Lengyel M et al. (2018), TRESK background potassium channel is not gated...

XB-ART-55537

PLoS One 2018 May 15;135:e0197622. doi: 10.1371/journal.pone.0197622.

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TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore.

Lengyel M , Czirják G , Enyedi P .

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Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK.

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Species referenced: Xenopus laevis
Genes referenced: kcnk18 mark2

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	Fig 1. Barium ions can be trapped in the Kv1.3 channel pore.Kv1.3 channels were expressed in HEK293T cells. The currents were recorded in inside-out excised patches. Voltage-dependent currents were measured by depolarizing the membrane to +40 mV for 250 ms. The holding potential was -80 mV. Currents were measured at the end of the +40 mV voltage step. Kv1.3 current was calculated by subtracting the current measured in K+-free bath solution from the value measured in the high K+ solution. The currents after the administration of Ba2+ were normalized to the value measured before the application of the blocker (on panel C). A, Representative recording showing the application of Ba2+ (1 mM) to the intracellular surface of the patches while keeping the holding potential at -80 mV (constant hyperpolarization). Afterwards the blocker was removed from the bath solution and the current was measured every 30 s by depolarizing steps to +40 mV. B, Representative recording showing the effect of Ba2+ (1 mM) applied to the intracellular surface of inside-out patches when the membrane was depolarized to +40 mV (transient depolarization). Barium was then removed from the bath solution while the holding potential was -80 mV. Voltage-gated K+ currents were measured every 30 s, as in panel A. C, The normalized currents after the application of Ba2+ are plotted as a function of time. The channels were opened by transient depolarization to +40 mV (black circles) or closed at constant -80 mV (grey squares) in the presence of Ba2+. Data are plotted as scatter plot, the averages of the different groups are plotted as columns. The differences between the groups were significant at all examined time points (p<0.05, statistical analysis was performed using Kruskal-Wallis ANOVA followed by pairwise comparison using the Mann-Whitney U test).
	Fig 2. The kinetics of Ba2+ block is not affected by the activation of TREK-1 channel.A, TREK-1 channels were expressed in HEK293T cells and recordings were done on excised inside-out patches. TREK-1 currents were measured at +60 mV by switching the K+-free bath solution to a high K+ solution (solution changes are marked with bars above the graphs). In this representative recording, TREK-1 channels were activated by perfusing the intracellular side of the patch with acidic solution (pH 6.1) as shown on the graph. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (application of Ba2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). B, The pH of the bath solution was 7.1 throughout the experiment. In this representative recording, Ba2+ (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow, application of Ba2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording.). C, The kinetics of Ba2+ induced TREK-1 block were determined for both the resting (perfused with pH 7.1 solution) and activated (pH 6.1) channel (n = 8 and 6 patches). The averaged normalized curves are plotted for both groups. The inset shows the onset of Ba2+ block at an early time period with higher temporal resolution. D, The current recordings of the Ba2+ block recorded at different pH values were fitted with a double exponential equation. The time constants of the fitted equations are plotted as a scatter plot. The average values are plotted as columns. Differences between the groups were not statistically significant.
	Fig 3. TRESK channel is inhibited by MARK2 and PKA phosphorylation.Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K+-free bath solution to a high K+ solution (solution changes are marked with bars above the graphs). Application of ATP and purified MARK2 (16 μg/ml) or separate application of ATP or MARK2 are marked by the bars above the recordings. A, Representative recording showing that application of MARK2 and 2 mM ATP leads to inhibition of TRESK current by phosphorylation. B, Representative recording showing that separate application of 2 mM ATP and MARK2 does not have an effect on TRESK current. C, Representative recording showing that application of PKA (30 U/ml) in the presence of 1 mM cAMP, 1 mM DTT and 2 mM ATP inhibits TRESK current by phosphorylating the channel. D, Representative recording showing that application of PKA (30 U/ml) in the presence of 1 mM cAMP, 1 mM DTT, but in the absence of ATP has no effect on TRESK current. E, The effects of ATP, MARK2 and ATP+MARK2 on TRESK current have been summarized as a scatter plot. The averages of each group are plotted as column graphs. Statistical significant differences between the groups (p<0.05, determined by Kruskal-Wallis test followed by multiple comparisons of mean ranks) are marked with asterisks. F, The effects of PKA and PKA+ATP on TRESK current have been summarized as a scatter plot. The averages of each group are plotted as column graphs. Statistical significant differences between the groups (p<0.05, determined by the Mann-Whitney U test) are marked with asterisks.
	Fig 4. The phosphorylation state of TRESK does not influence the kinetics of Ba2+ block.Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K+-free bath solution to a high K+ solution (solution changes are marked with bars above the graphs). Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV. TRESK channels were either dephosphorylated by application of 0.5 μM ionomycin to the bath solution before recording or phosphorylated by application of purified MARK2 (16 μg/ml), 30 U/ml PKA (1 mM cAMP and 1 mM DTT was added to ensure the enzymatic activity of PKA) and 2 mM ATP before the initiation of the Ba2+ block. A, Representative recording, TRESK channels were dephosphorylated before patch excision by application of 0.5 μM ionomycin to the bath solution. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow, application of Ba2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). B, Representative recording, TRESK channels were phosphorylated by perfusing the intracellular side of the patch with a bath solution containing both kinases (purified MARK2, PKA, 1 mM cAMP, 1 mM DTT and 2 mM ATP) as shown on the graph. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow, application of Ba2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). C, The kinetics of TRESK current inhibition by Ba2+ were determined for both the phosphorylated and dephosphorylated channel (n = 6 and 5 patches). The average normalized curves for both groups are plotted. The inset shows the onset of Ba2+ with a higher temporal resolution. D, The current recordings of the Ba2+ block recorded for both the phosphorylated and dephosphorylated groups were fitted with a double exponential equation. The time constants of the fitted equations are plotted as a scatter plot. The average values are plotted as columns. The difference between the groups was not statistically significant (Student’s t test).
	Fig 5. The rate of Ba2+ inhibition is similar in TRESK mutants mimicking the phosphorylated and dephosphorylated channel.Mutant mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K+-free bath solution to a high K+ solution. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV. A and B, The current recordings of the Ba2+ block recorded for the different mutants were fitted with a double exponential equation. The time constants of the fitted equations are illustrated as a scatter plot. The average values are plotted as columns. The differences between the groups were not statistically significant (ANOVA).

References [+] :

Armstrong, Block of squid axon K channels by internally and externally applied barium ions. 1982, Pubmed