BK channel properties correlate with neurobehavioral severity in three KCNMA1-linked channelopathy mouse models
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Evaluation Summary:
This study is of broad interest to neuroscientists interested in membrane excitability and translational biologists and physicians eager for robust animal models for disorders involving mutations in the KCNMA gene, such as paroxysmal nonkinesigenic dyskinesia PNKD3. Here, phenotypes of mouse models of three of the more common patient disease-related mutations in KCNMA are evaluated for similarities to patient phenotypes. This work establishes that BK channel mutations linked to human neurological disease can, on their own, cause similar pathology in mice, and it also begins to provide neurological bases for the associated behavioral deficits. Importantly, one of the mutant alleles expressed in mice most closely phenocopies the patient phenotype and will serve as an important animal model for studies seeking therapeutic treatments for the resulting debilitating disease moving forward.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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.
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Author Response
Reviewer #3 (Public Review):
The primary strength of this study is in establishing the N999S heterozygous mouse as a useful model system for debilitating paroxysmal non-kinesigenic dyskinesia (PKND), with or without epilepsy. This outcome was hard-won following a comprehensive analysis of biophysical, neurophysiological, and behavioral tests. Ultimately the convincing evidence was demonstrated through a clever application of a stress-related behavioral test (quite in alignment with triggers in patients) to elicit the hypo-motility associated with PKND. Like patients who exhibit variable penetrance, even highly inbred mice exhibit much variability, and uncovering a robust phenotype took a nuanced approach and perseverance.
To reach this point, several experiments provided mechanistic insights into the mutant channel …
Author Response
Reviewer #3 (Public Review):
The primary strength of this study is in establishing the N999S heterozygous mouse as a useful model system for debilitating paroxysmal non-kinesigenic dyskinesia (PKND), with or without epilepsy. This outcome was hard-won following a comprehensive analysis of biophysical, neurophysiological, and behavioral tests. Ultimately the convincing evidence was demonstrated through a clever application of a stress-related behavioral test (quite in alignment with triggers in patients) to elicit the hypo-motility associated with PKND. Like patients who exhibit variable penetrance, even highly inbred mice exhibit much variability, and uncovering a robust phenotype took a nuanced approach and perseverance.
To reach this point, several experiments provided mechanistic insights into the mutant channel behavior. First, whole-cell patch clamp experiments revealed shifts in the G-V consistent with gain-of-function behavior previously characterized using the N999S and D434G mutants expressed heterologously. Novel observations of H444Q revealed a loss-of-function (LOF) behavior with the G-V shifted to positive potentials but to a lesser degree. These electrophysiological phenotypes establish the rank of predicted severity as N999S>D434G>H444Q.
This prediction was tested in brain slices of heterozygous animals where the mutant channels would be normally spliced and associate with WT subunits and other components such as beta subunits. The investigators evaluated BK currents by patch clamp from hippocampal neurons where BK channels are known to play key functional roles. Both N999S and D434G showed the predicted increase in current magnitude, though interestingly the differences between them apparent in heterologous expression were lost in the native setting. Curiously, no differences in BK current magnitude were observed in neurons of heterozygotes carrying the putatively LOF mutation H444Q.
In terms of seizure susceptibility, D434G mutants different from WT and less severe than N999S mutants with respect to time to evoked seizure, although differences in "EEG power" were not statistically significant between D434G and WT. These observations support the conclusion that D434G represents an intermediate disease phenotype.
The behavioral studies were the most effective in revealing differences among the variants and in defining GOF N999S heterozygotes as a compelling animal model for PKND and providing evidence that the LOF mutation conferred the opposite effect of hyperkinetic mobility. The findings provide the new insight that KCNMA is the target of heritable, monogenic disease, a conclusion that was previously not forthcoming because known human mutations have arisen de novo. The dyskinetic phenotypes in response to stress induction are wholly consistent with patient symptoms.
With respect to rigor and reproducibility, it is commendable that the investigators were blinded to genotype during data collection and analysis. Moreover, the study provides an important confirmation of previous findings from another lab regarding the cellular phenotype of the N999S mutant. WT controls were compared to transgenic littermates within individual transgenic lines. In some cases, the sample sizes were rather low (see below), but otherwise the study seems rigorous.
The strengths of the manuscript far outweighed the weaknesses. The experiments interpreted to suggest a gene dosage effect with D434G were not compelling to this reviewer and might be better documented in the supplement with the conclusion that further work is required.
Due to pandemic-related animal and lab issues, we were unable to generate and surgically implant full Kcnma1D434G/D434G homozygous cohorts for the EEG/seizure portion of the study. We focused instead on using the limited mice of this genotype for the novel PNKD3 assays (n=7), leaving the seizure dataset at n=3.
To address the concern, the Kcnma1D434G/D434G data was removed from Figure 4 to avoid overinterpretation of a gene dosage effect. However, we did retain the individual measurements within the Results text (lines 383 and 385), on the basis of facilitating direct comparisons between our study and other D434G studies. For example, even with only three measurements, the trend toward the shortest seizure latencies in Kcnma1D434G/D434G mice is similar to the result obtained with an independently generated D434G mouse model (Dong et al, 2022). Yet seizure power and the presence of spontaneous seizures do not show a similar trend, suggesting our results differ from theirs in these important aspects. This is now stated more clearly in the revised conclusion for that paragraph, ‘While not conclusive and requiring substantiation in a larger cohort, the Kcnma1D434G/D434G seizure data raise the possibility of a gene dosage effect with D434G that qualitatively differs from an independently-generated D434G mouse model (Dong et al., 2022),’ (lines 388-390).
In contrast to the seizure part of the study, the increased severity of Kcnma1D434G/D434G PNKD-immobility is fully supported by the data with sufficient statistical power (Figure 5D). However, the idea that the increased severity with homozygous D434G in PNKD-immobility was consistent with gene dosage observations for seizure was removed for consistency (lines 549-550).
As a side note, we also added additional clinical descriptors (akinesia) and colloquial descriptions for PNKD3 (‘drop attack’) to disambiguate how a PNKD3 episode appears different from other types of motor dysfunction. This was to facilitate comparison with the two other KCNMA1-D434G models (mouse and fly; Dong et al, 2022; Kratschmer et al., 2021), which report aspects of dyskinesia in the setting of baseline locomotor dysfunction. To our knowledge, these models have not been evaluated for the striking ‘drop attack’ immobility presenting in patients (lines 84-85).
The consequences of the altered BK current levels were assessed on the voltage dependence of firing frequency in the hippocampal neurons, but it was not very clear how increased BK current would enhance neuronal excitability. Also, how might it lead to the PKND phenotype? A paragraph even speculating on these mechanistic links in the Discussion would be welcome.
The mechanism for how BK currents increase action potential firing are not fully identified in this study (see also response to reviewer #2). In the Results, a new paragraph was added at the end of action potential section to summarize the AHP changes in more detail and speculate an indirect mechanism of action for the increase in BK current, predicted from a similar ‘GOF’ BK current type, where β4 regulation of BK channels is lost (lines 294-304). Additional details have also been added to the Discussion regarding the factors contributing to lower seizure threshold (lines 675-680).
Additional re-organization of Discussion text addresses the basis for PNKD. A direct statement that it is not clear yet which neurons/circuits are the most critical for PNKD-like symptomology was added, and which of these express BK channels (lines 680-700). We follow with a succinct summary of phenotypically-relevant PNKD models. While there is a lot to unpack with respect to similarities and differences between different paroxysmal dyskinesia models in the literature, they ultimately shed little light the question of KCNMA1 PNKD3-related dysfunction. With the addition of the d-amp rescue control, we focus mainly on the amphetamine response predicting a CNS locus (lines 692-693). The d-amp response may even suggest dopaminergic pathways (some of which express BK channels) as a plausible to investigate in future studies, but due to the complex interplay of d-amp dosage and the novel motor assay, we don’t think speculating on a specific circuit is supported with enough actual data to add in the Discussion.
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Evaluation Summary:
This study is of broad interest to neuroscientists interested in membrane excitability and translational biologists and physicians eager for robust animal models for disorders involving mutations in the KCNMA gene, such as paroxysmal nonkinesigenic dyskinesia PNKD3. Here, phenotypes of mouse models of three of the more common patient disease-related mutations in KCNMA are evaluated for similarities to patient phenotypes. This work establishes that BK channel mutations linked to human neurological disease can, on their own, cause similar pathology in mice, and it also begins to provide neurological bases for the associated behavioral deficits. Importantly, one of the mutant alleles expressed in mice most closely phenocopies the patient phenotype and will serve as an important animal model for studies seeking …
Evaluation Summary:
This study is of broad interest to neuroscientists interested in membrane excitability and translational biologists and physicians eager for robust animal models for disorders involving mutations in the KCNMA gene, such as paroxysmal nonkinesigenic dyskinesia PNKD3. Here, phenotypes of mouse models of three of the more common patient disease-related mutations in KCNMA are evaluated for similarities to patient phenotypes. This work establishes that BK channel mutations linked to human neurological disease can, on their own, cause similar pathology in mice, and it also begins to provide neurological bases for the associated behavioral deficits. Importantly, one of the mutant alleles expressed in mice most closely phenocopies the patient phenotype and will serve as an important animal model for studies seeking therapeutic treatments for the resulting debilitating disease moving forward.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #3 (Public Review):
The primary strength of this study is in establishing the N999S heterozygous mouse as a useful model system for debilitating paroxysmal non-kinesigenic dyskinesia (PKND), with or without epilepsy. This outcome was hard-won following a comprehensive analysis of biophysical, neurophysiological, and behavioral tests. Ultimately the convincing evidence was demonstrated through a clever application of a stress-related behavioral test (quite in alignment with triggers in patients) to elicit the hypo-motility associated with PKND. Like patients who exhibit variable penetrance, even highly inbred mice exhibit much variability, and uncovering a robust phenotype took a nuanced approach and perseverance.
To reach this point, several experiments provided mechanistic insights into the mutant channel behavior. First, …
Reviewer #3 (Public Review):
The primary strength of this study is in establishing the N999S heterozygous mouse as a useful model system for debilitating paroxysmal non-kinesigenic dyskinesia (PKND), with or without epilepsy. This outcome was hard-won following a comprehensive analysis of biophysical, neurophysiological, and behavioral tests. Ultimately the convincing evidence was demonstrated through a clever application of a stress-related behavioral test (quite in alignment with triggers in patients) to elicit the hypo-motility associated with PKND. Like patients who exhibit variable penetrance, even highly inbred mice exhibit much variability, and uncovering a robust phenotype took a nuanced approach and perseverance.
To reach this point, several experiments provided mechanistic insights into the mutant channel behavior. First, whole-cell patch clamp experiments revealed shifts in the G-V consistent with gain-of-function behavior previously characterized using the N999S and D434G mutants expressed heterologously. Novel observations of H444Q revealed a loss-of-function (LOF) behavior with the G-V shifted to positive potentials but to a lesser degree. These electrophysiological phenotypes establish the rank of predicted severity as N999S>D434G>H444Q.
This prediction was tested in brain slices of heterozygous animals where the mutant channels would be normally spliced and associate with WT subunits and other components such as beta subunits. The investigators evaluated BK currents by patch clamp from hippocampal neurons where BK channels are known to play key functional roles. Both N999S and D434G showed the predicted increase in current magnitude, though interestingly the differences between them apparent in heterologous expression were lost in the native setting. Curiously, no differences in BK current magnitude were observed in neurons of heterozygotes carrying the putatively LOF mutation H444Q.
In terms of seizure susceptibility, D434G mutants different from WT and less severe than N999S mutants with respect to time to evoked seizure, although differences in "EEG power" were not statistically significant between D434G and WT. These observations support the conclusion that D434G represents an intermediate disease phenotype.
The behavioral studies were the most effective in revealing differences among the variants and in defining GOF N999S heterozygotes as a compelling animal model for PKND and providing evidence that the LOF mutation conferred the opposite effect of hyperkinetic mobility. The findings provide the new insight that KCNMA is the target of heritable, monogenic disease, a conclusion that was previously not forthcoming because known human mutations have arisen de novo. The dyskinetic phenotypes in response to stress induction are wholly consistent with patient symptoms.
With respect to rigor and reproducibility, it is commendable that the investigators were blinded to genotype during data collection and analysis. Moreover, the study provides an important confirmation of previous findings from another lab regarding the cellular phenotype of the N999S mutant. WT controls were compared to transgenic littermates within individual transgenic lines. In some cases, the sample sizes were rather low (see below), but otherwise the study seems rigorous.
The strengths of the manuscript far outweighed the weaknesses. The experiments interpreted to suggest a gene dosage effect with D434G were not compelling to this reviewer and might be better documented in the supplement with the conclusion that further work is required.
The consequences of the altered BK current levels were assessed on the voltage dependence of firing frequency in the hippocampal neurons, but it was not very clear how increased BK current would enhance neuronal excitability. Also, how might it lead to the PKND phenotype? A paragraph even speculating on these mechanistic links in the Discussion would be welcome.
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Reviewer #2 (Public Review):
The manuscript by Park et al focuses on cellular and behavioral effects of BK channel mutations in mouse models. This work is important, as it establishes that BK channel mutations linked to human neurological disease can, on their own, cause similar pathology in mice, and it also begins to provide neurological bases for the associated behavioral deficits (in terms of possible effects on action potential waveforms), and specifically, the N999S/WT mouse may serve as a model for understanding the neuronal correlates of paroxysmal dyskinesia (PNKD3) linked to this mutation in humans. Briefly, the authors find that BK currents and AP firing rates were increased in hippocampal dentate gyrus (DG) neurons in N999S/WT and D434/WT mice, with no changes observed in H444Q/WT mice. In behavioral assays, N999S/WT and …
Reviewer #2 (Public Review):
The manuscript by Park et al focuses on cellular and behavioral effects of BK channel mutations in mouse models. This work is important, as it establishes that BK channel mutations linked to human neurological disease can, on their own, cause similar pathology in mice, and it also begins to provide neurological bases for the associated behavioral deficits (in terms of possible effects on action potential waveforms), and specifically, the N999S/WT mouse may serve as a model for understanding the neuronal correlates of paroxysmal dyskinesia (PNKD3) linked to this mutation in humans. Briefly, the authors find that BK currents and AP firing rates were increased in hippocampal dentate gyrus (DG) neurons in N999S/WT and D434/WT mice, with no changes observed in H444Q/WT mice. In behavioral assays, N999S/WT and D434/D434 mice became immobile after stress, whereas H444Q/H444Q mice became hyperkinetic.
The manuscript is well written and well organized, and the experimental data are of high quality.
I have some comments pertaining to possible interpretations of the mutant data, specifically on the potential impact of the neuronal BK-beta4 subunit on the mutation effects. This can lead to alterations in channel activity that may be different in neurons that express the beta-4 subunit (like DG neurons) vs. non-beta4 expressing neurons (like midbrain dopaminergic neurons). It may ultimately be important to build on this work by assessing activity in different neurons in the brain.
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Reviewer #1 (Public Review):
Park et al investigated the association of three different mutations of the KCNMA1 gene (expressing the potassium channel BK) with paroxysmal nonkinesigenic dyskinesia (PNKD3). To this end they use electrophysiology in heterologous expression systems, neuronal cultures of dentate granule cells, and behavioural tests in transgenic mice harbouring the specific mutations. They find that two mutations (N999S and D434G) result in gain-of-function properties, associated to larger BK currents and increased action potential firing. Conversely, another mutation (H444Q) renders loss-of-function characteristics, which are correlated with non-significant changes in ionic currents or excitability. Behavioural tests were conducted with hetero- or homozygous transgenic mice harbouring the BK mutations. Results reveal …
Reviewer #1 (Public Review):
Park et al investigated the association of three different mutations of the KCNMA1 gene (expressing the potassium channel BK) with paroxysmal nonkinesigenic dyskinesia (PNKD3). To this end they use electrophysiology in heterologous expression systems, neuronal cultures of dentate granule cells, and behavioural tests in transgenic mice harbouring the specific mutations. They find that two mutations (N999S and D434G) result in gain-of-function properties, associated to larger BK currents and increased action potential firing. Conversely, another mutation (H444Q) renders loss-of-function characteristics, which are correlated with non-significant changes in ionic currents or excitability. Behavioural tests were conducted with hetero- or homozygous transgenic mice harbouring the BK mutations. Results reveal significantly decreased seizure threshold and higher immobility after stress for gain-of-function, but not loss-of-function mutants. The latter showed hyperkinetic behaviour. The data provide relevant evidence linking gain-of-function defects on BK function to the disease phenotype, and provide a novel mouse model for PNKD disease. The conclusions of this paper are mostly supported by data. Some aspects need to be revised and clarified.
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