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. 2009 Dec 15;587(Pt 24):5819-30.
doi: 10.1113/jphysiol.2009.180372.

AMP-activated protein kinase inhibits TREK channels

Orsolya Kréneisz  1 , Justin P Benoit, Douglas A Bayliss, Daniel K Mulkey
Affiliations

AMP-activated protein kinase inhibits TREK channels

Orsolya Kréneisz et al. J Physiol. .

Abstract

AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by conditions that increase the AMP : ATP ratio. In carotid body glomus cells, AMPK is thought to link changes in arterial O(2) with activation of glomus cells by inhibition of unidentified background K(+) channels. Modulation by AMPK of individual background K(+) channels has not been described. Here, we characterize effects of activated AMPK on recombinant TASK-1, TASK-3, TREK-1 and TREK-2 background K(+) channels expressed in HEK293 cells. We found that TREK-1 and TREK-2 channels but not TASK-1 or TASK-3 channels are inhibited by AMPK. AMPK-mediated inhibition of TREK involves key serine residues in the C-terminus that are also known to be important for PKA and PKC channel modulation; inhibition of TREK-1 requires Ser-300 and Ser-333 and inhibition of TREK-2 requires Ser-326 and Ser-359. Metabolic inhibition by sodium azide can also inhibit both TREK and TASK channels. The effects of azide on TREK occlude subsequent channel inhibition by AMPK and are attenuated by expression of a dominant negative catalytic subunit of AMPK (dnAMPK), suggesting that metabolic stress modulates TREK channels by an AMPK mechanism. By contrast, inhibition of TASK channels by azide was unaffected by expression of dnAMPK, suggesting an AMPK-independent mechanism. In addition, prolonged exposure (6-7 min) to hypoxia ( = 11 +/- 1 mmHg) inhibits TREK channels and this response was blocked by expression of dnAMPK. Our results identify a novel modulation of TREK channels by AMPK and indicate that select residues in the C-terminus of TREK are points of convergence for multiple signalling cascades including AMPK, PKA and PKC. To the extent that carotid body O(2) sensitivity is dependent on AMPK, our finding that TREK-1 and TREK-2 channels are inhibited by AMPK suggests that TREK channels may represent the AMPK-inhibited background K(+) channels that mediate activation of glomus cells by hypoxia.

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Figures

Figure 1
Figure 1. TASK-1 and TASK-3 channels are not modulated by activated AMPK
Whole cell currents were recorded from HEK 293 cells following transient transfection with TASK-1 or TASK-3. Traces of conductance (measured as a linear slope between −30 and +5 mV) as a function of time show characteristic responses of TASK-1 (A), TASK-3 (B) and heteromeric TASK channels (C) to changes in bath pH; acidification from pH 7.3 to pH 5.9 inhibits TASK channels whereas alkalization to pH 8.4 activates TASK channels. At pH 7.3, 3–5 min applications of AICAR (2 mm) had no discernable effect on TASK channel conductance. Insets, current–voltage (I–V) relationships characteristic of TASK-1 and TASK-3 were obtained from depolarizing voltage ramp commands (−130 to +20 mV at 0.2 V s−1) during control conditions (a) and exposure to AICAR (b). Bar graphs show averaged conductance (n= 5) of the relevant TASK channels at pH 7.3 in the absence and presence of AICAR. These results indicate that AICAR-mediated activation of AMPK does not affect activity of recombinant TASK channels.
Figure 2
Figure 2. TREK channels are inhibited by AMPK
A, trace of whole cell conductance shows a typical time course for inhibition of TREK-1 by bath application of AICAR (2 mm). Inset, I–V plots of whole cell TREK-1 current evoked by ramp commands (−130 to +20 mV at 0.2 V s−1) during control (a) and in the presence of AICAR (b). Bar graph of averaged data (n= 13) shows that 2 min exposure to AICAR decreases TREK-1 conductance. B, representative conductance trace shows the time course of TREK-2 inhibition by AICAR (2 mm). Inset, I–V plots of whole cell TREK-2 ramp currents during control (a) and in the presence of AICAR (b). Bar graph of averaged data (n= 7) shows that 2 min exposure to AICAR decreases TREK-2 conductance. The effect of AICAR on TREK-1 and TREK-2 was reversible in the majority of cells tested.
Figure 3
Figure 3. Select serine residues in the C-terminus of TREK-1 and TREK-2 are required for AMPK-modulation
A, representative conductance trace shows that Ala substitution at Ser-333 (S333A) blocks AICAR-mediated inhibition of TREK-1. Inset, I–V plot of the S333A mutant TREK-1 current during control (a) and exposure to AICAR (b). Averaged data (n= 6) shows that 2 min exposure to AICAR had no effect on S333A mutant TREK-1 conductance. B, conductance trace shows that Ala substitution at Ser-300 (S300A) also blocked AICAR-mediated inhibition of TREK-1. Inset, I–V plot of the S300A mutant TREK-1 current during control (a) and exposure to AICAR (b). Averaged data (n= 9) show that 2 min exposure to AICAR had no effect on S300A mutant TREK-1 conductance. C, representative conductance trace show that Ala substitutions at Ser-326 and Ser-359 (S326A/S359A) of TREK-2 channels blocks AICAR-mediated inhibition of TREK-2. Inset, I–V plot of the S326A/S359A mutant TREK-2 current during control (a) and exposure to AICAR (b). Averaged data (n= 5) show that 3 min exposure to AICAR had no effect on S326A/S359A mutant TREK-1 conductance.
Figure 4
Figure 4. Inhibition of TREK channels by sodium azide require AMPK phosphorylation sites
Whole cell currents were recorded from HEK cells transfected with wild-type or AICAR-resistant mutant TREK-1 (S333A, S300A) or TREK-2 (S326A/S359A) channels during exposure to metabolic stress in the form of sodium azide. Aa, trace of wild-type TREK-1 conductance and bath temperature show that heating from 24°C to 30°C increases TREK-1 activity. At 30°C bath application of sodium azide (10 μm) caused a rapid and reversible inhibition of TREK-1 conductance. Inset, I–V plots of TREK-1 current obtained at 30°C during control conditions (a) and in the presence of azide (b). Ab and Ac, bath temperature (top) and whole cell conductance from S300A (Ab) and S333A (Ac) mutant TREK-1 channels show that heating from 24°C to 30°C increases whole cell conductance of cells expressing either mutant channel. At 30°C bath application of sodium azide (10 μm) had no effect on S300A (Ab) or S333A (Ac) conductance. Inset, I–V relationships of S300A and S333A mutant TREK-1 channels obtained at 30°C during control conditions (a) and in the presence of azide (b). Ba, trace of wild-type TREK-2 conductance and bath temperature show that heating from 24°C to 30°C increases TREK-2 activity. At 30°C exposure to sodium azide (10 μm) caused a rapid and reversible inhibition of TREK-2 conductance. Inset, I–V plots of TREK-2 current obtained at 30°C during control conditions (a) and in the presence of azide (b). Bb, bath temperature (top) and conductance trace from a cell expressing the TREK-2 S326A/S359A mutant channel (bottom) show that the mutant channel is activated by warming but exposure to sodium azide (10 μm) at 30°C had no effect on conductance. Inset, I–V relationships of TREK-2 S326A/S359A mutant obtained at 30°C during control conditions (a) and in the presence of azide (b). C, average data summarize the effects of sodium azide on wild-type (n= 6) and mutant TREK-1 channels (S333A, n= 6; S300A, n= 3) as well as wild-type (n= 3) and S326A/S359A mutant (n= 4) TREK-2 channels.
Figure 5
Figure 5. Expression of dnAMPK blocks the effects of sodium azide on TREK channels
A, trace of wild-type TREK-1 conductance in the presence of dnAMPK and bath temperature show that heating from 24°C to 30°C increases TREK-1 activity. At 30°C bath application of sodium azide (10 μm) had no discernable effect on channel conductance. Inset, I–V plots of TREK-1 current in the presence of dnAMP obtained at 30°C during control conditions (a) and in the presence of azide (b). Bar graph of average data (n= 4) shows the percentage inhibition of TREK-1 by azide alone and in the presence of dnAMPK. B, trace of wild-type TREK-2 conductance in the presence of dnAMPK and bath temperature show that heating from 24°C to 30°C increases TREK-1 activity. At 30°C bath application of sodium azide (10 μm) had no discernable effect on channel conductance. Inset, I–V plots of TREK-2 current in the presence of dnAMP obtained at 30°C during control conditions (a) and in the presence of azide (b). Bar graph of average data (n= 3) shows the percentage inhibition of TREK-1 by azide alone and in the presence of dnAMPK.
Figure 6
Figure 6. Sodium azide occludes AICAR-mediated inhibition of TREK channels
A, traces of wild-type TREK-1 conductance and bath temperature show that heating from 24°C to 30°C increases TREK-1 conductance. At 30°C exposure to sodium azide (10 μm) inhibited TREK-1 conductance. In the continued presence of sodium azide, bath application of AICAR had no effect on TREK-1 conductance. Inset, I–V relationship of TREK-1 current obtained under control conditions at 30°C (a) and during exposure to azide alone (b) and azide in the presence of AICAR (c). Bar graph summarizes the percentage inhibition by azide alone and AICAR in the presence of azide. B, traces of wild-type TREK-2 conductance and bath temperature show that heating from 24°C to 30°C increases TREK-1 conductance. At 30°C exposure to sodium azide (10 μm) inhibited TREK-2 conductance. In the continued presence of sodium azide, bath application of AICAR had no effect on TREK-2 conductance. Inset, I–V relationship of TREK-2 current obtained under control conditions at 30°C (a) and during exposure to azide alone (b) and azide in the presence of AICAR (c). Bar graph summarizes the percentage inhibition by azide alone and AICAR in the presence of azide.
Figure 7
Figure 7. Sodium azide inhibits heteromeric TASK channels by an AMPK-independent mechanism
A, trace of TASK1–TASK-3 conductance and bath temperature show that heating from 24°C to 30°C increases activity of TASK channels. At 30°C 3 min exposure to sodium azide (10 μm) inhibits channel conductance. Inset, I–V plots of heteromeric TASK current obtained at 30°C during control conditions (a) and in the presence of sodium azide (b). B, trace of TASK1–TASK-3 conductance in the presence of dnAMPK and bath temperature show that heating from 24°C to 30°C increases activity of TASK channels. At 30°C exposure to sodium azide (10 μm) inhibits channel conductance. Inset, I–V plots of heteromeric TASK current in the presence of dnAMP obtained at 30°C during control conditions (a) and in the presence of sodium azide (b). C, bar graph of average data (n= 4) shows the percentage inhibition of heteromeric TASK channels by sodium azide alone and in the presence of dnAMPK.
Figure 8
Figure 8. Hypoxia inhibits TREK channels by an AMPK-dependent mechanism
A, trace of wild-type TREK-1 conductance and bath temperature show that heating from 24°C to 30°C increases TREK-1 activity. At 30°C exposure to hypoxia (measured bath formula image= 11 ± 1 mmHg) for 6 min inhibited channel conductance. Inset, I–V plots of TREK-1 current obtained at 30°C under control conditions (a) and during exposure to hypoxia (b). B, trace of wild-type TREK-1 conductance in the presence of dnAMPK and bath temperature show that heating from 24°C to 30°C increases TREK-1 activity. At 30°C exposure to hypoxia (formula image= 11 ± 1 mmHg) for 6 min had no effect on channel conductance. Inset, I–V plots of TREK-1 current in the presence of dnAMP obtained at 30°C under control conditions (a) and during exposure to hypoxia (b). C, bar graph of average data (n= 4) shows the percentage inhibition of TREK-1 by hypoxia alone and in the presence of dnAMPK.
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