Gain-of-function mutations in KCNK3 cause a developmental disorder with sleep apnea

Abstract

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K⁺ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the 'X-gate', a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K⁺ channels and their link with sleep apnea but also identify possible therapeutic strategies.

Fig. 1. DDSA mutations produce a gain-of-function phenotype in TASK-1.

a, Topological model of a TASK-1 subunit with the position of the DDSA variants labeled in red and the X-gate in dark gray. Two subunits co-assemble to form the K⁺ channel pore. b, Model showing the dimeric structure of TASK-1 (PDB: 6RV2), with one subunit shown in teal and the residues mutated in DDSA shown as red spheres. K⁺ ions within the selectivity filter are shown in purple. c, Representative TEVC recordings of WT TASK-1 and DDSA mutant currents in response to voltage steps from −120 to +50 mV in 20 mV steps from a holding potential of −80 mV. d, Current–voltage plot of WT TASK-1 (n = 38), L122V (n = 39), G129D (n = 44), N133S (n = 55), L241F (n = 42) and L239P (n = 57); data are presented as mean ± s.d. e, Currents for homomeric DDSA mutants, and ‘heterozygous’ channels formed from 1:1 coexpression of WT TASK-1 and DDSA mutants normalized to WT current at +50 mV: WT (n = 28), L122V (n = 32), L122V–WT (n = 32), G129D (n = 36), G129D–WT (n = 23), N133S (n = 29), N133S–WT (n = 23), L239P (n = 25), L239P–WT (n = 34), L241F (n = 42) and L241F–WT (n = 43), data are presented as mean ± s.d. With the exception of L239P, all mutant currents differ from WT (P < 0.01, two-paired t-test). f, WT or DDSA mutant TASK-1 coexpressed 1:1 with WT TASK-3. Currents normalized to WT heteromeric TASK-1–TASK-3 currents: WT (n = 50), L122V (n = 49), G129D (n = 44), N133S (n = 53), L239P (n = 41) and L241F (n = 30), data are presented as mean ± s.d. All mutant currents differ from WT (P < 0.01, two-paired t-test).

Fig. 2. Increased channel open probability due to destabilization of the X-gate.

a, Representative single-channel recordings of DDSA mutants at a holding potential of −160 mV. b, Single-channel P_o values for each mutant recorded at −160 mV. WT (n = 3), N133S (n = 3), L239P (n = 4), G129D (n = 4), L122V (n = 4), L241F (n = 4) and L122P (n = 5). All mutant P_o values differ from that for WT (P < 0.01, two-paired t-test). Single-channel conductance measurements for each mutant are also reported in Supplementary Table 1. c, Plot of the minimum pore radius at the lower X-gate during three independent repeats of molecular dynamics simulations of the WT TASK-1 structure compared to the N133S and L239P mutant structures. These variants destabilize the closed X-gate structure, allowing the channel to open more frequently.

Fig. 3. Dysfunctional GPCR-mediated inhibition in DDSA mutants.

a, Representative currents at +50 mV of WT TASK-1 channels (WT-WT) and ‘heterozygous’ channels from coexpressed WT and N133S subunits, over time while adding 10 µM carbachol. This concentration produces ~50% inhibition of WT TASK-1. b, Currents normalized to the initial WT current for WT TASK-1 coexpressed 1:1 with DDSA mutants before and after addition of 10 µM carbachol. WT TASK-1 (n = 6), L122V (n = 21), G129D (n = 19), N133S (n = 24), L239P (n = 18) and L241F (n = 26); data are presented as mean ± s.d. c,d, Equivalent recordings for WT TASK-1 coexpressed 1:1 with each DDSA mutant as indicated and the P2Y receptor (1:1:4). The current levels shown are before and after addition of 300 µM ATP normalized to the initial WT current. TASK-1 (n = 12), L122V (n = 13), G129D (n = 12), N133S (n = 12), L239P (n = 12) and L241F (n = 18); data are presented as mean ± s.d. The GPCR-mediated inhibition of mutant channel currents is reduced.

Fig. 4. Mutant channel pharmacology.

a, Representative excised membrane patch recordings of WT TASK-1 and N133S or L241F mutant channel activity in response to different concentrations of BAY1000493 applied to the intracellular side of the patch (10 nM, red). b, Corresponding dose–response curves for inhibition of either WT TASK-1 or DDSA mutants by BAY1000493; WT TASK-1 (n = 8), L122V (n = 10), G129D (n = 10), N133S (n = 11), L239P (n = 3) and L241F (n = 10); data are presented as mean ± s.e.m. Values for WT TASK-1 fitted with gray dashed line. c, Comparison of IC₅₀ values of various high-affinity TASK-1 inhibitors on either WT TASK-1 or the N133S mutant. BAY1000493: WT (n = 10), N133S (n = 11); PK-THPP: WT (n = 3), N133S (n = 9); A1899: WT (n = 4), N133S (n = 9); A239: WT (n = 4): N133S (n = 7); TPA: WT (n = 5), N133S (n = 9); doxapram: WT (n = 6), N133S (n = 6); carvedilol: WT (n = 4), N133S (n = 7), bupivacaine: WT (n = 9), N133S (n = 10). Data are presented as mean ± s.d.

Fig. 5. Proposed model for the effect of DDSA mutants on cellular electrical activity.

In WT cells, the activity of TASK-1 (that is, homomeric TASK-1 and/or heteromeric TASK-1–TASK-3) channels contributes to the hyperpolarized resting membrane potential (RMP). This activity can be inhibited by Gα_q-coupled receptor pathways and results in depolarization of the RMP. This gating process involves the cytoplasmic X-gate of TASK-1. However, in cells with a single heterozygous DDSA mutation affecting TASK-1, these variants (marked as X) result in defective closure of the X-gate (marked in red). Consequently, TASK-1 channel activity is increased and/or unresponsive to GPCR-mediated inhibition that amplifies the underlying gain of function. This increased channel activity keeps cells hyperpolarized near the RMP and also uncouples them from regulation by many different GPCR signaling pathways. Notably, mutant channels retain sensitivity to inhibition by several high-affinity small-molecule inhibitors, including BAY1000493. This offers a range of possible therapeutic strategies for these probands and strengthens the rationale for their proposed use in the treatment of sleep apnea.