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Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

ABSTRACT: Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

SUBMITTER: Levitz J

PROVIDER: S-EPMC4839437 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

Levitz Joshua J Royal Perrine P Comoglio Yannick Y Wdziekonski Brigitte B Schaub Sébastien S Clemens Daniel M DM Isacoff Ehud Y EY Sandoz Guillaume G

Proceedings of the National Academy of Sciences of the United States of America 20160328 15

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of ...[more]

PMID: 27035963

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Project description:BackgroundThe TWIK-related potassium channel (TREK-1) can be regulated by different stimuli. However, it is not clear whether some antiarrhythmics affect the activity of TREK-1. In the present study, the effect of bepridil on the TREK-1 currents is investigated.MethodsIn a TREK-1 stably-expressed HEK-293 cell line (HEK-TREK-1), U251MG cells, and isolated mouse ventricular myocytes, the TREK-1 current and action potentials were recorded by the patch-clamp technique. The standard voltage protocol was a 200 ms constant potential at 20 mV, followed bya 500 ms ramp from -90 to +20 mV (HEK-TREK-1) or +80 mV (U251MG cells and myocytes) every 10 s. The currents at +20 mV or +80 mV were used for analysis. The docking study of bepridil's binding model in the TREK-1 channel was performed using the Swissdock web service.ResultsIn HEK-TREK-1 cells, BL1249 induced a significantly large outwardly rectifying current with similar baseline TREK-1 current characteristic, with a reversal potential (-70 mV). The concentration of half-maximal activation (EC50) of BL1249 was 3.45 µM. However, bepridil decreased the baseline TREK-1 currents, with a concentration of half-maximal inhibition (IC50) 0.59 µM and a Hill coefficient of 1.1. Also, bepridil inhibited BL1249-activated TREK-1 currents, with an IC50 4.08 µM and a Hill coefficient of 3.22. The outside-out patch-clamp confirmed bepridil inhibited BL1249-activated TREK-1 currents. In U251MG cells and myocytes, BL1249 activated outwardly rectifying endogenous TREK-1 currents, which could be inhibited by bepridil. BL1249 (10 µM) could decrease the peak value and reduce the duration of the action potential. Bepridil (10 µM) prolonged the duration of action potential of myocytes. The docking study revealed that bepridil might affect the K+ pore domain and the M4 modulator pocket.ConclusionsBepridil may be a blocker for the TREK-1K+channel at a clinically therapeutic concentration, providing a new mechanism of TREK-1 regulation and bepridil's antiarrhythmic effect.

Project description:Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2-3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.

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Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

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Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

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