The voltage-dependent gate in MthK potassium channels is located at the selectivity filter

Abstract

Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. Blockers bind in an aqueous cavity between two putative gates: an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location--an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.

Figure 1

Block mechanism with two gate locations. (a, b) MthK pore cartoons illustrating gated access and blocker trapping (a) versus state-independent access of blockers (b). Selectivity filter is red with two K⁺ ions bound (green). Inner-pore helices (grey) may form a bundle-crossing gate. (c) Mean open probability (Po) measurements for MthK in symmetric 200 mM K⁺ (n = 3–8, mean ± S.D., error bars smaller than symbols). Inactivation gating (50 to 125 mV, red) is described by voltage-dependent transitions between one open (O) and one inactivated (I) state and was fit to a Boltzmann function (red line, Equation 1) with P_o^max = 0.994 ± 0.004, z = 1.44 ± 0.02, and V_½ = 103.3 ± 0.2 mV (Supplementary Figure 1A). The black line has no theoretical meaning.

Figure 2

Block of open MthK. (a) Representative recorded currents through single MthK channels at −100 mV with TBA, bTBA, and bbTBA (molecular models shown above traces). Closed level is indicated with a dotted line. (b) Closed and open dwell-time distributions for data in a. Control (top) was fit to a single closed-open model (right) to fit flicker gating (state F) (Supplementary Figure 1B). Blocker binding was fit with an additional closed state, OB (Scheme I). Fitted rates were used to calculate dissociation constants to MthK open state, K_D^open = k_off k_on⁻¹. The calculated K_D^open values are: TBA, 570 μM; bTBA, 27 μM; and bbTBA, 3.8 μM. Black and red lines are exponential fits to distributions using the schemes (right).

Figure 3

Voltage dependence of open-state block and apparent affinities during inactivation. (a) Single-channel traces without and with 25 μM bTBA at −25, −75, and −125 mV. (b) K_D^open (open symbols) and K_D^ap (filled symbols, Equation 3 for TBA (black squares), bTBA (red circles), and bbTBA (blue triangles), n = 3–6, mean ± S.D., error bars smaller than symbols. K_D^open data were fit to a model described in c (lines, Equation 2, TBA: K_D^low = 62 ± 5 μM, K_D^v = 20 ± 1 μM, z = 0.81 ± 0.01; bTBA: K_D^low = 2.0 ± 0.5 μM, K_D^v = 1.7 ± 0.2 μM, z = 0.67 ± 0.03; bbTBA: K_D^low = 0.37 ± 0.03 μM, K_D^v = 0.16 ± 0.01 μM, z = 0.78 ± 0.01). Dashed lines are extrapolated K_D^open values. (c) Cartoon of K⁺-blocker coupling model used to fit data in b (adapted from ref. 36). Left, pore with two conductive states differing in K⁺ (green) distributions in selectivity filter (S1–S3 and S2–S4). Right, TBA (cyan) blocked MthK pore with K⁺ in S1–S3. Membrane voltage, ΔV_m, is shown only across selectivity filter. TBA binding with K⁺ in S1–S3 is voltage-independent with dissociation constant K_D^low (horizontal transition) and TBA binding with K⁺ in S2–S4 is voltage-dependent with dissociation constant $K_D^V{e}^{-\frac{\mathit{zFV}}{RT}}$ (diagonal transition). Cartoon of ion movement between S1–S3 and S2–S4 is a simplification drawn merely for visualization of the ion-coupling concept.

Figure 4

Inactivation does not gate TBA access to the binding site. (a) Schemes used to fit single-channel kinetic data. (b) Example closed dwell-time distributions from single MthK recordings with fits to the models in a: open channel block (Scheme IIA, red dashed line), blocker trapping (Scheme IIB, green dotted line), and state-independent access models (Scheme III, black line). Fit parameters are in Supplementary Tables (see also Supplementary Figure 4). (c) TBA state-independent access (Scheme III) fit results. Black squares are TBA K_D^ap measurements from Figure 3 (n = 3–6, mean ± S.D.) and the black dotted line is the predicted K_D^ap using fitted parameters with Scheme III (Equation 4, Methods). The green dashed line is the TBA K_D^open fit from Figure 3 (Equation 2). The black solid and dashed lines are K_D^open (63.5 ± 9.9 μM) and K_D^inact (5.6 ± 0.6 μM), respectively, from fits to Scheme III (n = 3, mean ± S.D.).

Figure 5

bTBA and bbTBA block are consistent with state-independent access. (a) Example closed dwell-time distributions from single MthK recordings are shown for bTBA (top) and bbTBA (bottom) at 125 mV with fits to the open channel block (Scheme IIA, red dashed line) and the state-independent access (Scheme III, black line) models. Fit parameters are in Supplementary Tables (see Supplementary Figures 5 and 6). (b) bTBA (red) and bbTBA (blue) state-independent access (Scheme III) fit results. Red circles (bTBA) and blue triangles (bbTBA) are K_D^ap measurements from Figure 3 (n = 3–6, mean ± S.D.) and solid red and blue lines are calculated bTBA and bbTBA K_D^ap values using fitted parameters from Scheme III (Equation 4, Methods). Red and blue dotted lines are bTBA and bbTBA K_D^open fits from Figure 3 (Equation 2). The red and blue dashed lines are K_D^open for bTBA (2.9 ± 0.3 μM) and bbTBA (0.44 ± 0.02 μM), respectively, and grey dashed lines are K_D^inact values for bTBA (7.5 ± 1.3 μM) and bbTBA (4.0 ± 0.9 μM) from fits to Scheme III (n = 3, mean ± S.D.).

Figure 6

Summary of state-dependent MthK block. (a–c) Block kinetics from the state-independent access model (Scheme III) for TBA (black), TPeA (green), bTBA (red), and bbTBA (blue) are plotted for the open state (filled bars) and the inactivated state (dashed bars). Shown are the mean ± S.D. from three fitted datasets (Supplementary Table 3) for blocker on-rate (a), off-rate (b), and dissociation constants, K_D^open and K_D^inact (c).

Figure 7

TBSb difference density in the cavity supports blocker binding immediately below the selectivity filter. Diffraction data for crystals of the isolated MthK pore with and without the heavy TBSb blocker were used to generate a F_o^TBSb – F_o^no-blocker difference map (red mesh), shown in the channel cavity, contoured at 4.5 σ. This peak precisely overlapped with a previously modeled TBA molecule (shown right) found in the cavity of KcsA, positioned by aligning the selectivity filter structures of the TBA-KcsA complex (2HVK) with MthK. Two opposing subunits of the MthK channel pore structure are shown in grey with four potassium ions in the selectivity filter (green spheres).