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 + (K 2P ) 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 K 2P channels.potassium channels | single-molecule fluorescence | leak current | combinatorial diversity | heteromerization

Formation of Functional Heterodimers by TREK-1 and TREK-2 Two-pore Domain Potassium Channel Subunits

“…Note Added in Proof-While this paper was under review, other authors have published results showing heteromerization of channels in the TREK K 2P subfamily (38,39). These studies reached the same conclusion by using different methods.…”

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

“…We and others Hwang et al, 2014;Lengyel et al, 2016;Levitz et al, 2016) have recently shown that K 2P channels are able to form both homodimeric and heterodimeric potassium channels. In this study, we show that TREK and TRESK readily assemble as heteromers.…”

“…Despite the fact that K 2P channels share a similar architecture and global function, they share a low level of sequence identity, even between members of the same subfamily. Surprisingly, this low level of identity does not preclude heteromerization, as we and others recently showed within the TREK subfamily Hwang et al, 2014;Lengyel et al, 2016;Levitz et al, 2016). Based on this and the fact that TG neurons express many K 2P channels (TREK1, TREK2, TRAAK, TASK1, and TASK3; Bautista et al, 2008;Yamamoto et al, 2009), we hypothesized that the difference between TRESK mutants is due to their differential ability to modify the function of other K 2P chan-nels through heteromerization.…”

“…Xenopus leavis oocytes were collected from female Xenopus leavis (agreement # 35382015121816318324V7) and dissociated using collagenase type IV (Levitz et al, 2016). Oocytes were injected with 50 nL of a cRNA at a concentration of 1mg/mL and maintained at 18 C in ND96 solution (96 mM NaCl, 2 mM KCl, 2 mM MgCl 2 , 1.8 mM CaCl 2 , 5mM HEPES, pH 7.4).…”

Two-pore Domain Potassium Channels in Astrocytes

“…In general, K 2P channels form homodimers of two of the same isoforms, and the characteristics of the channels are determined by the single isoforms. Recently some K 2P isoforms were discovered that form heterodimers between two different isoforms, and the heterodimers showed different channel properties from those of the homodimeric channels of each single isoform [3435363738394041]. The heterodimerization of the channels therefore provides diversity in channel properties.…”