrow). B, the arachidonic acid-activated (8 μ

m

) I–V curves from control cells, and from cells exposed to 100 μ

m

ATP for 5 min (n = 5 in both cases). C, the effects of exposure to ATP (100 μ

m

) on the mean inward arachidonic acid-activated currents measured at −80 mV in control cells, and in cells pretreated with PPADS plus suramin (100 μ

m

and 50 μ

m

, respectively) or cyclosporin A (1 μ

m

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.

Figure 3

Figure 3. PKA-dependent phosphorylation modulates ARC channel activity following ATP-pretreatment

The effect of forskolin (10 μ

m

) and incubation in the presence of the PKA inhibitor myr-PKI (20 μ

m

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 μ

m

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 μ

m

for 5 min) and the subsequent addition of forskolin (10 μ

m

) on the mean inward currents measured at −80 mV in cells activated by addition of a low concentration (0.5 μ

m

) of carbachol. Control, n = 3; ATP-pretreated cells, n = 7; forskolin-treated cells, n = 5.

Figure 4

Figure 4. Following dephosphorylation, a spontaneous PKA-dependent rephosphorylation reactivates ARC channels

A, cells were exposed to ATP (100 μ

m

) 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 μ

m

; ○, 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 μ

m

) 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 μ

m

ATP and 6 min later whole-cell patch clamp was initiated. Addition of arachidonic acid (8 μ

m

) 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 μ

m

) 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.

Figure 5

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 μ

m

), in such cells subsequently exposed to ATP (100 μ

m

for 5 min) and following addition of forskolin (10 μ

m

). 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.

Figure 6

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 RIIβ subunit of PKA (D).

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