Gain-of-function KCNJ6 Mutation in a Severe Hyperkinetic Movement Disorder Phenotype

Abstract

Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen-Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient's DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K⁺ selectivity and gained Ca²⁺ permeability. Notably, the inward current was inhibited by the Na⁺ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen-Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.

Keywords: K(IR)3; KCNJ6; channelopathy; inward rectifier; movement disorder; weaver mouse.

Figure 1:. EEG samples in hyperkinetic movements.

(A) Paroxysmal hyperkinetic movement (*) with no ictal epileptiform electrographic correlate. (B) Occasional poorly-formed bilateral frontal sharp waves (*) in non-REM sleep. (C) Normal 9 Hz alpha posterior dominant rhythm (*) during relaxed wakefulness with eyes closed. (D) Normal symmetric and synchronous sleep spindles (SS) and vertex waves (*) in non-REM sleep. Ceegraph EEG: anterior-posterior bipolar montage, sensitivity 15 μV/mm, time scale 30 mm/s, LFF 0.05 Hz, HFF 70 Hz.

Figure 2:. Identification of mutation in human KCNJ6.

(A) Sequencing of patient and parent DNA revealed a mutation in KCNJ6. Cartoon shows position of L171R mutation in GIRK2 ion channel (M1, M2: transmembrane domains; P: pore). (B) 3D structural model of human GIRK2 obtained by homology modeling from mouse GIRK2 channel (PDB: 3SYO). The mutation L171R allows Arg171 to potentially form H-bonds with Glu148 in the adjacent subunit. This electrostatic interaction could produce a more stable structure and impair conformational movements, i.e., opening/closing of the channel, or alter ion selectivity. (C) A detail of the 3D structural model of human KCNJ6 shows the network of interactions (H-bonds) that stabilize the conformation of the outer part (the selectivity filter) of the KCNJ6 channel.

Figure 3:. Change in gating and ion selectivity of mouse GIRK2(L173R) channels.

(A) Cartoon shows similarity between human and mouse GIRK2 protein, with homologous Leu highlighted (red). (B,C) Whole-cell patch-clamp currents are plotted as a function of time for wild-type GIRK2 (B) and mutant GIRK2(L173R) (C) channels. Currents were measured at −120 mV from HEK293T cells transfected with GABA_B1b/B2 receptor cDNA and either GIRK1, GIRK2 and/or GIRK2(L173R) cDNAs. Plots show the response to bath application of PrOH (100 mM, orange bar), Baclofen (100 μM, red bar), the sodium channel inhibitor QX-314 (100 μM, blue bar) and the K_ir potassium channel inhibitor Ba²⁺ (1 mM, green bar). Note that the mutant GIRK2(L173R) channel shows no activation with Baclofen or PrOH, lack sensitivity to inhibition with Ba²⁺, but is inhibited with QX-314 (100 μM). Similar results were observed for HEK293T cells expressing heterotetramers of GIRK1+GIRK2 (D) or GIRK1+GIRK2(L173R). (E) channels. Dashed line indicates zero current level. Bar graphs show average percentage change (± SEM) in current with indicated compounds (N=8–9). A negative percentage change indicates activation.

Figure 4:. GIRK1 and GIRK2(L173R) form dominant negative heterotetramers.

(A,B) Basal currents were elicited with a voltage step from −40 mV to −120 mV for GIRK1+GIRK2 channels (Ba²⁺-sensitive, light blue), GIRK2 channels (Ba²⁺-sensitive, blue) and mutant GIRK1+GIRK2(L173R) (QX-314-sensitive, pink). The mean current (black line) is shown with ± SEM (N=9). Dashed line indicates zero current level. Note similarity in kinetics for GIRK1+GIRK2(L173R) and GIRK2 alone. (C) Plots show the response to bath application of PrOH (100 mM, orange bar), Baclofen (100 μM, red bar), QX-314 (100 μM, blue bar) and Ba²⁺ (1 mM, green bar) for HEK293T cells expressing the GIRK1-GIRK2 dimer and GIRK1-GIRK2(L173R) dimer. Note the mutant heterotetramers show no activation or inhibition. (D) Bar graph shows average basal current density (± SEM) for GIRK1-GIRK2 dimer (Ba²⁺-sensitive) and GIRK1-GIRK2(L173R) dimer (QX-314 sensitive).

Figure 5:. Altered ion selectivity of GIRK2(L173R) channels.

(A) Current-voltage plots (I-V) show Ba²⁺-sensitive basal currents for wild-type channels and QX-314-sensitive basal current for GIRK2(L173R) channels with ‘2K’ (green trace), ‘5K’ (blue trace), and ‘20K’ (black trace) solutions. (B) Reversal potentials for wild-type (blue circles) and mutant (orange squares) GIRK2 channels are plotted as a function of extracellular [K⁺] (mean ± SEM, N=5). (C) Bar graph shows normalized FRET ratio measured in a fluorometric plate reader with ACSF for wild-type and L173R channels (± SEM, N=48). QX: 100 μM QX-314 (**P < 0.001; *P < 0.05, One-way ANOVA with Tukey’s post hoc test). HEK293T cells coexpressing FRET-based Ca²⁺ detector, Twitch 2B. (D) Bar graph shows average basal current density for wild-type (Ba²⁺-sensitive) and mutant (QX-314 sensitive) channels measured at −70 mV using ‘5K’ solution (± SEM). Outward current indicated as positive number and inward current as negative number (*P < 0.01, Student’s t test). (E) Bar graph shows average rectification ratio for wild-type and mutant channels with ‘5K’ solution. Rectification ratio was calculated as inward current (−20 mV + E_rev / outward current (+20 mV + E_rev) (± SEM) (*P < 0.001, Student’s t test).