Calcium ions open a selectivity filter gate during activation of the MthK potassium channel

Abstract

Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K(+) channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca(2+)-activated K(+) channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K(+). Thus, Ca(2+)-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.

Figure 1. Blockers as probes for Ca²⁺-dependent gate location in K⁺ channels.

(a) Comparison of BK (left) and MthK (right) channel architectures. The gating rings (red ribbons) with bound Ca²⁺ (green spheres) are structurally conserved between BK (pdb 3U6N) and MthK (pdb ILNQ). The unknown transmembrane structure for BK is illustrated with voltage-sensor domains (VSDs, brown) and pore domain (grey) with selectivity filter (blue). MthK lacks VSDs and the full-length protein structure is available, except for linkers between pore and gating ring (dashed lines). Locations of two putative gates are indicated by arrows. Only two subunits for the transmembrane domains are illustrated for clarity. (b) The gated access model: the pore domain has a bundle-crossing gate that prevents QA blocker binding to the aqueous vestibule. Rapid activation by Ca²⁺ results in immediate channel activity (purple circles and arrow) followed by blocker binding just below the selectivity filter. (c) The selectivity filter gate model: blocker has access to the binding site in both closed and open channels and steady-state block is reached before MthK is activated by Ca²⁺.

Figure 2. MthK is closed in 0 Ca²⁺ and is activated quickly with Ca²⁺.

(a) Schematic representation of the sequential-mixing stopped-flow device. The mixing sequence for a closed-state block assay is shown from left to right (Supplementary Fig. 1). MthK-reconstituted liposomes are mixed with blocker and incubated in a delay loop for a defined time interval followed by mixing with activating Ca²⁺ and fluorescence-quenching Tl⁺ into an optical cell for fluorescence readout. (b) Open MthK channels allow Tl⁺ entry (red arrow) into the liposomes, quenching the fluorescence of the encapsulated ANTS dye. (c) Fluorescence quench curves for MthK liposomes after 10 or 100 ms (grey and black, respectively) incubation with 2 mM Ca²⁺. Flux rates were from fits to stretched exponentials (red lines). Control fluorescence is in the absence of Tl⁺ (green). A small leak of Tl⁺ into liposomes is observed in experiments without Ca²⁺ (red), similar to the leak in MthK-free liposomes (cyan). The non-specific Tl⁺ leak in MthK liposomes was also measured in the presence of 100 μM TPeA (pink). A linear fit was used to analyse the slow Tl⁺ leak signals (black dotted lines). (d) Relative Tl⁺ flux rates as a function of Ca²⁺ incubation time for 0 (red), 2 (black) and 17.2 mM (green) Ca²⁺. Symbols are the mean±s.d. from three (or two for 0 mM Ca²⁺)-independent measurements. (e) Histogram of 49 independent estimates of apparent open probability in the absence of Ca²⁺ (P_o^ap(0 Ca²⁺), calculated using equations (4, 5, 6, 7, 8) in the Methods section). The average value was −0.00004±0.0002 (mean±s.e.m.). The histogram was fit with a Gaussian distribution (black line) with mean at P_o^ap(0 Ca²⁺)=0.0001±0.0003 (0.01±0.03%) and s.d. σ=0.0013±0.0003.

Figure 3. TPeA blocks closed and open MthK.

(a) Fluorescence quench traces after closed-state incubation with 3 μM TPeA for 0.1 (black), 10 s (cyan) and no blocker control (green) (average of 4–7 repeats). (b) Relative Tl⁺ flux rates versus incubation time from data as in a. Red line is a fit with equations (10 and 11) (τ_eq=1.6±0.2 s, =2.1±0.2 μM, =0.14±0.02 μM⁻¹ s⁻¹). (c) Dose–response curve for closed-state TPeA equilibrium block after 10-s blocker incubation. Red line is a Hill equation fit (Equation 12, =2.0±0.2 μM, n_H=0.84±0.08). Dose–response curve for open-state block after the 2 ms mixing time (grey circles). The grey line has no theoretical meaning. (d) Fluorescence quench traces after 30-ms incubation with 17.2 mM Ca²⁺ to measure open-state block by 0 (green), 1 (cyan), 3 (black) or 10 μM (red) TPeA (average of 4–7 repeats). (e) Relative Tl⁺ flux rates versus incubation time with open MthK for three [TPeA], coloured as in d. The results were simultaneously fit to equation (11) (lines; =4.6±0.2 μM, k_on^closed=20±3 μM⁻¹ s⁻¹). (f) Dose–response curve for TPeA binding to open MthK after 30-ms incubation with blocker (results from e) was fit by the Hill equation (red line; =5.2±0.3 μM, n_H=0.98±0.07). Mean±s.d. from at least three independent samples, except for experiments marked (*) in e where n=2.

Figure 4. Br-bTBA and bbTBA block closed MthK more slowly than TPeA.

(a) Relative Tl⁺ flux rates as a function of Br-bTBA and bbTBA (structures inset) incubation with closed MthK channels. Blocker equilibrations were fit to exponential functions, τ_Br-bTBA=9±3 s, τ_bbTBA=32±7 s (Equation 10, black and red solid lines, respectively). The fitted blocker time course for TPeA (from Fig. 3b) is shown for comparison (black dashed line). (b) Relative Tl⁺ flux rates versus incubation time with open MthK for 1 (black squares) and 3 μM (red circles) bbTBA. The results were simultaneously fit to equation (11) (lines; =2.1±0.2 μM, =17±2 μM⁻¹ s⁻¹). (c) Closed-state Br-bTBA dose–response curve for 45-s blocker incubations before activation of MthK by 17.2 mM Ca²⁺ (black squares). The results were fit to the Hill equation (red line; =2.5±0.2, n_H=1.16±0.09). Dose response for open-state Br-bTBA block after the mixing dead time (grey circles). Grey line has no theoretical meaning. Symbols are the mean±s.d. from at least three (only two for bbTBA data in b) independent measurements.

Figure 5. Closure at the MthK selectivity filter is accompanied by a conformational change near the intracellular entryway.

(a) Model for QA block of the closed and open MthK channel. The selectivity filter is non-conductive in the absence of Ca²⁺ and the intracellular entryway (grey) is narrowed but still allows blocker/K⁺ entry into vestibule (left). Channel activation opens the selectivity filter gate (purple K⁺ inside blue filter) and increases blocker access rate into the pore (right). (b) The radius of a hydrated K⁺ is smaller than the extended structures in TPeA⁺, Br-bTBA⁺ and bbTBA⁺, suggesting that K⁺ can access the closed MthK intracellular entryway despite the constriction.