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ARTICLE

Protein Kinase C Disrupts Cannabinoid Actions by Phosphorylation of the CB1 Cannabinoid Receptor

D. E. Garcia, S. Brown, B. Hille and K. Mackie
Journal of Neuroscience 15 April 1998, 18 (8) 2834-2841; DOI: https://doi.org/10.1523/JNEUROSCI.18-08-02834.1998
D. E. Garcia
1Departments of Physiology and Biophysics and
3Departamento de Fisiologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, CP 04510 Mexico DF, Mexico
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S. Brown
2Anesthesiology, University of Washington, Seattle, Washington 98195, and
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B. Hille
1Departments of Physiology and Biophysics and
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K. Mackie
1Departments of Physiology and Biophysics and
2Anesthesiology, University of Washington, Seattle, Washington 98195, and
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    Fig. 1.

    Stimulation of protein kinase C attenuates activation of Kir current by cannabinoids but not by somatostatin. Cells were exposed to 100 nm 4α-phorbol or 100 nm PMA for 10 min at room temperature and transferred to the recording chamber, and potassium currents were recorded. Kir current was defined as the component of the current inhibited by 1 mm Ba2+. A, Left, Mean current activated by a 250 msec hyperpolarization to −100 mV from a holding potential of −45 mV is plotted versus time in a cell preincubated with 100 nm 4α-phorbol for 10 min. Bath application of 100 nm of the cannabimimetic WIN 55,212-2 (WIN) for the indicated time greatly increases the component of the current sensitive to 1 mmBa2+. The increase in inwardly rectifying potassium current is defined as indicated to the left(Kir current). Right, Individual current traces taken at the times indicated. B, Left, In a cell pretreated with 100 nm PMA (see Results), 100 nm WIN 55,212-2 slightly increases the barium-sensitive current. Right, Individual current traces taken at the times indicated. C, Bath application of 10 nm somatostatin (SOM) to a cell pretreated with 100 nm 4α-phorbol for 10 min elicits a large increase in the barium-sensitive inward current.D, In a cell pretreated with 100 nm PMA, 10 nm somatostatin still results in a large increase in the barium-sensitive current.

  • Fig. 2.
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    Fig. 2.

    Protein kinase C stimulation markedly reduces the activation of Kir current by cannabinoids but not by somatostatin. Summary of the experiments illustrated in Figure 1. The Kir current activated by 100 nm WIN 55,212-2 (WIN) and 10 nm somatostatin (SOM) was normalized to cell capacitance and compared for the following conditions: 100 nm 4α-phorbol, 100 nm PMA (PMA), and 1 μmstaurosporine (STAU). The number of cells tested for each condition is in parentheses.

  • Fig. 3.
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    Fig. 3.

    Activation of protein kinase C greatly reduces inhibition of P/Q-type calcium currents by cannabinoids but not by somatostatin. Cells were exposed to 100 nm 4α-phorbol or 100 nm PMA for 10 min at room temperature and transferred to the recording chamber, and barium currents flowing through calcium channels were recorded. A, Left, Mean current activated by a 25 msec depolarization to 0 mV from a holding potential of −80 mV is plotted against time in a cell preincubated for 10 min with 100 nm 4α-phorbol. Bath application of 100 nm of the cannabimimetic WIN 55,212-2 (WIN) for the indicated time inhibits ∼40% of the barium current [defined as the fraction of the current sensitive to 100 μmCdCl2 (Cd)]. Right,Individual current traces taken at the times indicated. B, Left, In a cell pretreated with 100 nm PMA (see text), 100 nm WIN 55,212-2 has a minimal effect on the barium current. Right, Individual current traces taken at the times indicated. C, Bath application of 10 nm somatostatin (SOM) to a cell pretreated with 100 nm 4α-phorbol for 10 min inhibits ∼50% of the barium current. D, In a cell pretreated with 100 nm PMA, 10 nm somatostatin still inhibits a significant fraction of the barium current.

  • Fig. 4.
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    Fig. 4.

    Protein kinase C stimulation greatly reduces the inhibition of barium current by cannabinoids but not by somatostatin. Summary of the experiments illustrated in Figure 3. The barium current activated by 100 nm WIN 55,212-2 (WIN) and 10 nm somatostatin (SOM) was compared for the following conditions: 100 nm 4α-phorbol, 100 nm PMA (PMA), 1 μm staurosporine (STAU), 100 nm bisindolylmaleimide I (BIS), and 100 nm bisindolylmaleimide V (IBIS). The number of cells tested for each condition is indicated in parentheses.

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    Fig. 5.

    Protein kinase C phosphorylates GST-IC3 fusion proteins on the serine corresponding to serine 317 in rat CB1. Fusion proteins were phosphorylated by purified rat brain PKC (50 ng) for 30 min and separated by SDS-PAGE. Phosphorylated proteins were identified by autoradiography of the gel. Wild-type third intracellular loop, S304A, and S323A fusion proteins were phosphorylated to a similar extent, whereas the S317A fusion protein was minimally phosphorylated.Arrowhead, GST-IC3 fusion protein.

  • Fig. 6.
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    Fig. 6.

    The CB1-S317A mutant receptor activation of Kir current is not attenuated by protein kinase C.A, Time course of Kir current activation by 100 nm WIN 55,212-2 in a CB1-S317A cell after preincubation with 100 nm 4α-phorbol (Con).Inset, Current traces from the indicated times.B, Time course of Kir current activation by 100 nm WIN 55,212-2 after preincubation with 100 nm PMA to stimulate protein kinase C. Inset, Current traces from the indicated times. C, Comparison of Kir current activation in cells expressing CB1 or CB1-S317A. The number of cells for each condition is inparentheses.

  • Fig. 7.
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    Fig. 7.

    The CB1-S317A mutant receptor inhibition of calcium channels is resistant to PKC. A, Time course of barium current inhibition by 100 nm WIN 55,212-2 in a CB1-S317A cell after preincubation with 100 nm 4α-phorbol (Con). Inset, Current traces from the indicated times. B, Time course of barium current inhibition by 100 nm WIN 55,212-2 after preincubation with 100 nm PMA to stimulate protein kinase C.Inset, Current traces from the indicated times.C, Comparison of barium current inhibition (Inh) in cells expressing CB1 or CB1-S317A. The small residual PMA effect in the S317A mutant was abolished by ω-conotoxin GVIA (GVIA). The number of cells for each condition is in parentheses.

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The Journal of Neuroscience: 18 (8)
Journal of Neuroscience
Vol. 18, Issue 8
15 Apr 1998
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Protein Kinase C Disrupts Cannabinoid Actions by Phosphorylation of the CB1 Cannabinoid Receptor
D. E. Garcia, S. Brown, B. Hille, K. Mackie
Journal of Neuroscience 15 April 1998, 18 (8) 2834-2841; DOI: 10.1523/JNEUROSCI.18-08-02834.1998

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Protein Kinase C Disrupts Cannabinoid Actions by Phosphorylation of the CB1 Cannabinoid Receptor
D. E. Garcia, S. Brown, B. Hille, K. Mackie
Journal of Neuroscience 15 April 1998, 18 (8) 2834-2841; DOI: 10.1523/JNEUROSCI.18-08-02834.1998
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Keywords

  • cannabinoid
  • G-protein-coupled receptor
  • calcium channel
  • inwardly rectifying potassium channel
  • protein kinase C
  • phosphorylation

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