BK channel dysfunction disrupts attention-controlled behaviors and altered perseverative responses in murine instrumental learning.

This study examined the effect of knockout of KCNMA1 gene, coding for the BK channel, on cognitive and attentional functions in mice, with an aim to better understand its implications for human neurodevelopmental disorders. The study used the 3-choice serial reaction time task (3-CSRTT) to assess the learning performance, attentional abilities, and repetitive behaviors in mice lacking the KCNMA1 gene (KCNMA1-/-) compared to wild-type (WT) controls. Results showed no significant differences in learning accuracy between the two groups. However, KCNMA1-/- mice were more prone to omitting responses to stimuli. In addition, when the timing of cue presentation was randomized, the KCNMA1-/- showed premature responses. Notably, these mice also demonstrated a marked reduction in perseverative responses, which include repeated nose-poke behaviors following decisions. These findings highlight the involvement of the KCNMA1 gene in managing attention, impulsivity, and potentially moderating repetitive actions.

BK Channelopathies and KCNMA1-Linked Disease Models.

Novel KCNMA1 variants, encoding the BK K+ channel, are associated with a debilitating dyskinesia and epilepsy syndrome. Neurodevelopmental delay, cognitive disability, and brain and structural malformations are also diagnosed at lower incidence. More than half of affected individuals present with a rare negative episodic motor disorder, paroxysmal nonkinesigenic dyskinesia (PNKD3). The mechanistic relationship of PNKD3 to epilepsy and the broader spectrum of KCNMA1-associated symptomology is unknown. This review summarizes patient-associated KCNMA1 variants within the BK channel structure, functional classifications, genotype-phenotype associations, disease models, and treatment. Patient and transgenic animal data suggest delineation of gain-of-function (GOF) and loss-of-function KCNMA1 neurogenetic disease, validating two heterozygous alleles encoding GOF BK channels (D434G and N999S) as causing seizure and PNKD3. This discovery led to a variant-defined therapeutic approach for PNKD3, providing initial insight into the neurological basis. A comprehensive clinical definition of monogenic KCNMA1-linked disease and the neuronal mechanisms currently remain priorities for continued investigation.

Structural mapping of patient-associated KCNMA1 gene variants.

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.

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

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.

Structural mapping of patient-associated KCNMA1 gene variants.

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 cryo-EM 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 Ca 2+ 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 (VUS), 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), consisting of a weighted summation of this trained dataset combined with a structural component derived from the Ca 2+ 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 VUS, suggesting strategies for improving channel-level predictions in future studies by building on ensemble algorithms such as KMS.

Effect of an autism-associated KCNMB2 variant, G124R, on BK channel properties.

BK K+ channels are critical regulators of neuron and muscle excitability, comprised of a tetramer of pore-forming αsubunits from the KCNMA1 gene and cell- and tissue-selective ß subunits (KCNMB1-4). Mutations in KCNMA1 are associated with neurological disorders, including autism. However, little is known about the role of neuronal BK channel ß subunits in human neuropathology. The ß2 subunit is expressed in central neurons and imparts inactivation to BK channels, as well as altering activation and deactivation gating. In this study, we report the functional effect of G124R, a novel KCNMB2 mutation obtained from whole-exome sequencing of a patient diagnosed with autism spectrum disorder. Residue G124, located in the extracellular loop between TM1 and TM2, is conserved across species, and the G124R missense mutation is predicted deleterious with computational tools. To investigate the pathogenicity potential, BK channels were co-expressed with ß2WT and ß2G124R subunits in HEK293T cells. BK/ß2 currents were assessed from inside-out patches under physiological K+ conditions (140/6 mM K+ and 10 µM Ca2+) during activation and inactivation (voltage-dependence and kinetics). Using ß2 subunits lacking inactivation (ß2IR) revealed that currents from BK/ß2IRG124R channels activated 2-fold faster and deactivated 2-fold slower compared with currents from BK/ß2IRWT channels, with no change in the voltage-dependence of activation (V1/2). Despite the changes in the BK channel opening and closing, BK/ß2G124R inactivation rates (τinact and τrecovery), and the V1/2 of inactivation, were unaltered compared with BK/ß2WT channels under standard steady-state voltage protocols. Action potential-evoked current was also unchanged. Thus, the mutant phenotype suggests the ß2G124R TM1-TM2 extracellular loop could regulate BK channel activation and deactivation kinetics. However, additional evidence is needed to validate pathogenicity for this patient-associated variant in KCNMB2.

Lisdexamfetamine Therapy in Paroxysmal Non-kinesigenic Dyskinesia Associated with the KCNMA1-N999S Variant.

BACKGROUND: KCNMA1-linked channelopathy is a rare movement disorder first reported in 2005. Paroxysmal non-kinesigenic dyskinesia (PNKD) in KCNMA1-linked channelopathy is the most common symptom in patients harboring the KCNMA1-N999S mutation. PNKD episodes occur up to hundreds of times daily with significant morbidity and limited treatment options, often in the context of epilepsy. CASES: We report 6 cases with the KCNMA1-N999S variant treated with lisdexamfetamine (0.7-1.25 mg/kg/day), a pro-drug of dextroamphetamine. Data were collected retrospectively from interviews and chart review. Parent-reported daily PNKD episode counts were reduced under treatment, ranging from a 10-fold decrease to complete resolution. CONCLUSION: Our findings suggest that lisdexamfetamine is an effective therapy for PNKD3 (KCNMA1-associated PNKD). Treatment produced dramatic reductions in debilitating dyskinesia episodes, without provocation or exacerbation of other KCNMA1-associated symptoms such as seizures.

Contributions of CaV1.3 Channels to Ca2+ Current and Ca2+-Activated BK Current in the Suprachiasmatic Nucleus.

Daily regulation of Ca2+ - and voltage-activated BK K+ channel activity is required for action potential rhythmicity in the suprachiasmatic nucleus (SCN) of the hypothalamus, the brain's circadian clock. In SCN neurons, BK activation is dependent upon multiple types of Ca2+ channels in a circadian manner. Daytime BK current predominantly requires Ca2+ influx through L-type Ca2+ channels (LTCCs), a time when BK channels are closely coupled with their Ca2+ source. Here we show that daytime BK current is resistant to the Ca2+ chelator BAPTA. However, at night when LTCCs contribute little to BK activation, BK current decreases by a third in BAPTA compared to control EGTA conditions. In phase with this time-of-day specific effect on BK current activation, LTCC current is larger during the day. The specific Ca2+ channel subtypes underlying the LTCC current in SCN, as well as the subtypes contributing the Ca2+ influx relevant for BK current activation, have not been identified. SCN neurons express two LTCC subtypes, CaV1.2 and CaV1.3. While a role for CaV1.2 channels has been identified during the night, CaV1.3 channel modulation has also been suggested to contribute to daytime SCN action potential activity, as well as subthreshold Ca2+ oscillations. Here we characterize the role of CaV1.3 channels in LTCC and BK current activation in SCN neurons using a global deletion of CACNA1D in mouse (CaV1.3 KO). CaV1.3 KO SCN neurons had a 50% reduction in the daytime LTCC current, but not total Ca2+ current, with no difference in Ca2+ current levels at night. During the day, CaV1.3 KO neurons exhibited oscillations in membrane potential, and most neurons, although not all, also had BK currents. Changes in BK current activation were only detectable at the highest voltage tested. These data show that while CaV1.3 channels contribute to the daytime Ca2+ current, this does not translate into a major effect on the daytime BK current. These data suggest that BK current activation does not absolutely require CaV1.3 channels and may therefore also depend on other LTCC subtypes, such as CaV1.2.

An emerging spectrum of variants and clinical features in KCNMA1-linked channelopathy.

KCNMA1-linked channelopathy is an emerging neurological disorder characterized by heterogeneous and overlapping combinations of movement disorder, seizure, developmental delay, and intellectual disability. KCNMA1 encodes the BK K+ channel, which contributes to both excitatory and inhibitory neuronal and muscle activity. Understanding the basis of the disorder is an important area of active investigation; however, the rare prevalence has hampered the development of large patient cohorts necessary to establish genotype-phenotype correlations. In this review, we summarize 37 KCNMA1 alleles from 69 patients currently defining the channelopathy and assess key diagnostic and clinical hallmarks. At present, 3 variants are classified as gain-of-function with respect to BK channel activity, 14 loss-of-function, 15 variants of uncertain significance, and putative benign/VUS. Symptoms associated with these variants were curated from patient-provided information and prior publications to define the spectrum of clinical phenotypes. In this newly expanded cohort, seizures showed no differential distribution between patients harboring GOF and LOF variants, while movement disorders segregated by mutation type. Paroxysmal non-kinesigenic dyskinesia was predominantly observed among patients with GOF alleles of the BK channel, although not exclusively so, while additional movement disorders were observed in patients with LOF variants. Neurodevelopmental and structural brain abnormalities were prevalent in patients with LOF mutations. In contrast to mutations, disease-associated KCNMA1 single nucleotide polymorphisms were not predominantly related to neurological phenotypes but covered a wider set of peripheral physiological functions. Together, this review provides additional evidence exploring the genetic and biochemical basis for KCNMA1-linked channelopathy and summarizes the clinical repository of patient symptoms across multiple types of KCNMA1 gene variants.

BK channel activation by L-type Ca2+ channels CaV1.2 and CaV1.3 during the subthreshold phase of an action potential.

Mammalian circadian (24 h) rhythms are timed by the pattern of spontaneous action potential firing in the suprachiasmatic nucleus (SCN). This oscillation in firing is produced through circadian regulation of several membrane currents, including large-conductance Ca2+- and voltage-activated K+ (BK) and L-type Ca2+ channel (LTCC) currents. During the day steady-state BK currents depend mostly on LTCCs for activation, whereas at night they depend predominantly on ryanodine receptors (RyRs). However, the contribution of these Ca2+ channels to BK channel activation during action potential firing has not been thoroughly investigated. In this study, we used a pharmacological approach to determine that both LTCCs and RyRs contribute to the baseline membrane potential of SCN action potential waveforms, as well as action potential-evoked BK current, during the day and night, respectively. Since the baseline membrane potential is a major determinant of circadian firing rate, we focused on the LTCCs contributing to low voltage activation of BK channels during the subthreshold phase. For these experiments, two LTCC subtypes found in SCN (CaV1.2 and CaV1.3) were coexpressed with BK channels in heterologous cells, where their differential contributions could be separately measured. CaV1.3 channels produced currents that were shifted to more hyperpolarized potentials compared with CaV1.2, resulting in increased subthreshold Ca2+ and BK currents during an action potential command. These results show that although multiple Ca2+ sources in SCN can contribute to the activation of BK current during an action potential, specific BK-CaV1.3 partnerships may optimize the subthreshold BK current activation that is critical for firing rate regulation.NEW & NOTEWORTHY BK K+ channels are important regulators of firing. Although Ca2+ channels are required for their activation in excitable cells, it is not well understood how BK channels activate using these Ca2+ sources during an action potential. This study demonstrates the differences in BK current activated by CaV1.2 and CaV1.3 channels in clock neurons and heterologous cells. The results define how specific ion channel partnerships can be engaged during distinct phases of the action potential.

Comparative Ca2+ channel contributions to intracellular Ca2+ levels in the circadian clock.

Circadian rhythms in mammals are coordinated by the central clock in the brain, located in the suprachiasmatic nucleus (SCN). Multiple molecular and cellular signals display a circadian variation within SCN neurons, including intracellular Ca2+, but the mechanisms are not definitively established. SCN cytosolic Ca2+ levels exhibit a peak during the day, when both action potential firing and Ca2+ channel activity are increased, and are decreased at night, correlating with a reduction in firing rate. In this study, we employ a single-color fluorescence anisotropy reporter (FLARE), Venus FLARE-Cameleon, and polarization inverted selective-plane illumination microscopy to measure rhythmic changes in cytosolic Ca2+ in SCN neurons. Using this technique, the Ca2+ channel subtypes contributing to intracellular Ca2+ at the peak and trough of the circadian cycle were assessed using a pharmacological approach with Ca2+ channel inhibitors. Peak (218 ± 16 nM) and trough (172 ± 13 nM) Ca2+ levels were quantified, indicating a 1.3-fold circadian variance in Ca2+ concentration. Inhibition of ryanodine-receptor-mediated Ca2+ release produced a larger relative decrease in cytosolic Ca2+ at both time points compared to voltage-gated Ca2+channels. These results support the hypothesis that circadian Ca2+ rhythms in SCN neurons are predominantly driven by intracellular Ca2+ channels, although not exclusively so. The study provides a foundation for future experiments to probe Ca2+ signaling in a dynamic biological context using FLAREs.

Status Dystonicus, Oculogyric Crisis and Paroxysmal Dyskinesia in a 25 Year-Old Woman with a Novel KCNMA1 Variant, K457E.

The diagnosis of a paroxysmal dyskinesia is difficult and status dystonicus is a rare life threatening movement disorder characterised by severe, frequent or continuous episodes of dystonic spasms. A 25 year old woman with chronic ataxia and paroxysmal dyskinesia presented with facial twitching, writhing of arms, oculogyric crisis and visual and auditory hallucinations. She developed respiratory failure and was ventilated. No cause was found so whole exome sequencing was performed and this revealed a novel, non-synonymous heterozygous variant in exon 11 of the KCNMA1 gene, K457E (c 1369A>G) in the patient but not her parents. This variant has not been previously reported in gnomAD or ClinVar. The finding of a de novo variant in a potassium channel gene guided a trial of the potassium channel antagonist 3,4 diaminopyridine resulting in significant improvement, discharge from the intensive care unit and ultimately home.

Ion Channels Controlling Circadian Rhythms in Suprachiasmatic Nucleus Excitability.

Animals synchronize to the environmental day-night cycle by means of an internal circadian clock in the brain. In mammals, this timekeeping mechanism is housed in the suprachiasmatic nucleus (SCN) of the hypothalamus and is entrained by light input from the retina. One output of the SCN is a neural code for circadian time, which arises from the collective activity of neurons within the SCN circuit and comprises two fundamental components: 1) periodic alterations in the spontaneous excitability of individual neurons that result in higher firing rates during the day and lower firing rates at night, and 2) synchronization of these cellular oscillations throughout the SCN. In this review, we summarize current evidence for the identity of ion channels in SCN neurons and the mechanisms by which they set the rhythmic parameters of the time code. During the day, voltage-dependent and independent Na+ and Ca2+ currents, as well as several K+ currents, contribute to increased membrane excitability and therefore higher firing frequency. At night, an increase in different K+ currents, including Ca2+-activated BK currents, contribute to membrane hyperpolarization and decreased firing. Layered on top of these intrinsically regulated changes in membrane excitability, more than a dozen neuromodulators influence action potential activity and rhythmicity in SCN neurons, facilitating both synchronization and plasticity of the neural code.

Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing.

KEY POINTS: Circadian oscillations in spontaneous action potential firing in the suprachiasmatic nucleus (SCN) translate time-of-day throughout the mammalian brain. The ion channels that regulate the circadian pattern of SCN firing have not been comprehensively identified. Ca2+ channels regulate action potential activity across many types of excitable cells, and the activity of L-, N-, P/Q- and R-type channels are required for normal daytime firing frequency in SCN neurons and circuit rhythms. Only the L-type Ca2+ current exhibits a day versus night difference in current magnitude, providing insight into the mechanism that produces rhythmic action potential firing in SCN. ABSTRACT: The mammalian circadian clock encodes time via rhythmic action potential activity in the suprachiasmatic nucleus (SCN) of the hypothalamus, which governs daily rhythms in physiology and behaviour. SCN neurons exhibit 24 h oscillations in spontaneous firing, with higher firing during day compared to night. Several ionic currents have been identified that regulate SCN firing, including voltage-gated Ca2+ currents, but the circadian regulation of distinct voltage-gated Ca2+ channel (VGCC) components has not been comprehensively addressed. In this study, whole-cell L- (nimodipine-sensitive), N- and P/Q- (ω-agatoxin IVA, ω-conotoxin GVIA, ω-conotoxin MVIIC-sensitive), R- (Ni2+ -sensitive) and T-type (TTA-P2-sensitive) currents were recorded from day and night SCN slices. Using standard voltage protocols, Ni2+ -sensitive currents comprised the largest proportion of total VGCC current, followed by nimodipine-, ω-agatoxin IVA-, ω-conotoxin GVIA- and TTA-P2-sensitive currents. Only the nimodipine-sensitive current exhibited a diurnal difference in magnitude, with daytime current larger than night. No diurnal variation was observed for the other Ca2+ current subtypes. The difference in nimodipine-sensitive current was due to larger peak current activated during the day, not differences in inactivation, and was eliminated by Bay K8644. Blocking L-type channels decreased firing selectively during the day, consistent with higher current magnitudes, and reduced SCN circuit rhythmicity recorded by multi-electrode arrays. Yet blocking N-, P/Q- and R-type channels also decreased daytime firing, with little effect at night, and decreased circuit rhythmicity. These data identify a unique diurnal regulation of L-type current among the major VGCC subtypes in SCN neurons, but also reveal that diurnal modulation is not required for time-of-day-specific effects on firing and circuit rhythmicity.

Comparative gain-of-function effects of the KCNMA1-N999S mutation on human BK channel properties.

KCNMA1, encoding the voltage- and calcium-activated potassium channel, has a pivotal role in brain physiology. Mutations in KCNMA1 are associated with epilepsy and/or dyskinesia (PNKD3). Two KCNMA1 mutations correlated with these phenotypes, D434G and N999S, were previously identified as producing gain-of-function (GOF) effects on BK channel activity. Three new patients have been reported harboring N999S, one carrying a second mutation, R1128W, but the effects of these mutations have not yet been reported under physiological K+ conditions or compared to D434G. In this study, we characterize N999S, the novel N999S/R1128W double mutation, and D434G in a brain BK channel splice variant, comparing the effects on BK current properties under a physiological K+ gradient with action potential voltage commands. N999S, N999S/R1128W, and D434G cDNAs were expressed in HEK293T cells and characterized by patch-clamp electrophysiology. N999S BK currents were shifted to negative potentials, with faster activation and slower deactivation compared with wild type (WT) and D434G. The double mutation N999S/R1128W did not show any additional changes in current properties compared with N999S alone. The antiepileptic drug acetazolamide was assessed for its ability to directly modulate WT and N999S channels. Neither the WT nor N999S channels were sensitive to the antiepileptic drug acetazolamide, but both were sensitive to the inhibitor paxilline. We conclude that N999S is a strong GOF mutation that surpasses the D434G phenotype, without mitigation by R1128W. Acetazolamide has no direct modulatory action on either WT or N999S channels, indicating that its use may not be contraindicated in patients harboring GOF KCNMA1 mutations.NEW & NOTEWORTHYKCNMA1-linked channelopathy is a new neurological disorder characterized by mutations in the BK voltage- and calcium-activated potassium channel. The epilepsy- and dyskinesia-associated gain-of-function mutations N999S and D434G comprise the largest number of patients in the cohort. This study provides the first direct comparison between D434G and N999S BK channel properties as well as a novel double mutation, N999S/R1128W, from another patient, defining the functional effects during an action potential stimulus.

Effects of Single Nucleotide Polymorphisms in Human KCNMA1 on BK Current Properties.

BK Ca2+-activated K+ channels are important regulators of membrane excitability. Multiple regulatory mechanisms tailor BK current properties across tissues, such as alternative splicing, posttranslational modifications, and auxiliary subunits. Another potential mechanism for modulating BK channel activity is genetic variation due to single nucleotide polymorphisms (SNPs). The gene encoding the human BK α subunit, KCNMA1, contains hundreds of SNPs. However, the variation in BK channel activity due to SNPs is not well studied. Here, we screened the effects of four SNPs (A138V, C495G, N599D, and R800W) on BK currents in HEK293T cells, selected based on predicted protein pathogenicity or disease linkage. We found that the SNPs C495G and R800W had the largest effects on BK currents, affecting the conductance-voltage relationship across multiple Ca2+ conditions in the context of two BK channel splice variants. In symmetrical K+, C495G shifted the V1/2 to more hyperpolarized potentials (by -15 to -20 mV) and accelerated activation, indicating C495G confers some gain-of-function properties. R800W shifted the V1/2 to more depolarized potentials (+15 to +35 mV) and slowed activation, conferring loss-of-function properties. Moreover, the C495G and R800W effects on current properties were found to persist with posttranslational modifications. In contrast, A138V and N599D had smaller and more variable effects on current properties. Neither application of alkaline phosphatase to patches, which results in increased BK channel activity attributed to channel dephosphorylation, nor bidirectional redox modulations completely abrogated SNP effects on BK currents. Lastly, in physiological K+, C495G increased the amplitude of action potential (AP)-evoked BK currents, while R800W had a more limited effect. However, the introduction of R800W in parallel with the epilepsy-linked mutation D434G (D434G/R800W) decreased the amplitude of AP-evoked BK currents compared with D434G alone. These results suggest that in a physiological context, C495G could increase BK activation, while the effects of the loss-of-function SNP R800W could oppose the gain-of-function effects of an epilepsy-linked mutation. Together, these results implicate naturally occurring human genetic variation as a potential modifier of BK channel activity across a variety of conditions.

KCNMA1-linked channelopathy.

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.

Glutamate-activated BK channel complexes formed with NMDA receptors.

The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.

Expression and Activation of BKCa Channels in Mice Protects Against Ischemia-Reperfusion Injury of Isolated Hearts by Modulating Mitochondrial Function.

Aims: Activation and expression of large conductance calcium and voltage-activated potassium channel (BKCa) by pharmacological agents have been implicated in cardioprotection from ischemia-reperfusion (IR) injury possibly by regulating mitochondrial function. Given the non-specific effects of pharmacological agents, it is not clear whether activation of BKCa is critical to cardioprotection. In this study, we aimed to decipher the mechanistic role of BKCa in cardioprotection from IR injury by genetically activating BKCa channels. Methods and Results: Hearts from adult (3 months old) wild-type mice (C57/BL6) and mice expressing genetically activated BKCa (Tg-BKCa R207Q, referred as Tg-BKCa) along with wild-type BKCa were subjected to 20 min of ischemia and 30 min of reperfusion with or without ischemic preconditioning (IPC, 2 times for 2.5 min interval each). Left ventricular developed pressure (LVDP) was recorded using Millar's Mikrotip® catheter connected to ADInstrument data acquisition system. Myocardial infarction was quantified by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Our results demonstrated that Tg-BKCa mice are protected from IR injury, and BKCa also contributes to IPC-mediated cardioprotection. Cardiac function parameters were also measured by echocardiography and no differences were observed in left ventricular ejection fraction, fractional shortening and aortic velocities. Amplex Red® was used to assess reactive oxygen species (ROS) production in isolated mitochondria by spectrofluorometry. We found that genetic activation of BKCa reduces ROS after IR stress. Adult cardiomyocytes and mitochondria from Tg-BKCa mice were isolated and labeled with Anti-BKCa antibodies. Images acquired via confocal microscopy revealed localization of cardiac BKCa in the mitochondria. Conclusions: Activation of BKCa is essential for recovery of cardiac function after IR injury and is likely a factor in IPC mediated cardioprotection. Genetic activation of BKCa reduces ROS produced by complex I and complex II/III in Tg-BKCa mice after IR, and IPC further decreases it. These results implicate BKCa-mediated cardioprotection, in part, by reducing mitochondrial ROS production. Localization of Tg-BKCa in adult cardiomyocytes of transgenic mice was similar to BKCa in wild-type mice.

Differential contribution of Ca2+ sources to day and night BK current activation in the circadian clock.

Large conductance K+ (BK) channels are expressed widely in neurons, where their activation is regulated by membrane depolarization and intracellular Ca2+ (Ca2+i). To enable this regulation, BK channels functionally couple to both voltage-gated Ca2+ channels (VGCCs) and channels mediating Ca2+ release from intracellular stores. However, the relationship between BK channels and their specific Ca2+ source for particular patterns of excitability is not well understood. In neurons within the suprachiasmatic nucleus (SCN)-the brain's circadian clock-BK current, VGCC current, and Ca2+i are diurnally regulated, but paradoxically, BK current is greatest at night when VGCC current and Ca2+i are reduced. Here, to determine whether diurnal regulation of Ca2+ is relevant for BK channel activation, we combine pharmacology with day and night patch-clamp recordings in acute slices of SCN. We find that activation of BK current depends primarily on three types of channels but that the relative contribution changes between day and night. BK current can be abrogated with nimodipine during the day but not at night, establishing that L-type Ca2+ channels (LTCCs) are the primary daytime Ca2+ source for BK activation. In contrast, dantrolene causes a significant decrease in BK current at night, suggesting that nighttime BK activation is driven by ryanodine receptor (RyR)-mediated Ca2+i release. The N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC causes a smaller reduction of BK current that does not differ between day and night. Finally, inhibition of LTCCs, but not RyRs, eliminates BK inactivation, but the BK ß2 subunit was not required for activation of BK current by LTCCs. These data reveal a dynamic coupling strategy between BK channels and their Ca2+ sources in the SCN, contributing to diurnal regulation of SCN excitability.