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Disease-associated KCNMA1 variants decrease circadian clock robustness in channelopathy mouse models.

ABSTRACT: KCNMA1 encodes the voltage- and calcium-activated K+ (BK) channel, which regulates suprachiasmatic nucleus (SCN) neuronal firing and circadian behavioral rhythms. Gain-of-function (GOF) and loss-of-function (LOF) alterations in BK channel activity disrupt circadian behavior, but the effect of human disease-associated KCNMA1 channelopathy variants has not been studied on clock function. Here, we assess circadian behavior in two GOF and one LOF mouse lines. Heterozygous Kcnma1N999S/WT and homozygous Kcnma1D434G/D434G mice are validated as GOF models of paroxysmal dyskinesia (PNKD3), but whether circadian rhythm is affected in this hypokinetic locomotor disorder is unknown. Conversely, homozygous LOF Kcnma1H444Q/H444Q mice do not demonstrate PNKD3. We assessed circadian behavior by locomotor wheel running activity. All three mouse models were rhythmic, but Kcnma1N999S/WT and Kcnma1D434G/D434G showed reduced circadian amplitude and decreased wheel activity, corroborating prior studies focused on acute motor coordination. In addition, Kcnma1D434G/D434G mice had a small decrease in period. However, the phase-shifting sensitivity for both GOF mouse lines was abnormal. Both Kcnma1N999S/WT and Kcnma1D434G/D434G mice displayed increased responses to light pulses and took fewer days to re-entrain to a new light:dark cycle. In contrast, the LOF Kcnma1H444Q/H444Q mice showed no difference in any of the circadian parameters tested. The enhanced sensitivity to phase-shifting stimuli in Kcnma1N999S/WT and Kcnma1D434G/D434G mice was similar to other Kcnma1 GOF mice. Together with previous studies, these results suggest that increasing BK channel activity decreases circadian clock robustness, without rhythm ablation.

SUBMITTER: Dinsdale RL

PROVIDER: S-EPMC10510740 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Disease-associated KCNMA1 variants decrease circadian clock robustness in channelopathy mouse models.

Dinsdale Ria L RL Roache Cooper E CE Meredith Andrea L AL

The Journal of general physiology 20230920 11

KCNMA1 encodes the voltage- and calcium-activated K+ (BK) channel, which regulates suprachiasmatic nucleus (SCN) neuronal firing and circadian behavioral rhythms. Gain-of-function (GOF) and loss-of-function (LOF) alterations in BK channel activity disrupt circadian behavior, but the effect of human disease-associated KCNMA1 channelopathy variants has not been studied on clock function. Here, we assess circadian behavior in two GOF and one LOF mouse lines. Heterozygous Kcnma1N999S/WT and homozygo ...[more]

PMID: 37728576

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Project description:KCNMA1 encodes the pore-forming α subunit of the "Big K+" (BK) large conductance calcium and voltage-activated K+ channel. BK channels are widely distributed across tissues, including both excitable and nonexcitable cells. Expression levels are highest in brain and muscle, where BK channels are critical regulators of neuronal excitability and muscle contractility. A global deletion in mouse (KCNMA1-/- ) is viable but exhibits pathophysiology in many organ systems. Yet despite the important roles in animal models, the consequences of dysfunctional BK channels in humans are not well characterized. Here, we summarize 16 rare KCNMA1 mutations identified in 37 patients dating back to 2005, with an array of clinically defined pathological phenotypes collectively referred to as "KCNMA1-linked channelopathy." These mutations encompass gain-of-function (GOF) and loss-of-function (LOF) alterations in BK channel activity, as well as several variants of unknown significance (VUS). Human KCNMA1 mutations are primarily associated with neurological conditions, including seizures, movement disorders, developmental delay, and intellectual disability. Due to the recent identification of additional patients, the spectrum of symptoms associated with KCNMA1 mutations has expanded but remains primarily defined by brain and muscle dysfunction. Emerging evidence suggests the functional BK channel alterations produced by different KCNMA1 alleles may associate with semi-distinct patient symptoms, such as paroxysmal nonkinesigenic dyskinesia (PNKD) with GOF and ataxia with LOF. However, due to the de novo origins for the majority of KCNMA1 mutations identified to date and the phenotypic variability exhibited by patients, additional evidence is required to establish causality in most cases. The symptomatic picture developing from patients with KCNMA1-linked channelopathy highlights the importance of better understanding the roles BK channels play in regulating cell excitability. Establishing causality between KCNMA1-linked BK channel dysfunction and specific patient symptoms may reveal new treatment approaches with the potential to increase therapeutic efficacy over current standard regimens.

Project description:BackgroundRobustness is a central property of living systems, enabling function to be maintained against environmental perturbations. A key challenge is to identify the structures in biological circuits that confer system-level properties such as robustness. Circadian clocks allow organisms to adapt to the predictable changes of the 24-hour day/night cycle by generating endogenous rhythms that can be entrained to the external cycle. In all organisms, the clock circuits typically comprise multiple interlocked feedback loops controlling the rhythmic expression of key genes. Previously, we showed that such architectures increase the flexibility of the clock's rhythmic behaviour. We now test the relationship between flexibility and robustness, using a mathematical model of the circuit controlling conidiation in the fungus Neurospora crassa.ResultsThe circuit modelled in this work consists of a central negative feedback loop, in which the frequency (frq) gene inhibits its transcriptional activator white collar-1 (wc-1), interlocked with a positive feedback loop in which FRQ protein upregulates WC-1 production. Importantly, our model reproduces the observed entrainment of this circuit under light/dark cycles with varying photoperiod and cycle duration. Our simulations show that whilst the level of frq mRNA is driven directly by the light input, the falling phase of FRQ protein, a molecular correlate of conidiation, maintains a constant phase that is uncoupled from the times of dawn and dusk. The model predicts the behaviour of mutants that uncouple WC-1 production from FRQ's positive feedback, and shows that the positive loop enhances the buffering of conidiation phase against seasonal photoperiod changes. This property is quantified using Kitano's measure for the overall robustness of a regulated system output. Further analysis demonstrates that this functional robustness is a consequence of the greater evolutionary flexibility conferred on the circuit by the interlocking loop structure.ConclusionsOur model shows that the behaviour of the fungal clock in light-dark cycles can be accounted for by a transcription-translation feedback model of the central FRQ-WC oscillator. More generally, we provide an example of a biological circuit in which greater flexibility yields improved robustness, while also introducing novel sensitivity analysis techniques applicable to a broader range of cellular oscillators.

Project description:KCNMA1-linked channelopathy is a neurological disorder characterized by seizures, motor abnormalities, and neurodevelopmental disabilities. The disease mechanisms are predicted to result from alterations in KCNMA1-encoded BK K+ channel activity; however, only a subset of the patient-associated variants have been functionally studied. The localization of these variants within the tertiary structure or evaluation by pathogenicity algorithms has not been systematically assessed. In this study, 82 nonsynonymous patient-associated KCNMA1 variants were mapped within the BK channel protein. Fifty-three variants localized within cryoelectron microscopy-resolved structures, including 21 classified as either gain of function (GOF) or loss of function (LOF) in BK channel activity. Clusters of LOF variants were identified in the pore, the AC region (RCK1), and near the Ca2+ bowl (RCK2), overlapping with sites of pharmacological or endogenous modulation. However, no clustering was found for GOF variants. To further understand variants of uncertain significance (VUSs), assessments by multiple standard pathogenicity algorithms were compared, and new thresholds for sensitivity and specificity were established from confirmed GOF and LOF variants. An ensemble algorithm was constructed (KCNMA1 meta score (KMS)), consisting of a weighted summation of this trained dataset combined with a structural component derived from the Ca2+-bound and unbound BK channels. KMS assessment differed from the highest-performing individual algorithm (REVEL) at 10 VUS residues, and a subset were studied further by electrophysiology in HEK293 cells. M578T, E656A, and D965V (KMS+;REVEL-) were confirmed to alter BK channel properties in voltage-clamp recordings, and D800Y (KMS-;REVEL+) was assessed as benign under the test conditions. However, KMS failed to accurately assess K457E. These combined results reveal the distribution of potentially disease-causing KCNMA1 variants within BK channel functional domains and pathogenicity evaluation for VUSs, suggesting strategies for improving channel-level predictions in future studies by building on ensemble algorithms such as KMS.

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Disease-associated KCNMA1 variants decrease circadian clock robustness in channelopathy mouse models.

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Disease-associated KCNMA1 variants decrease circadian clock robustness in channelopathy mouse models.

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