BK channel properties correlate with neurobehavioral severity in three KCNMA1-linked channelopathy mouse models

Abstract

KCNMA1 forms the pore of BK K⁺ channels, which regulate neuronal and muscle excitability. Recently, genetic screening identified heterozygous KCNMA1 variants in a subset of patients with debilitating paroxysmal non-kinesigenic dyskinesia, presenting with or without epilepsy (PNKD3). However, the relevance of KCNMA1 mutations and the basis for clinical heterogeneity in PNKD3 has not been established. Here, we evaluate the relative severity of three KCNMA1 patient variants in BK channels, neurons, and mice. In heterologous cells, BK^N999S and BK^D434G channels displayed gain-of-function (GOF) properties, whereas BK^H444Q channels showed loss-of-function (LOF) properties. The relative degree of channel activity was BK^N999S > BK^D434G>WT > BK^H444Q. BK currents and action potential firing were increased, and seizure thresholds decreased, in Kcnma1^N999S/WT and Kcnma1^D434G/WT transgenic mice but not Kcnma1^H444Q/WT mice. In a novel behavioral test for paroxysmal dyskinesia, the more severely affected Kcnma1^N999S/WT mice became immobile after stress. This was abrogated by acute dextroamphetamine treatment, consistent with PNKD3-affected individuals. Homozygous Kcnma1^D434G/D434G mice showed similar immobility, but in contrast, homozygous Kcnma1^H444Q/H444Q mice displayed hyperkinetic behavior. These data establish the relative pathogenic potential of patient alleles as N999S>D434G>H444Q and validate Kcnma1^N999S/WT mice as a model for PNKD3 with increased seizure propensity.

Keywords: BK channel; KCNMA1; calcium-activated potassium channel; dentate gyrus; epilepsy; mouse; neuroscience; paroxysmal non-kinesigenic dyskinesia.

Figure 1.. Location and consequence of KCNMA1 variants in the BK K⁺ channel.

(A) KCNMA1 forms the homotetrameric BK channel. Each α subunit is comprised of seven transmembrane domains (S0‒S6) and an intracellular gating ring with hydrophobic segments (S7–S10, black). Pore (+) opening and closing is regulated by voltage-sensitive residues in S2‒S4 (VSD), the AC domain (βA to αC), and two Regulators of Conductance of Potassium (RCK) domains in the gating ring (gray), each containing a Ca²⁺ binding site (green) (Yang et al., 2015; Giraldez and Rothberg, 2017). (B) BK channel structure showing two opposing subunits with Ca²⁺ bound in the gating ring (PDB 6V38). H444Q (purple) and D434G (blue) are located within the βB-αB and αA and βB of the AC domain, respectively, a region within RCK1 affecting Ca²⁺-dependent gating (Du et al., 2005; Tao and MacKinnon, 2019). N999S (red) is located at the helix bend in the middle of the S10 domain within RCK2 (Tao and MacKinnon, 2019). (C) Representative inside-out patch-clamp recordings from BK^WT, BK^N999S, BK^D434G, BK^H444Q channels expressed in HEK293 cells. Macroscopic BK currents were recorded in symmetrical K⁺ and 1 μM intracellular Ca²⁺ by holding patches at −100 mV, stepping from −100 to 250 mV for 30 ms, followed by a tail step −100 mV for 15 ms. Scale bars: 1 nA, 5 ms. (D) Normalized conductance-voltage (G-V) relationships fit with Boltzmann functions (solid lines). There was no change in the slope factor (z) for any of the variants (p=0.06, one-way ANOVA). BK^WT (n=12), BK^N999S (n=9), BK^D434G (n=12), and BK^H444Q (n=12). (E) Voltage of half-maximal activation (V_1/2) obtained from Boltzmann fits for individual patches. *p<0.0001. One-way ANOVA with Dunnett’s post hoc. (F) Activation time constants (τ_act). BK^N999S and BK^D434G channels had decreased τ_act compared to BK^WT, either across all voltage steps (mixed effects model for repeated measures with Bonferroni post hoc, p<0.01) or above 120 mV (p<0.05), respectively. At lower voltages, BK^D434G channels were more steeply voltage dependent, but did not exceed the fast activation time constants of BK^N999S channels. BK^H444Q channels had increased τ_act compared to BK^WT between 160 and 250 mV (p<0.05). Inset: Representative current traces from 170 mV step, scaled to the maximal current to illustrate activation timecourse (x-axis scale bar: 10 ms). τ_act for BK^WT currents was 3.8±0.3 ms, while BK^N999S and BK^D434 currents activated faster (0.9±9.1 and 1.8±0.1 ms, respectively) and BK^H444Q activated slower (6.5±0.5 ms). (G) Deactivation time constants (τ_deact). BK^N999S and BK^D434G channels had increased τ_deact compared to BK^WT, across all voltage steps (mixed effects model for repeated measures with Bonferroni post hoc, p<0.01), with the exception of ‒160 (p>0.05), respectively. BK^H444Q channels had decreased τ_deact compared to BK^WT between ‒190 mV and between ‒140 and ‒20 mV (p<0.05). Inset: Representative current traces from ‒20 mV step, scaled to the maximal current to illustrate deactivation timecourse (x-axis scale bar: 10 ms). τ_deact for BK^WT currents was 0.7±0.01 ms, while BK^N999S and BK^D434 currents deactivated slower (4.5±0.7 and 1.5±0.1 ms, respectively) and BK^H444Q deactivated more quickly (0.4±0.01 ms). Data are presented as mean ± SEM. Additional data on the effects of stimulants on BK^WT and BK^N999S channels appears in Figure 1—figure supplement 1.

Figure 1—figure supplement 1.. Sequences for CRISPR/Cas9 editing of the mouse Kcnma1 gene.

(A) Kcnma1^N999S/WT mice were generated by introducing a non-synonymous mutation within the codon AAT→ AgT (red boxes) in exon 25. WT sequence is C57BL/6J. Underlined nucleotides are the gRNA sequence. Lowercase letters denote mutations. Chromatogram from an N1 heterozygous mouse. (B) Kcnma1^D434G/WT mice were generated by mutation within the codon GAT→ GgT in exon 10. Chromatogram from a founder mouse. (C) Kcnma1^H444Q/WT mice were generated by mutation within the codon CAC → CAg in exon 10 (same guide RNA as D434G). Chromatogram from a founder mouse.

Figure 1—figure supplement 2.. Effects of lisdexamfetamine (lis) and dextroamphetamine (d-amp) on BK^WT and BK^N999S channels expressed in HEK293 cells.

D-amp and lis, a prodrug of D-amphetamine and L-lysine, have been reported to treat paroxysmal non-kinesigenic dyskinesia (PNKD) episodes in six children harboring N999S variants, as well as one child harboring another gain-of-function (GOF) variant (Miller et al., 2021; Zhang et al., 2020). To test if either drug has a direct effect on BK currents in heterologous cells, as has been suggested for methamphetamine (Lin et al., 2016; Fu et al., 2021; Tatro et al., 2013; Wang et al., 2013), d-amp and lis were applied to patches from HEK293 cells expressing BK^WT or BK^N999S. Macroscopic BK currents were evaluated after perfusion of each drug and compared to pre-drug control current levels. Normalized data presented as the proportion of the maximal current (I/I_max) for each patch, before (control) and after drug application. Neither 155 ng/ml lis (BK^N999S: n=7, p=0.98; one-way ANOVA) nor 155 ng/ml d-amp (BK^WT: n=4, p=0.99; one-way ANOVA and BK^N999S: n=6, p=0.15; one-way ANOVA) produced a decrease in BK current levels. However, the BK channel inhibitor paxilline (pax, 100 nM), applied as a control, fully abrogated BK^WT or BK^N999S currents at the end of each experiment (p<0.001; one-way ANOVA for all). Data are presented as mean ± SEM.

Figure 2.. Increased BK current in Kcnma1^N999S/WT and Kcnma1^D434G/D434G granule neurons.

Whole-cell macroscopic BK currents were recorded in 1 μM tetrodotoxin (TTX) and 2 mM 4-aminopyridine (4-AP), isolated with 10 μM paxilline, and normalized to cell capacitance. Activating voltage steps were applied from V_h of ‒90 mV, stepping from ‒100 to +30 mV for 150 ms, and back to ‒90 mV for 130 ms. (A‒C) Peak BK current density versus voltage relationships. Data are presented as mean ± SEM. * and ^†, p<0.05, two-way repeated measures ANOVA with Bonferroni post hoc. Insets: Representative BK current traces at 30 mV. Scale bars: 500 pA, 5 ms. (A) BK current density was larger in Kcnma1^N999S/WT neurons (n=16 neurons, 5 mice) compared to Kcnma1^WT/WT (n=14 neurons, 4 mice) at ‒30 mV (p=0.0114), ‒20 (p=0.0210), ‒10 (p=0.0426) voltage steps (indicated with *). (B) BK current density was larger in Kcnma1 ^D434G/D434G neurons (n=12 neurons, 3 mice) compared to Kcnma1^WT/WT (n=10 neurons, 4 mice) at density at ‒40 mV (p=0.0112), ‒30 (p=0.0026), ‒20 (p=0.0031), ‒10 (p=0.0038), 0 (p=0.0078), 10 (p=0.0068), 20 (p=0.0071), 30 (p=0.0088) voltage steps (*). Kcnma1^D434G/WT mice (n=9 neurons, 3 mice) had higher BK current density compared to Kcnma1^WT/WT at ‒30 mV only (^†p=0.0321). (C) BK current density was not different in Kcnma1^H444Q/WT neurons (n=7 neurons, 2 mice) compared to Kcnma1^WT/WT (n=6 neurons, 3 mice).

Figure 3.. Increased intrinsic excitability in Kcnma1^N999S/WT, Kcnma1^D434G/WT, and Kcnma1^D434G/D434G granule neurons.

In current-clamp mode, step currents from 0 to 400 pA were applied to dentate granule neurons under the same ionic conditions used to record BK currents. (Ai–Aiii) Representative AP trains elicited from the 200 pA current injection step in WT and transgenic neurons. Scale bar: 20 mV, 100 ms. (Bi–Biii) Input-output relationship for firing frequency versus step current injection. Data are presented as mean ± SEM. *p<0.05, two-way repeated measures ANOVA with Bonferroni post hoc. (Bi) Kcnma1^N999S/WT (n=23 neurons, 5 mice) firing was higher than Kcnma1^WT/WT (n=16 neurons, 5 mice) at 160 pA (p=0.0426), 180 pA (p=0.0143), 200 (p=0.0068), 220 pA (p=0.0009), and 240 pA (p=0.0337) current steps. (Bii) Kcnma1^D434G/WT (n=27 neurons, 5 mice) firing was higher than Kcnma1^WT/WT (n=22 neurons, 5 mice) at 260 pA (p=0.0452), 280 (p=0.0314), 300 (p=0.0351), 320 (p=0.0177), 340 (p=0.0309), 360 (p=0.0358), 380 (p=0.0312), and 400 (p=0.0444) current steps. Kcnma1^D434G/D434G (n=19 neurons, 4 mice) firing was higher than Kcnma1^WT/WT at 40 pA (p=0.0266), 60 (p=0.0233), 80 (p=0.0277), 100 (p=0.0130), 120 (p=0.0074), 140 (p=0.0119), 160 (p=0.0084), 180 (p=0.0063), 200 (p=0.0059), and 220 (p=0.0261) current steps. (Biii) Kcnma1^H444Q/WT (n=8 neurons, 2 mice) and Kcnma1^H444Q/H444Q (n=11 neurons, 2 mice) firing was not different than Kcnma1^WT/WT (n=7 neurons, 1 mouse) at any current step (p=0.3222). (Ci–Ciii) Initial slope for the firing rate gain between 0 and 160 pA current injections. Data are presented as mean ± SEM, with individual data points. (Ci) Kcnma1^N999S/WT firing slope was increased compared to WT (*p=0.0034; t-test). (Cii) Kcnma1 ^D434G/D434G firing slope was increased compared to WT (*p=0.0051; one-way ANOVA), Kcnma1 ^D434G/WT slopes were unchanged (p=0.9774). (Ciii) Kcnma1^H444Q/WT and/or Kcnma1^H444Q/H444Q firing slopes were not different than WT (p=0.9658). (Di–Diii) Maximum firing frequency. Data are presented as mean ± SEM. (Di) Maximal firing from Kcnma1^N999S/WT neurons was increased compared to WT (*p<0.0001; t-test). (Dii) Maximal firing from Kcnma1 ^D434G/WT and Kcnma1 ^D434G/D434G neurons was increased compared to WT (*p=0.0387 and p=0.0111, respectively; one-way ANOVA). (Diii) Maximal firing from Kcnma1^H444Q/WT and/or Kcnma1^H444Q/H444Q neurons was not different than WT (p=0.4625; one-way ANOVA). Passive membrane properties for this dataset appear in Figure 3—figure supplement 1. Action potential waveform analysis for this dataset appears in Figure 3—figure supplement 2.

Figure 3—figure supplement 1.. Passive membrane properties.

Data are presented as mean ± SEM. Resting membrane potential (RMP) was ‒80 to ‒82 mV across WT controls. No significant depolarizations were observed between littermate controls and Kcnma1^N999S/WT, Kcnma1^D434G/D434G, Kcnma1^H444Q/WT, and Kcnma1^H444Q/H444Q dentate granule neurons (p>0.05, unpaired t-test and one-way ANOVA, respectively). Similarly, the range for input resistance (R_i) was 320–364 MΩ for WT neurons and was not different in any transgenic condition. These results are consistent with previous studies in dentate granule cells finding no effect of BK channel inhibition on RMP or R_i (Bock and Stuart, 2016; Brenner et al., 2005). The only condition showing a difference from the respective WT control was a greater membrane capacitance (C_m) in Kcnma1^D434G/WT neurons (*p=0.0036). An explanation for this change in C_m is unclear from the data. However, this difference could have the potential to reduce the firing gain at lower current injections, preventing the Kcnma1^D434G/WT input-output curve from looking similar to Kcnma1^N999S/WT (Figure 3Bi, Bii).

Figure 3—figure supplement 2.. Kcnma1^N999S/WT, Kcnma1^D434G/WT, and Kcnma1^D434G/D434G action potential waveforms.

(A) Superimposed Kcnma1^N999S/WT and Kcnma1^WT/WT waveforms from the 10th action potential at the 200 pA current injection from data in Figure 3. (B) Action potential half-width (t_1/2) was not different in Kcnma1^N999S/WT neurons versus WT controls (p=0.6617; Mann-Whitney test). (C) Fast afterhyperpolarizations (fAHP) amplitude was not different in Kcnma1^N999S/WT neurons versus WT controls (p=9214; Mann-Whitney test). (D) AHP decay 3 ms after the peak was faster in Kcnma1^N999S/WT neurons (Kcnma1^N999S/WT 0.60±0.03 mV/ms) compared to WT controls (0.41±0.03 mV/ms, *p=0.0002; t-test). In panels B-D, Kcnma1^N999S/WT (n = 23 neurons) and Kcnma1^WT/WT (n = 16 neurons). (E) Superimposed Kcnma1^D434G/D434G, Kcnma1^D434G/WT, and Kcnma1^WT/WT waveforms from the 10th action potential at the 200 pA current injection from data in Figure 3. (F) t_1/2 was different in Kcnma1^D434G/WT (*p=0.0002; Kruskal-Wallis test), but not Kcnma1^D434G/D434G neurons (p=0 > 0.9999). (G) fAHP amplitudes were comparable in Kcnma1^D434G mice (p=0.3441; Kruskal-Wallis test). (H) AHP decay 3 ms after the peak was faster in Kcnma1^D434G/D434G neurons (0.69±0.04 mV/ms) compared to WT controls (0.46±0.04 mV/ms, *p=0.0002), but not in Kcnma1^D434G/WT (0.56±0.02 mV/ms, p=0.0620; one-way ANOVA). In panels F-H, Kcnma1^D434G/D434G (n = 19 neurons), Kcnma1^D434G/WT (n = 27 neurons), and Kcnma1^WT/WT (n = 22 neurons). Data are presented as mean ± SEM, with individual data points.

Figure 4.. Pentylenetetrazol (PTZ)-induced seizures in mice.

(Ai–Aiv) Latency to initial seizure after PTZ injection. Data are individual mice with median and inter-quartile range. (Ai) Latency was decreased in Kcnma1^N999S/WT mice (n=13) compared to Kcnma1^WT/WT (n=18, *p=0.0006; Mann-Whitney test). (Aii) Latency was decreased in Kcnma1^D434G/WT mice (n=7) compared to Kcnma1^WT/WT (n=11, *p=0.0041; Mann-Whitney test). (Aiii) Seizure latency was comparable between Kcnma1^H444Q/WT (n=7) and Kcnma1^WT/WT (n=4, p=0.5273; Mann-Whitney test). (Aiv) No differences were found in seizure latency between Kcnma1^‒/‒ (n=9) and Kcnma1^+/+ mice (n=13, p=0.2282; Mann-Whitney test). (Bi–iv) Total EEG power after PTZ injection (y-axis in µV²/Hz × 10²). Data are individual mice with median and inter-quartile range. (Bi) EEG power was not different between Kcnma1^N999S/WT (n=7) and Kcnma1^WT/WT (n=11, p=0.0619; t-test). (Bii) Kcnma1^D434G/WT (n=4) was not different from Kcnma1^WT/WT (n=6, p=0.7563; t-test). (Biii) Kcnma1^H444Q/WT (n=6) was not different from Kcnma1^WT/WT (n=4, p=0.9641; t-test). (Biv) Kcnma1^‒/‒ (n=10) was not different from Kcnma1^+/+ (n=9, p=0.2134; t-test). (C) Representative EEG traces over 45 min at baseline and after PTZ injection (red line). (D) Expanded EEG traces for the first seizure indicated with the red boxes in (C). Representative videos for this dataset appear in Figure 4—videos 1–4.

Figure 5.. Stress-induced paroxysmal dyskinesia.

(A) Control: Without restraint stress, there was no difference in the time spent immobile between Kcnma1^WT/WT (n=10) and Kcnma1^N999S/WT mice (n=6, p>0.9999; two-way ANOVA with Bonferroni post hoc). Restraint stress: Immobility time was longer for restrained Kcnma1^N999S/WT mice (n=11) compared to Kcnma1^WT/WT (n=7, *p=0.0001; one-way ANOVA), and between restrained Kcnma1^N999S/WT mice (n=11) compared to unrestrained Kcnma1^N999S/WT mice (n=6, *p<0.0001). In contrast, unrestrained Kcnma1^WT/WT mice (n=10) had no differences from restrained Kcnma1^WT/WT mice (n=7, p=0.1174). (B) Grooming behavior increased in restrained Kcnma1^WT/WT mice (n=7) compared to unrestrained Kcnma1^WT/WT mice (n=10, *p=0.0300; t-test), and in restrained Kcnma1^N999S/WT mice (n=11) compared to unrestrained Kcnma1^N999S/WT mice (n=6, *p=0.0174; t-test). (C) Immobility time was longer for saline-treated Kcnma1^N999S/WT mice (n=7) compared to Kcnma1^WT/WT (n=7, *p=0.0018) and d-amp-treated Kcnma1^N999S/WT mice (n=6, *p=0.0053; two-way ANOVA with Bonferroni post hocs). There was no difference between d-amp-treated Kcnma1^WT/WT mice (n=7), d-amp-treated Kcnma1^N999S/WT mice (n=6, p>0.9999), and saline-treated Kcnma1^WT/WT mice (n=7, p>0.9999). (D) After restraint, Kcnma1^D434G/D434G mice (n=7) spent more time immobile compared to Kcnma1^WT/WT mice (n=14, *p=0.0166; one-way ANOVA). However, Kcnma1^D434G/WT mice were not different (n=18, p=0.7174). (E) Immobility time was shorter in restrained Kcnma1^H444Q/H444Q mice (n=8) compared to Kcnma1^H444Q/WT mice (n=11, *p=0.0081; t-test). Kcnma1^WT/WT mice were not included in the statistical analysis due to small sample size (n=3). (F) Kcnma1^–/– mice (n=8) had reduced immobility compared to Kcnma1^–/+ mice (n=8) and Kcnma1^+/+ mice (n=11, p=0.0535; Kruskal-Wallis test). Data are individual mice with median and inter-quartile range. Representative videos for this dataset appear in Figure 5—video 5‒1.

Figure 5—figure supplement 1.. Locomotor, stress, and immobility analysis methodology controls in Kcnma1^N999S/WT mice.

(A) Unrestrained WT C57BL6J-background mice were used to test for any baseline effects of the injection procedures (saline) or d-amp (0.5 mg/kg) on immobility in the beaker assay, assessed 30 min after injection. No difference between saline (n=5) and d-amp-injected mice (n=5, p=0.1429, Mann-Whitney test) was observed. (B) Unrestrained WT C57BL6J-background mice were used to test for any baseline effects of d-amp (0.5 mg/kg) on locomotor wheel running activity. Normalized activity after injection was comparable between saline (n=3) and d-amp-injected mice (n=3, p=0.7000, Mann-Whitney test). (C) Kcnma1^WT/WT and Kcnma1^N999S/WT mice used in the restraint-induced immobility assay in main Figure 5C were scored for grooming behavior after d-amp treatment. No significant differences were present between saline-injected Kcnma1^WT/WT mice (n=7), saline-injected Kcnma1^N999S/WT mice (n=7, p=0.1456), and d-amp-injected Kcnma1^WT/WT mice (n=7, p>0.9999, two-way ANOVA with Bonferroni post hoc). Additionally, d-amp-injected Kcnma1^N999S/WT mice (n=6) were not different from d-amp-injected Kcnma1^WT/WT mice (n=7, p>0.9999) and saline-injected Kcnma1^N999S/WT mice (n=7, p=0.1461). (D) Center point movement parameters were calculated using automated analysis (EthoVision software) from the same videos of restraint stress-induced immobility in main Figure 5C. Immobility time was longer for saline-injected Kcnma1^N999S/WT mice (n=7) compared to Kcnma1^WT/WT (n=7, *p=0.0077) and d-amp-injected Kcnma1^N999S/WT mice (n=6, *p=0.0223; two-way ANOVA with Bonferroni post hoc). In contrast, d-amp-injected Kcnma1^N999S/WT mice (n=6) were not different from d-amp-injected Kcnma1^WT/WT mice (n=7, p>0.9999) or saline-injected Kcnma1^WT/WT mice (n=7, p>0.9999). All data are median and inter-quartile range.

Figure 6.. Motor coordination in Kcnma1^N999S/WT mice.

(A) Locomotor wheel running parameters calculated from average activity counts over 48 hr from singly housed mice with free access to wheels. (Ai) Distance covered was reduced for Kcnma1^N999S/WT (n=12) compared to Kcnma1^WT/WT mice (n=11, *p=0.0411; t-test). (Aii) Maximum speed was comparable between Kcnma1^N999S/WT (n=11) and Kcnma1^WT/WT mice (n=12, p=0.3618; t-test). (Aiii) Duration of time off wheels (gap duration) was comparable between Kcnma1^N999S/WT (n=11) and Kcnma1^WT/WT mice (n=12, p=0.8281; t-test). (Aiv) Number of times the mouse was off the wheel (gap events) was higher for Kcnma1^N999S/WT (n=12) compared to Kcnma1^WT/WT mice (n=11, *p=0.0040; t-test). (B) Open field assay. Kcnma1^N999S/WT mice (n=8) covered the same distance as Kcnma1^WT/WT mice (n=8) in a 15 min trial (p=0.6973; t-test). (C) Acute muscle strength was tested by hanging mice from a stationary platform (cage lid) for 120 s. Fall latency was lower in Kcnma1^N999S/WT (n=11) compared to Kcnma1^WT/WT mice (n=10, *p=0.0014; Mann-Whitney test) indicating weaker grip strength. (D) Rotarod assay. Fall latency was lower for Kcnma1^N999S/WT mice (n=11) on day 2 (*p=0.0045) and day 7 (*p=0.0124) compared to Kcnma1^N999S/WT mice (n=12). Motor learning was observable as an improvement in fall latency times across the three trials on each day (data not shown), suggesting the overall impairment was related to motor coordination and not learning. Data are presented as individual data points with median and inter-quartile range (A–C) and mean ± SEM (D). Results for these assays with Kcnma1^D434G, Kcnma1^H444Q, and Kcnma1^–/– mice appear in Figure 6—figure supplements 1 and 2. For these assays, the baseline motor coordination severity fell in the series Kcnma1^‒/‒>Kcnma1^D434G/D434G > Kcnma1^N999S/WT>Kcnma1^H444Q/H444Q.

Figure 6—figure supplement 1.. Motor coordination in Kcnma1^D434G, Kcnma1^H444Q, and Kcnma1^‒/‒ mice.

(Ai) Distance was comparable between Kcnma1^D434G/WT (n=11) and Kcnma1^WT/WT mice (n=12, p=0.8118; t-test). (Aii) Distance was comparable between Kcnma1^H444Q/H444Q (n=7) and Kcnma1^H444Q/WT (n=6, p=0.4880; t-test). Kcnma1^WT/WT mice were not included in statistical analysis in panels Aii–Dii due to small sample size. (Aiii) Distance was reduced for Kcnma1^–/– (n=10) compared Kcnma1^+/+ (n=9, *p=0.0032), but not for Kcnma1^–/+ mice (n=15, p=0.9057; one-way ANOVA). (Bi) Maximum speed was lower for Kcnma1^D434G/WT (n=11) compared to Kcnma1^WT/WT mice (n=12, *p=0.0085; t-test). (Bii) Maximum speed was comparable between Kcnma1^H444Q/H444Q (n=7) and Kcnma1^H444Q/WT (n=6, p=0.3634; t-test). (Biii) Maximum speed was lower for Kcnma1^–/– (n=10) compared to Kcnma1^+/+ (n=9, *p=0.0024), but not for Kcnma1^–/+ mice (n=15, p=0.9871; one-way ANOVA). (Ci) Gap duration was reduced for Kcnma1^D434G/WT (n=11) compared to Kcnma1^WT/WT mice (n=12, *p=0.0467; t-test). (Cii) Gap duration was comparable between Kcnma1^H444Q/H444Q (n=7) and Kcnma1^H444Q/WT mice (n=6, p=0.8326; t-test). (Ciii) Gap duration was higher in Kcnma1^–/– (n=10) compared to Kcnma1^+/+ mice (n=9, *p=0.0026), but not for Kcnma1^–/+ mice (n=15, p=0.8987; one-way ANOVA). (Di) Gap events were comparable between Kcnma1^D434G/WT (n=11) and Kcnma1^WT/WT mice (n=12, p=0.7425; t-test). (Dii) Gap events were comparable between Kcnma1^H444Q/H444Q (n=7) and Kcnma1^H444Q/WT (n=6, p=0.9341; t-test). (Diii) Gap events were comparable for Kcnma1^–/– (n=10), Kcnma1^–/+ (n=15), and Kcnma1^+/+ mice (n=9, p=0.3047; one-way ANOVA). All data are presented as individual data points with median and inter-quartile range.

Figure 6—figure supplement 2.. Hanging wire and rotarod in Kcnma1^D434G, Kcnma1^H444Q, and Kcnma1^‒/‒ mice.

(Ai-iii) Hanging wire assay. Data are presented as individual data points with median and inter-quartile range. (Ai) Fall latency was comparable for Kcnma1^D434G/D434G (n=4), Kcnma1^D434G/WT (n=11), and Kcnma1^WT/WT mice (n=11, p=0.8329; Kruskal-Wallis test), providing no evidence for acute differences in strength. (Aii) Fall latency was reduced for Kcnma1^H444Q/H444Q (n=8) compared to Kcnma1^H444Q/WT mice (n=14, *p=0.0465; Mann-Whitney test). Kcnma1^WT/WT mice were not included in the statistical analysis due to small sample size (n=3). (Aiii) Fall latency times were lower for Kcnma1^‒/‒ mice (n=10) compared to Kcnma1^+/+ mice (n=10, *p=0.0036), but not for Kcnma1^‒/+ mice (n=17, p>0.9999; Kruskal-Wallis test). No Kcnma1^‒/‒ mouse had the ability to hang on longer than 60 s, consistent with previous reports (Meredith et al., 2004; Sausbier et al., 2004; Wang et al., 2020; Yao et al., 2021). (Bi-iii) Time to fall in rotarod assay. Data are presented as mean ± SEM. (Bi) Fall latency was lower for Kcnma1^D434G/D434G mice (n=4) on day 2 (p<0.0001), day 4 (p=0.0030), day 6 (p<0.0001), and day 7 (p=0.0009) compared to Kcnma1^WT/WT mice (n=21), but were comparable in Kcnma1^D434G/WT mice (n=18; *p<0.05, repeated measures ANOVA with Bonferroni post hoc). (Bii) Fall latency was not different between Kcnma1^H444Q/H444Q (n=7) and Kcnma1^H444Q/WT (n=10; repeated measures ANOVA with Bonferroni post hoc); however, performance was highly variable, reducing the ability to make a firm conclusion from this data. Kcnma1^WT/WT mice were not included in the statistical analysis due to small sample size (n=2). (Biii) Fall latency was lower for Kcnma1^‒/‒ mice (n=6) on day 1 (p=0.0277), day 2 (p=0.0056), day 3 (p=0.0122), day 4 (p=0.0081), day 5 (p=0.0166), day 6 (p=0.0071), and day 7 (p=0.0168) compared to Kcnma1^+/+ mice (n=6), but were comparable in Kcnma1^‒/+ mice (n=13; *p<0.05, repeated measures ANOVA with Bonferroni post hoc).