XB-ART-57744
J Biol Chem
2020 Aug 28;29535:12408-12425. doi: 10.1074/jbc.RA120.014125.
Show Gene links
Show Anatomy links
TRESK and TREK-2 two-pore-domain potassium channel subunits form functional heterodimers in primary somatosensory neurons.
Lengyel M, Czirják G, Jacobson DA, Enyedi P.
???displayArticle.abstract???
Two-pore-domain potassium channels (K2P) are the major determinants of the background potassium conductance. They play a crucial role in setting the resting membrane potential and regulating cellular excitability. These channels form homodimers; however, a few examples of heterodimerization have also been reported. The K2P channel subunits TRESK and TREK-2 provide the predominant background potassium current in the primary sensory neurons of the dorsal root and trigeminal ganglia. A recent study has shown that a TRESK mutation causes migraine because it leads to the formation of a dominant negative truncated TRESK fragment. Surprisingly, this fragment can also interact with TREK-2. In this study, we determined the biophysical and pharmacological properties of the TRESK/TREK-2 heterodimer using a covalently linked TRESK/TREK-2 construct to ensure the assembly of the different subunits. The tandem channel has an intermediate single-channel conductance compared with the TRESK and TREK-2 homodimers. Similar conductance values were recorded when TRESK and TREK-2 were coexpressed, demonstrating that the two subunits can spontaneously form functional heterodimers. The TRESK component confers calcineurin-dependent regulation to the heterodimer and gives rise to a pharmacological profile similar to the TRESK homodimer, whereas the presence of the TREK-2 subunit renders the channel sensitive to the selective TREK-2 activator T2A3. In trigeminal primary sensory neurons, we detected single-channel activity with biophysical and pharmacological properties similar to the TRESK/TREK-2 tandem, indicating that WT TRESK and TREK-2 subunits coassemble to form functional heterodimeric channels also in native cells.
???displayArticle.pubmedLink??? 32641496
???displayArticle.pmcLink??? PMC7458809
???displayArticle.link??? J Biol Chem
???displayArticle.grants??? [+]
Species referenced: Xenopus laevis
Genes referenced: kcnk10 kcnk18 ppp3ca
GO keywords: potassium channel activity
???displayArticle.disOnts??? migraine
???attribute.lit??? ???displayArticles.show???
|
|
|
|
|
|
|
|
|
|
|
|
Figure 1T2A3 is a selective activator of TREK-2. A, normalized currents of oocytes expressing TREK-2, measured by the two-electrode voltage-clamp technique. Currents were measured at the end of 300-ms voltage steps to −100 mV applied every 4 s from a holding potential of 0 mV. The extracellular K+ concentration was increased from 2 to 80 mm as indicated by the bars above the graph. Oocytes were challenged with T2A3 (3, 10, and 30 μm; see the horizontal bars on the recordings). Currents were normalized to the value measured in 80 mm K+ before the application of the drug. Data are plotted as mean ± S.D. (error bars). B, concentration–response relationship of T2A3 and TREK-2 current. The effects of different concentrations of A2764 (in a range from 3 to 30 μm) on TREK-2 current are plotted. Each data point represents the average of seven oocytes. C and D, mouse K2P channels were expressed in Xenopus oocytes. The effect of T2A3 (10 μm in C and 30 μm in D) on the inward current in 80 mm extracellular K+ was determined in 5–6 oocytes/channel type as in A and plotted as a scatter plot. The mean for each channel type is plotted as a column graph. |
|
Figure 2Dominant negative action of the pore mutant TRESK subunit on both TRESK and TREK-2 currents. Channels (mouse TRESK, mouse TREK-2, and human Kv1.3) were coexpressed with a nonfunctional TRESK subunit, TRESK-G131E (TRESKDN-pCD8), or pCD8 at a ratio of 1:3 in HEK293T cells. The WT TRESK/TREK-2 tandem and TREK-2/TRESK-G131E tandem were coexpressed with pCD8. Currents were measured in the whole-cell configuration every 4 s at the end of 1-s voltage steps ranging from −100 to +50 mV. The holding potential was −80 mV. Currents were normalized to cell capacitance. A, D, and G, representative current traces from cells expressing TRESK (A), TREK-2 (D), or human Kv1.3 in combination with either pCD8 (left traces) or TRESKDN-pCD8 (right). Zero current levels are marked with a dash on the left side of the recordings. B, E, and H, current–voltage relationship of the different channels with the coexpression of pCD8 (black circles) or TRESKDN-pCD8 (white circles) is summarized as mean ± S.D. (error bars). The number of cells in each experimental group is shown in parentheses. C, F, and I, current density measured at 0 mV of cells coexpressing either pCD8 (black circles) or TRESKDN-pCD8 (white circles) is plotted as a scatter plot. The average values for each group are plotted as a column. The difference between the two groups was statistically significant (Mann–Whitney test). J, representative current traces from cells expressing the WT TRESK/TREK-2 tandem (left) or the TREK-2/TRESK-G131E tandem (right). Zero current levels are marked with a dash on the left side of the recordings. K, current–voltage relationship of the WT TRESK/TREK-2 tandem (black circles) or the TREK-2/TRESK-G131E tandem (white circles) is summarized as mean ± S.D. The number of cells in each experimental group is shown in parentheses. L, current density measured at 0 mV of cells coexpressing either the WT TRESK/TREK-2 tandem (black circles) or the TREK-2/TRESK-G131E tandem (white circles) is plotted as a scatter plot. The average values for each group are plotted as a column. The difference between the two groups was statistically significant (Student's t test). |
|
Figure 3Unique pharmacological profile of the TRESK/TREK-2 tandem channel. The TRESK/TREK-2 tandem channel was expressed in Xenopus oocytes. Currents were measured as described in the legend to Fig. 1. Currents were normalized to the value measured in 80 mm K+ before the application of the drug(s). Changes of the extracellular K+ concentration and application of the different pharmacological agents are indicated by the horizontal bars above the graph. Data are plotted as mean ± S.D. (error bars). A, oocytes were stimulated with ionomycin (0.5 μm), leading to an activation of the tandem channel (n = 7 oocytes). B, application of A2764 (100 μm) inhibits the current of the tandem channel (n = 6 oocytes). C, TRESK/TREK-2 channels were activated via ionomycin prior to perfusion of A2764 (n = 6 oocytes). D, T2A3 activates the TRESK/TREK-2 tandem channel in a concentration-dependent manner. E and F, TRESK/TREK-2 tandem channels were activated by ionomycin (n = 7 oocytes) and cloxyquin (n = 6 oocytes), respectively. Subsequent application of T2A3 (30 μm) further increased the current. |
|
Figure 4Phosphorylation state of TRESK channel regulates the open probability of the channel. Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. Currents were measured at +60, 0, and −60 mV in symmetrical 140 mm KCl solutions. The current level corresponding to the closed state of the channel is marked with a dash on the left side of the recordings. A, representative recording of a dephosphorylated TRESK channel. To obtain channels in the dephosphorylated state, HEK293T cells expressing mouse TRESK were pretreated with 0.5 μm ionomycin (Iono) before patch excision. B, representative recording of a phosphorylated TRESK channel. To obtain channels in the phosphorylated state, HEK293T cells expressing mouse TRESK were pretreated overnight with 1 μm cyclosporine A (CsA). C, open probability of dephosphorylated (Iono) and phosphorylated (CsA) TRESK channels determined at +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. The difference between the two groups was statistically significant (Student's t test). D, single-channel conductance of dephosphorylated (Iono) and phosphorylated (CsA) TRESK channels determined at +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. The difference between the two groups was not statistically significant (Student's t test). |
|
Figure 5Phosphorylation state of the TRESK/TREK-2 tandem channel regulates the open probability of the tandem channel. Mouse TRESK/TREK-2 channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. Currents were measured at +60, 0, and −60 mV in symmetrical 140 mm KCl solutions. The current level corresponding to the closed state of the channel is marked with a dash on the left side of the recordings. A, representative recording of a dephosphorylated TRESK/TREK-2 channel. To obtain channels in the dephosphorylated state, HEK293T cells expressing mouse TRESK/TREK-2 were pretreated with 0.5 μm ionomycin (Iono) before patch excision. B, representative recording of a phosphorylated TRESK/TREK-2 channel. To obtain channels in the phosphorylated state, HEK293T cells expressing mouse TRESK/TREK-2 were pretreated overnight with 1 μm cyclosprine A (CsA). C, open probability of dephosphorylated (Iono) and phosphorylated (CsA) TRESK/TREK-2 channels determined at −60 and +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. The difference between the two groups was statistically significant (Student's t test). D, single-channel conductance of dephosphorylated (Iono) and phosphorylated (CsA) TRESK/TREK-2 channels determined at −60 and +60 mV is displayed as a scatter plot. The average values for each group is plotted as a column. The difference between the two groups was not statistically significant (Student's t test). |
|
Figure 6Mutations mimicking different phosphorylation states of TRESK channel affect the open probability of the channel. Mutant mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. Currents were measured at +60, 0, and −60 mV in symmetrical 140 mm KCl solutions. The current level corresponding to the closed state of the channel is marked with a dash on the left side of the recordings. A–D, representative recordings of TRESK channels containing different point mutations (the exact point mutation is marked above each recording). E, open probability of the different mutant TRESK channels determined at +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. Statistically significant differences between the groups are marked with asterisks (p < 10−4, one-way ANOVA followed by Tukey's post hoc test). F, single-channel conductance of the different mutant TRESK channels determined at +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. The difference between the different groups was not statistically significant (p = 0.63, one-way ANOVA). |
|
Figure 7Mutations mimicking different phosphorylation states of TRESK channel affect the open probability of the TRESK/TREK-2 tandem channel. TRESK/TREK-2 tandem channels (containing different mutant TRESK subunits) were expressed in HEK293T cells. Experiments were done on excised inside-out patches. Currents were measured at +60, 0, and −60 mV in symmetrical 140 mm KCl solutions. The current level corresponding to the closed state of the channel is marked with a dash on the left side of the recordings. A–D, representative recordings of TRESK/TREK-2 tandem channels containing different point mutations (the exact point mutation is marked above each recording). E, open probability of the different mutant TRESK/TREK-2 channels determined at −60 and +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. Statistically significant differences between the groups are marked with asterisk (p < 10−4 for both −60 and +60 mV, one-way ANOVA followed by Tukey's post hoc test). F, single-channel conductance of the different mutant TRESK channels determined at −60 and +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. The difference between the different groups was not statistically significant (p = 0.07 and p = 0.06 for −60 and +60 mV, respectively, one-way ANOVA). |
|
Figure 8TRESK, TREK-2, and the TRESK/TREK-2 tandem channel have distinct single-channel conductances. Mouse TRESK, TREK-2, or TRESK/TREK-2 tandem channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. Currents were measured at +60, 0, and −60 mV in symmetrical 140 mm KCl solutions. The current level corresponding to the closed state of the channel is marked with a dash on the left side of the recordings. A, top, representative recordings of TREK-2, TRESK, and TRESK/TREK-2 tandem channels. Bottom, representative recordings of a TRESK and a TRESK/TREK-2 tandem channel at higher magnification. B, single-channel conductance of the TRESK, TREK-2, and TRESK/TREK-2 tandem channels determined at −60 and +60 mV is displayed as a scatter plot. The average values for each group are plotted as a column. The difference between all three groups was found to be statistically significant (p < 10−4, one-way ANOVA followed by Tukey's post hoc test). |
|
Figure 9TRESK and TREK-2 subunits form functional heterodimers. Channels were expressed in HEK293T cells. Currents were measured at +60, 0, and −60 mV in symmetrical 140 mm KCl solutions in excised inside-out membrane patches. Single-channel conductances were determined at −60 and +60 mV. Conductance values are plotted as a scatter plot. A, TRESK and TREK-2 subunits were expressed in HEK293T cells in different ratios while keeping the total amount of DNA constant at 100 ng of channel DNA/35-mm dish. Single-channel conductances were determined at −60 and +60 mV. The average single-channel conductance of the TRESK-TREK2 tandem is plotted for comparison (as mean ± S.D. (error bars)). Conductance values corresponding to different channels are plotted as follows: TREK-2 homodimers are plotted with gray triangles, TRESK homodimers are marked with white circles, and TRESK/TREK-2 heterodimers are plotted as black diamonds. B, the TRESK-T2A-TREK2 construct was expressed in HEK293T cells. Single-channel conductances were determined and plotted as in A. |
|
Figure 10The TRESK/TREK-2 tandem channel is sensitive to both T2A3 and A2764. The TRESK/TREK-2 tandem construct was expressed in HEK293T cells. Experiments were done on excised inside-out patches. Currents were measured at −60 mV in symmetrical 140 mm KCl solutions. The current levels corresponding to the closed state and the different open levels of the channels in the patch are marked with dashes on the left side of the recordings. Channel activity (NPo) was determined before, during, and after application of the drugs. A, representative recording showing that 100 μm A2764 inhibits TRESK/TREK-2 tandem channel activity in excised inside-out patches. B, channel activity after application of A2764 is displayed as a scatter plot, whereas the average values are plotted as a column. C, representative recording showing that 30 μm T2A3 increases TRESK/TREK-2 tandem channel activity in excised inside-out patches. D, channel activity after application of T2A3 is displayed as a scatter plot, whereas the average values are plotted as a column. |
|
Figure 11TRESK and TREK-2 subunits heterodimerize in trigeminal ganglion neurons. Membrane patches were excised from trigeminal ganglion neurons. Single-channel currents were recorded in symmetrical 140 mm KCl in the inside-out configuration. The effects of T2A3 (10 μm) and A2764 (100 μm) application were determined at a membrane potential of −60 mV. Application of T2A3 and A2764 is indicated by the horizontal bars. Channel activity (NPo) was determined from 30–60 s of recording before, during, and after application of T2A3 and A2764. Channels resistant to both T2A3 and A2764 were not plotted. A, representative current recording of a patch containing two T2A3-sensitive but A2764-resistant channels. B, representative current recording of a patch containing a channel activated by T2A3 but inhibited by A2764 (regarded as a TRESK/TREK-2 heterodimer) and a channel resistant to both compounds. Representative portions of the recording (corresponding to control periods and application of the drugs) are shown at a higher temporal resolution (i–iv). C, effects of T2A3 (10 μm) and A2764 (100 μm) on channel activity were plotted against each other. Gray triangles represent T2A3-sensitive but A2764-resistant channels. Channels sensitive to both A2764 and T2A3 (i.e. TRESK/TREK-2 heterodimers) are marked by diamonds. D, single-channel conductances of the channels plotted in C were determined at −60 mV and plotted as a scatter plot. |
References [+] :
Aller,
Changes in expression of some two-pore domain potassium channel genes (KCNK) in selected brain regions of developing mice.
2008, Pubmed
Aller, Changes in expression of some two-pore domain potassium channel genes (KCNK) in selected brain regions of developing mice. 2008, Pubmed
Andres-Enguix, Functional analysis of missense variants in the TRESK (KCNK18) K channel. 2012, Pubmed
Bautista, Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels. 2008, Pubmed
Berg, Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits. 2004, Pubmed
Blin, Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties. 2016, Pubmed
Blin, Tandem pore domain halothane-inhibited K+ channel subunits THIK1 and THIK2 assemble and form active channels. 2014, Pubmed , Xenbase
Braun, TRESK background K(+) channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes. 2011, Pubmed , Xenbase
Braun, Differential sensitivity of TREK-1, TREK-2 and TRAAK background potassium channels to the polycationic dye ruthenium red. 2015, Pubmed , Xenbase
Coetzee, Molecular diversity of K+ channels. 1999, Pubmed , Xenbase
Czirják, TRESK background K(+) channel is inhibited by phosphorylation via two distinct pathways. 2010, Pubmed , Xenbase
Czirják, Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K+ channel, TRESK. 2006, Pubmed , Xenbase
Czirják, Characterization of the heteromeric potassium channel formed by kv2.1 and the retinal subunit kv8.2 in Xenopus oocytes. 2007, Pubmed , Xenbase
Czirják, The two-pore domain K+ channel, TRESK, is activated by the cytoplasmic calcium signal through calcineurin. 2004, Pubmed , Xenbase
Czirják, Zinc and mercuric ions distinguish TRESK from the other two-pore-domain K+ channels. 2006, Pubmed , Xenbase
Czirják, Formation of functional heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits. 2002, Pubmed , Xenbase
Dadi, Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx. 2017, Pubmed
Enyedi, TRESK: the lone ranger of two-pore domain potassium channels. 2012, Pubmed
Enyedi, Molecular background of leak K+ currents: two-pore domain potassium channels. 2010, Pubmed
Feliciangeli, The family of K2P channels: salient structural and functional properties. 2015, Pubmed
Guo, Nonmigraine-associated TRESK K+ channel variant C110R does not increase the excitability of trigeminal ganglion neurons. 2014, Pubmed , Xenbase
Hwang, A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes. 2014, Pubmed
Kang, TREK-2 (K2P10.1) and TRESK (K2P18.1) are major background K+ channels in dorsal root ganglion neurons. 2006, Pubmed
Lafrenière, A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. 2010, Pubmed
Lengyel, Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel. 2019, Pubmed , Xenbase
Lengyel, Formation of Functional Heterodimers by TREK-1 and TREK-2 Two-pore Domain Potassium Channel Subunits. 2016, Pubmed , Xenbase
Lengyel, TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore. 2018, Pubmed , Xenbase
Lengyel, Selective and state-dependent activation of TRESK (K2P 18.1) background potassium channel by cloxyquin. 2017, Pubmed , Xenbase
Liu, Functional analysis of a migraine-associated TRESK K+ channel mutation. 2013, Pubmed , Xenbase
Mathie, Two-pore domain potassium channels: potential therapeutic targets for the treatment of pain. 2015, Pubmed
Patel, Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes. 1997, Pubmed
Pereira, Role of the TREK2 potassium channel in cold and warm thermosensation and in pain perception. 2014, Pubmed
Pettingill, A causal role for TRESK loss of function in migraine mechanisms. 2019, Pubmed
Plant, SUMOylation silences heterodimeric TASK potassium channels containing K2P1 subunits in cerebellar granule neurons. 2012, Pubmed
Preisig-Müller, Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome. 2002, Pubmed , Xenbase
Royal, Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK. 2019, Pubmed , Xenbase
Simkin, Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation. 2008, Pubmed
Suzuki, Heterodimerization of two pore domain K+ channel TASK1 and TALK2 in living heterologous expression systems. 2017, Pubmed
Szymczak, Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. 2004, Pubmed
Talley, Cns distribution of members of the two-pore-domain (KCNK) potassium channel family. 2001, Pubmed
Tóth, BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation. 2016, Pubmed
Usoskin, Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. 2015, Pubmed
Weir, The Role of TRESK in Discrete Sensory Neuron Populations and Somatosensory Processing. 2019, Pubmed
Wright, Cloxyquin (5-chloroquinolin-8-ol) is an activator of the two-pore domain potassium channel TRESK. 2013, Pubmed
Yamamoto, Immunohistochemical colocalization of TREK-1, TREK-2 and TRAAK with TRP channels in the trigeminal ganglion cells. 2009, Pubmed
Yoo, Regional expression of the anesthetic-activated potassium channel TRESK in the rat nervous system. 2009, Pubmed