Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K⁺ Channel

Abstract

Two-pore domain (K2P) K + channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these various potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating the underlying mechanism, we observed that the low activity of the SF gate appears to arise from the inefficiency of K + in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb + , NH4 + , and Cs + , strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via a SF-mediated gating mechanism, but we found here that only very strong, non-physiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb + currents are potently inhibited by intracellular K + (IC50 = 2.8 mM). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K + appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.From the Supplemental Fig. S1. Example currents for WT TWIK-1* and the three key mutations shown in Figure 1B. These mutants all produce increases in whole-cell current that are equivalent to the effect of the previously reported K274E mutation (8). Currents were recorded by TEVC in response to voltage steps from -120 to +60 mV from a holding potential of -80 mV. Reported values were measured at 0 mV. Supplemental Fig. S2. A) Example of the fits used (red) on Rb + activated TWIK-1 currents. These fits were used to obtain the rate constants shown in Fig.3C which demonstrate that activation is not voltagedependent (see methods for details). B) Currents recorded in excised patches from a mutant version of TREK-2 (Gly 67 -Glu 340 ) (26) at both 23° and 3° under the same conditions shown in Fig. 3D. At 23° no tail currents are visible, but upon cooling to 3° the rate of activation slows, as well as the tail currents so they become more visible. An overlay of these currents at +100 mV is shown in the right hand panel. Supplemental Fig. S3Example of the fits used (in red) to obtain the rate constants for K + block and unblock that are shown in Fig. 5D. See experimental procedures for details.

Multiple Mechanisms Underlie State-Independent Inhibitory Effects of Norfluoxetine on TREK-2 K2P Channels

“…However, most K2P channels, including TREK channels, do not have a lower gate (Brohawn et al, 2012;Miller and Long, 2012;Lolicato et al, 2014;Dong et al, 2015). Instead they appear to gate primarily within their selectivity filter (Zilberberg et al, 2001;Bagriantsev et al, 2011;Piechotta et al, 2011;Schewe et al, 2016;Nematian-Ardestani et al, 2020) and current models for TREK channel gating propose that movement of the TM helices can regulate this filter gating mechanism but do not constrict enough at this lower region to prevent K + permeation (Brohawn et al, 2012;Lolicato et al, 2014;Dong et al, 2015). This current model for TREK channel gating is shown in Figure 1A.…”

Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate

“…K + channels that lack a gating mechanism though an S6 helix bundle-crossing usually feature a gate formed by the selectivity filter (32,33,53,54,57). It has been proposed that the structure of the selectivity filter changes as a result of allosteric coupling with the activation gate, facilitated by pore-lining transmembrane helices, to facilitate ion conduction (33).…”

“…It has been proposed that the structure of the selectivity filter changes as a result of allosteric coupling with the activation gate, facilitated by pore-lining transmembrane helices, to facilitate ion conduction (33). Importantly, a threonine residue at the cytoplasmic end of the selectivity filter is thought to be critical in the gating of MthK, K2P, K Ca 1.1 and KcsA channels (32,33,53,57). This threonine residue is conserved in K Na 1.1 and mutation T314C also disrupted rectification.…”

“…Lowering of the activation barrier by mutations also explains the increase in co-operative channel opening, compared to WT channels, observed in excised patches containing multiple K Na 1.1 channels 3 . K + channels that lack a gating mechanism though an S6 helix bundle-crossing usually feature a gate formed by the selectivity filter 32,33,53,54,57 . It has been proposed that the structure of the selectivity filter changes as a result of allosteric coupling with the activation gate, facilitated by pore-lining transmembrane helices, to facilitate ion conduction 33 .…”

“…The voltage-and Na + -independent activation time constants were reminiscent of K + channel subtypes that exhibit voltage-dependent selectivity filter gating, such as several mammalian tandem pore domain K + channels (K2P) (32,33,(53)(54)(55). In outwardly-rectifying K2P channels, mutation of a threonine at the cytoplasmic end of the selectivity filter motif that forms the intracellular-most K + binding site resulted in voltage-and ligand-independent channel currents (32).…”