Two-pore domain K+ (K2P) 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 potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, 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 an SF-mediated gating mechanism, but we found here that only very strong nonphysiological 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.

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.