Insights into the structure and modulation of human TWIK-2

Figures

• 

  Fig. 1. Function and structure of TWIK-2. A Functional analysis of TWIK-2 channel and Y111H mutant channel at various pH levels. Representative whole-cell current traces were recorded from cells expressing both wild-type TWIK-2 channels and the Y111H mutant channels. These traces were obtained using voltage-clamp steps that ranged from −90 to +100 mV in 10 mV increments, with pH levels set at 7.4 and 5.5. B A comparative analysis of peak current densities (pA/pF) at +100 mV and the reversal potential (VRev) was performed for TWIK-2 (n = 32; n = 35, respectively) and Y111H (n = 24; n = 28, respectively) at pH 7.4, as well as for TWIK-2 (n = 38; n = 45, respectively) and Y111H (n = 33; n = 54, respectively) at pH 5.5. The current–voltage (I–V) relationships recorded from both TWIK-2 and Y111H channels showed no significant differences across the voltage range of −90 to +100 mV, including at the reversal potential (VRev). The data in (B) are presented as mean ± standard deviation (SD), with some error bars smaller than the data points. Statistical significance was assessed by an unpaired t-test with Welch’s correction. C Topology and model of TWIK-2 protomer showing transmembrane helices…

• 

  Fig. 2. Ion conductance pathway of TWIK-2. A A cross-section of TWIK-2 along the membrane plane, with hydrophobic and hydrophilic amino acids colored in orange and cyan, respectively. Critical amino acids flanking the entry and exit of the ion permeation pore, at the hydrophobic cuff and EIP, respectively, are highlighted. B Pore radius of the TWIK-2 channels without or with hydrocarbon tail (yellow) modeled as distance along the ion permeation pathway calculated by HOLE80, shown in light blue (left) or light gray (right). C Cryo-EM density for the selectivity filters and K+ ions is highlighted in gray. The green (top) and black (bottom) dashed lines indicate the widest and narrowest pore diameters of the SF. D The cross-section views of the narrowest and widest planes of the SF as viewed from the extracellular cap domain. The distance between the amino acids indicates the pore diameters at that position. E TWIK-2 with lipid-like tubular density (yellow) and tetrahedral solvent or ion blobs (blue) highlighted. The center panel highlights the presence of lipid-like densities along the groove formed by M1, M4, and PH2 of one protomer with M2 of the other protomer.

• 

  Fig. 3. Effects of mutants along the ion permeation pathway on TWIK-2 conductance and channel activation. A The residues in TWIK-2 along the ion-permeation pore (light blue) used for subsequent analysis are highlighted in yellow. A close-up view of the residues T106A, Y111H, M135A, and T214A is shown: side-view (middle panel), top-down and bottom-up views (right panel). B TWIK-2 and its mutants were tested under conditions of high extracellular potassium (K+) at a pH of 7.4. Representative whole-cell current recordings were obtained from cells expressing both wild-type TWIK-2 channels and various mutants. These recordings were generated through voltage-clamp steps ranging from −90 to +100 mV in 10 mV increments. C Activation time constants (τ) were calculated using a single exponential fit during a 350 ms pulse to +60 mV for TWIK-2 (n = 28), TWIK-1 (n = 15), and the TWIK-2 mutants (M135A, n = 18; T106A, n = 14; T214A, n = 18; Y111H, n = 32; M135N, n = 15; T106A/T214A, n = 15; F98A, n = 12; S213A, n = 15; Y111A, n = 15). Deactivation time constants (τ) were calculated using a single exponential fit during a 350 ms pulse to -90 mV for TWIK-2 (n = 15), TWIK-1 (n = 15), and the TWIK-2…

• 

  Fig. 4. Small molecule modulation of TWIK-2 by known K2P channel modulators. A Chemical structure of ML365, NPBA, ML335, and BL1249. B Concentration-response curves of various K2P modulators on TWIK-2 channel inhibition. Data are presented as mean ± SEM: ML365 (n = 8), NPBA (n = 8), ML335 (n = 8), and BL1249 (n = 8). Data from non-T cells (traced lines, empty symbols) are also presented for ML365 (n = 7) and NPBA (n = 16). C Changes in TWIK-2 currents in response to ML365 or NPBA (50 μM) were measured over time using a voltage-step protocol, applying +60 mV for 350 ms after holding at −90 mV. Data are presented as the mean ± SEM: ML365 (n = 8), NPBA (n = 8). D Comparison of concentration-response curves of ML365 on wild-type and mutant TWIK-2 channels. Data are presented as mean ± SEM: ML365 (n = 8). Right panel. Scatter dot plots show the current inhibition percentage at 50 µM for ML365 for TWIK-2 (ML365, n = 9) and the mutant channels (M135A: n = 9; T106A: n = 9; T214A: n = 9; Y111H: n = 9). The data are presented as mean ± SD. A one-way ANOVA with Dunnett’s multiple comparisons test was performed to compare the wild-type channel against each mutant. E Comparison of…

• 

  Fig. 5. Comparison of TWIK-1 and TWIK-2 functional and structural properties. A TWIK-1 channels showed a pH-dependent function, unlike TWIK-2. Whole-cell current traces were recorded from cells expressing both wild-type TWIK-1 and TWIK-2 channels. These traces were obtained using voltage-clamp steps from −90 to +100 mV in 10 mV increments, with the pH held at 7.4. B A comparison of the reversal potential (VRev) was conducted for TWIK-1 (at pH 7.4, n = 18; at pH 5.5, n = 24) and TWIK-2 (at pH 7.4, n = 30; at pH 5.5, n = 36), confirming TWIK-1’s pH sensitivity. The current–voltage (I–V) relationships for both TWIK-1 and TWIK-2 exhibited significant differences across the range of −90 to +100 mV at the reversal potential (VRev). Data in (B) were expressed as mean ± SD, with some error bars smaller than the data points. An unpaired two-tailed t-test with Welch’s correction was used to assess statistical significance. C Superposition of TWIK-1 (pH 7.4, mint, PDB:7SK0 and pH 5.5, moss, PDB:7SK1) and TWIK-2 (pH 7.5, gray, this study). The extracellular cap and selectivity filter are highlighted by orange and blue dashed boxes, respectively. D Cap domain in TWIK-2 (gray) resembles the…