Rare Gain-of-Function KCND3 Variant Associated with Cerebellar Ataxia, Parkinsonism, Cognitive Dysfunction, and Brain Iron Accumulation

Loss-of-function mutations in the KV4.3 channel-encoding KCND3 gene are linked to neurodegenerative cerebellar ataxia. Patients suffering from neurodegeneration associated with iron deposition may also present with cerebellar ataxia. The mechanism underlying brain iron accumulation remains unclear. Here, we aim to ascertain the potential pathogenic role of KCND3 variant in iron accumulation-related cerebellar ataxia. We presented a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive impairment, and iron accumulation in the basal ganglia and the cerebellum. Whole exome sequencing analyses identified in the patient a heterozygous KCND3 c.1256G>A (p.R419H) variant predicted to be disease-causing by multiple bioinformatic analyses. In vitro biochemical and immunofluorescence examinations revealed that, compared to the human KV4.3 wild-type channel, the p.R419H variant exhibited normal protein abundance and subcellular localization pattern. Electrophysiological investigation, however, demonstrated that the KV4.3 p.R419H variant was associated with a dominant increase in potassium current amplitudes, as well as notable changes in voltage-dependent gating properties leading to enhanced potassium window current. These observations indicate that, in direct contrast with the loss-of-function KCND3 mutations previously reported in cerebellar ataxia patients, we identified a rare gain-of-function KCND3 variant that may expand the clinical and molecular spectra of neurodegenerative cerebellar disorders associated with brain iron accumulation.


Introduction
In neurons, the voltage-gated potassium (K + ) channel subunit K V 4.3 is localized in the somatodendritic compartment and contributes to the generation of A-type K + currents essential for regulating neuronal excitability and action potential firing [1,2]. The K V 4.3 channel is also significantly expressed in the heart, where it plays a crucial role in mediating the transient outward K + current that shapes the early repolarization phase of the cardiac action potential [3][4][5]. Perturbation of K V 4.3 channel properties may therefore substantially affect neuronal and/or cardiac functions.
In the brain, iron homeostasis is crucial for maintaining key physiological functions such as the synthesis of myelin and neurotransmitters [15]. In healthy aging and neurodegenerative diseases, excessive concentration of free iron leads to iron accumulation in brain regions including the basal ganglia and the cerebellum [15][16][17]. Patients suffering from neurodegeneration associated with iron deposition, which can be detected by magnetic resonance imaging (MRI), may develop movement disorders, cognitive decline, and cerebellar ataxia [15,18].
The detailed mechanisms underlying brain iron deposition remain unresolved, as only two of the 15 known causal genes for neurodegeneration with brain iron accumulation are directly linked to iron homeostasis (MIM#117700 and MIM#606159) [15,19]. Intriguingly, excessive iron accumulation has also been observed in patients suffering from neurodegenerative cerebellar ataxia disorders [20][21][22]. It remains unclear, however, whether ataxia-related KCND3 variants may be associated with brain iron accumulation. In this study, we report the identification of a rare KCND3 c.1256G>A (p.R419H) variant in a patient with cerebellar ataxia, parkinsonism, cognitive impairment, and brain iron accumulation. Further biochemical and electrophysiological analyses suggest that the KCND3 variant leads to a gain-of-function K V 4.3 channel phenotype. Our findings expand the clinical and molecular spectra of neurodegenerative cerebellar ataxia associated with iron deposition in the brain.

Case Presentation
The proband is a male with a past medical history of anxiety disorder who first presented to the University of Pennsylvania (UPenn) Movement Disorders Clinic at the age of 69 years with a chief complaint of gait dysfunction. He reported normal development with the exception of mild gait instability with occasional falls while playing sports as a child and throughout adulthood. In his mid-60s, the patient's balance deteriorated with an increased frequency of falls, and he developed urinary urgency with incontinence, and cognitive changes characterized by poor recall, naming, personality changes, disinhibition, and inappropriate joking. The patient was born in a non-consanguineous pedigree with no reported family history of neurological diseases ( Figure 1A). His parents are deceased due to non-neurological causes. No significant neurological deficit was noted for the patient's sibling and offspring.
The neurological examination revealed cerebellar ataxia and mild parkinsonism, characterized by masked facial expression, perioral dyskinesias, mild intention tremor and mild dysmetria, bilateral limb ataxia with dysdiadochokinesia, lower greater than upper extremity bradykinesia, paratonia, upright rigid posture, wide-based gait with short stride length, and mild tandem gait impairment (Supplementary Video S1; Supplementary Figure S1). He achieved 25/30 points on the Montreal Cognitive Assessment. Neuropsychological evaluation at the age of 69 demonstrated average overall intellectual functioning and verbal skills, with high average perceptual reasoning abilities. Executive dysfunction was also evident with notable impairments in processing speed, complex sequencing, inhibition, novel problem-solving, and conceptual reasoning and perseveration, as well as set loss errors, impulsivity, confabulation, verbosity, tangentiality, conflation, poor organization/planning, and variable self-monitoring. Memory retention was intact. In this pedigree, the heterozygous KCND3 variant was detected in the male individual II.3, who is now 70 years old. The patient's father (I.1) passed away in his seventies due to heart failure, and his mother (I.2) in her eighties due to chronic obstructive pulmonary disease. He has a sister (II.2) who is five years older than him. His two sons (III.4 and III.5) are now in their thirties. Arrow denotes the index case who harbors the KCND3 c.1256G>A (p.R419H) variant. Filled symbol represents symptomatic member. Open symbols indicate unaffected individuals. Circles stand for females. Squares correspond to males. Diagonal lines refer to the deceased. (B) Neuroimages of the patient. The axial T2-weighted (B1-B3) and the corresponding gradient-echo sequence (B1 -B3 ) images demonstrate hypointensity in bilateral caudate nuclei (B1,B1 ) and lentiform nuclei (B2,B2 ) of the basal ganglia, as well as in bilateral dentate nuclei of the cerebellum (B3,B3 ). (C) Standard 12-lead ECG indicates normal sinus rhythm.
Prior to presenting to the clinic, a presumptive diagnosis of normal pressure hydrocephalus prompted high-volume cerebrospinal fluid drainage (25 cc, opening pressure 15 cm-H 2 O) without improvement in gait or memory problems. Serological evaluation for ataxia was unrevealing. MRI of the brain on a 1.5 T scanner demonstrated two main features: (i) diffuse cerebral atrophy with ventriculomegaly and mild white matter disease; (ii) iron deposition in pallidal, caudate, and dentate nuclei ( Figure 1B). A standard electrocardiogram (ECG) examination showed normal sinus rhythm ( Figure 1C).

In Silico Pathogenicity
We employed population databases and bioinformatics analyses to evaluate the pathogenicity of the identified KCND3 variant (Table 1). In both the total and non-Finnish European population in the genome Aggregation Database (gnomAD), the estimated allele frequency of the c.1256G>A (p.R419H) variant is less than 0.0001, suggesting that this is a rare KCND3 variant. The c.1256G>A (p.R419H) variant was predicted to be damaging/disease-causing based on several in silico prediction tools. The CADD program [23] estimated a Phred score of 28.2, suggesting the variant may be more deleterious than 99.85% of the other variants in the genome. Polyphen-2 predicted a probability score as high as 1, implying that the variant is probably damaging [24]. The programs SNPs&GO [25] and SIFT [26] predicted the variant is disease-related and deleterious, respectively. Moreover, MutationTaster [27] also revealed a high probability score close to 1, consistent with the idea that the rare KCND3 c.1256G>A (p.R419H) variant may be disease-associated.

Lack of Effect of the p.R419H Variant on K V 4.3 Protein Expression and Localization
We went on to investigate the potential effect of the p.R419H variant on the in vitro property of human K V 4.3 channel. The K V 4.3 subunit comprises an intracellular aminoterminal domain, six transmembrane segments (S1-S6) containing a K + -conducting pore loop in the S5-S6 linker region, and a cytoplasmic carboxyl-terminal domain ( Figure 2A). The p.R419 residue is localized in the intracellular carboxyl-terminal region, close to the S6 transmembrane segment. p.R419 is a highly conserved residue across various K V 4 channel protein orthologs from multiple animal species ( Figure 2B), implying an evolutionary importance in the structure and function of K V 4.3 channel. Substitution of the positively charged, aliphatic amino acid arginine at residue 419 with the imidazolecontaining histidine results in a sizable reduction in the mean side-chain volume by about 17.3% (from 202 Å 3 to 167 Å 3 ) ( Figure 2C-E).
To assess the pathophysiological significance of the p.R419H variant, we began by determining whether the p.R419H affects protein homeostasis of K V 4.3 channels. K V 4.3 wild-type (WT) and p.R419H proteins were individually expressed in HEK293T cells. As shown by the immunoblots depicted in Figure 3A-C, regardless of the absence or presence of the auxiliary K + channel interacting protein 2 and 3 (KChIP2/KChIP3) subunits, no significant difference in the total protein level was observed between K V 4.3 WT and the p.R419H variant. Moreover, surface biotinylation analyses demonstrated that K V 4.3 WT and the p.R419H variant displayed comparable protein abundance at the plasma membrane in the absence/presence of the auxiliary KChIP2/KChIP3 subunits ( Figure 3D-F). Consistent with these biochemical observations, immunofluorescence analyses also indicated that, whether the auxiliary KChIP2/KChIP3 subunits were present or not, the majority of K V 4.3 WT and the p.R419H variant exhibited similar punctate staining pattern at the cell surface, consistent with the presence of effective plasma membrane-localization for both proteins ( Figure 3G,H). Together, these results suggest that the p.R419H variant does not appear to detectably affect protein expression and subcellular localization of K V 4.3 channels.

Dominant Gain-of-Function Effect of the p.R419H Variant on K V 4.3 Channel Function
Next, we examined the impact of the p.R419H variant on K V 4.3 channel function by performing electrophysiological analyses. Surprisingly, compared to its WT counterpart, K V 4.3 channels harboring the p.R149H variant were associated with a more than three-fold increase in the K + current level ( Figure 4A-C), with no apparent change in the channel activation and inactivation kinetics. This result implies that the p.R419H variant may substantially promote K V 4.3 channel function. (A-C) Compared to K V 4.3 WT, the p.R419H variant is associated with a significantly higher functional expression level. (A) Representative K + current traces recorded from homotetrameric K V 4.3 WT and p.R419H channels. Current traces were induced by a voltage protocol comprising test potentials ranging from -60 mV to +60 mV in 10-mV steps. (B) Normalized peak current amplitudes were plotted against matching test pulse potentials. For each current trace induced by a test pulse potential, the peak current amplitude was measured, followed by normalization with respect to the corresponding mean peak current amplitude at +60 mV of K V 4.3 WT. (C) Statistical comparison of the normalized peak current amplitude at +60 mV. Mean normalized peak current amplitude at +60 mV: WT, 1 ± 0.1; p.R419H, 3.2 ± 0.3. Asterisks denote significant difference from the WT control (p < 0.05). Numbers in parentheses refer to the amount of cells analyzed for each K V 4. Both the WT/R419H heterotetramer and the R419H/R419H homotetramer are associated with a significant increase in the window current size of K V 4.3 channels. Table 2. Comparison of the K V 4.3 voltage-dependent gating parameters of the wild type (WT) and the p.R419H variant. Steady-state activation and inactivation curves were generated from the averages of 9-12 cells expressing the indicated K V 4.3 constructs. Data were subject to fitting with the Boltzmann equation as described in the Materials and Methods section and depicted in Figure 4. V 0 . 5a : half-activation voltage. k a : activation slope factor. V 0 . 5i : half-inactivation voltage. k i : inactivation slope factor. A functional voltage-gated K + channel is formed by the assembly of four K + channel protein subunits (tetramer). Given the fact that the proband carries the heterozygous c.1256G>A (p.R419H) variant in the KCND3 gene and that K V 4.3 WT and the p.R419H variant appear to display comparable protein expression levels ( Figure 3), it is likely that K V 4.3 WT and p.R419H variant subunits may co-assemble and form heterotetrameric K V 4.3 channels in native cells in the patient. We therefore asked whether the p.R419H variant may exert a dominant effect on the functional expression of its K V 4.3 WT counterpart. To address this important issue, we co-expressed K V 4.3 WT and WT (WT/WT homotetramer), WT and p.R419H (WT/R419H heterotetramer), or p.R419H and p.R419H (R419H/R419H homotetramer) in the same cell, followed by comparing their functional properties. As outlined in Figure 4D-F, the mean current amplitude of K V 4.3 WT/R419H heterotetramers is more than two-fold lager than that of WT/WT homotetramers; moreover, the K + current level of R419H/R419H homotetramers is substantially higher than that of WT/R419H heterotetramers. This R419H-dependent, progressive increase in K + current level strongly argues that, in WT/R419H heterotetramers, the p.R419H variant is associated with a dominant effect on the functional expression of K V 4.3 channels.
To further explore the potential mechanism underlying the observed enhanced current amplitude associated with the p.R419H variant, we analyzed the voltage-dependent gaiting property of K V 4.3 channels comprising WT/WT homotetramers, WT/R419H heterotetramers, or R419H/R419H homotetramers. As clearly illustrated in Figure 4G,H and Table 2, a notable R419H-dependent modification effect was observed for both steady-state activation (G/G max ) and inactivation (I/I max ) properties of K V 4.3 channels. For example, compared to the WT/WT homotetramer control, the steady-state activation (G/G max ) curve of the WT/R419H heterotetramer and the R419H/R419H homotetramer was leftshifted by about 2.6 and 8.2 mV, respectively ( Figure 4G), implying that increasing the relative proportion of the p.R419H variant in WT/R419H heterotetramers may lead to progressively higher K V 4.3 channel open probability at the resting membrane potential. Similarly, the R419H-dependent right-shift of the steady-state K V 4.3 channel inactivation (I/I max ) curve ( Figure 4H) suggests that increasing the relative proportion of the p.R419H variant may render WT/R419H heterotetramers less likely to be inactivated at the resting membrane potential. Together, these observations are consistent with the idea that, in WT/R419H heterotetramers, the p.R419H variant may exert a dominant effect on the voltage-dependent gating function of K V 4.3 WT.
The superimposed region of steady-state activation and inactivation curves is known as the window current ( Figure 4I-L), which correlates with the voltage range in which a significant fraction of inactivating ion channels may remain open with minimal inactivation, and therefore provides an estimate of the effective K V 4.3 channel conductance under physiological conditions in neurons. In agreement with the foregoing R419H-dependent increase in K + current amplitudes ( Figure 4F), the WT/R419H heterotetramer and the R419H/R419H homotetramer displayed an enhancement of the size of the window current by about 2.8-fold and 3.6-fold, respectively ( Figure 4J,L), indicating that the observed K + current-potentiating effect can be in part attributed to the dominant gain-of-function voltage-dependent properties conferred by the p.R419H variant.

Discussion
Based on the standards and guidelines set forth by the American College of Medical Genetics (ACMG) [28], we employed multiple criteria to assess the clinical significance of the heterozygous KCND3 c.1256G>A (p.R419H) variant identified in a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive dysfunction, and brain ion accumulation. Several lines of evidence support the pathogenicity of this KCND3 variant: (i) this nonsynonymous variant in the KCND3 gene, whose missense variants are frequently linked to cerebellar ataxia, contributes to a low allele frequency (<0.0001) in the population database gnomAD (Table 1) (criterion PP2 in the ACMG guideline); (ii) prediction of deleterious or damaging effects by multiple in silico bioinformatics analyses (Table 1) (criterion PP3); and (iii) the patient's presentation of specific neurological symptoms relevant to KCND3-mutation-related SCA19/22 (criterion PP4). In addition, we provided the direct in vitro functional evidence ( Figure 4) showing that the p.R419H variant is associated with a dominant gain-of-function effect on the K + current amplitude and voltage-dependent gating of its K V 4.3 WT counterpart, a strong indication of the variant's pathogenicity (criterion PS3). Taken together, we propose that we have identified a "likely pathogenic" gain-of-function KCND3 variant. Table 3 outlines the genotype-phenotype relationship of known disease-associated KCND3 variants. Patients with KCND3-related neurological disorders are characterized by heterogeneous clinical presentations including cerebellar ataxia, cognitive dysfunction, and movement disorders such as parkinsonism [10]. KCND3-related ataxia is further known to be associated with a wide range of disease onset (from very early ages to later stages of life), as well as distinctly different clinical courses (including episodic, non-progressive, and slowly progressive). Consistent with these notions, in the current study the patient harboring the heterozygous KCND3 p.R419H variant presented with mild gait instability in his childhood and did not display complex neurological features until late adulthood. Moreover, the proband appears to be the only person in his family showing significant neurological disorders ( Figure 1A), suggesting that the case is seemingly sporadic. Despite the fact that most KCND3-related disorders are autosomal dominant, de novo mutation and incomplete penentrance have been reported as well [6,9]. Therefore, the presence of four persons carrying the c.1256G>A (p.R419H) variant in gnomAD (Table 1) may additionally imply that this is a rare KCND3 variant with incomplete penetrance. Table 3. The genotype-phenotype relationship for KCND3 variants associated with neurological or cardiac disorders. Notation for in vitro functional phenotype: GOF, gain of function; LOF, loss of function; NSFC, no significant function change; n.a., not available. See Figure 5 for topographic representation.
An asymptomatic mother carried the variant, suggesting incomplete penetrance. n.a. [29] p.F227 deletion Slowly progressive cerebellar ataxia, onset from teenage to middle age; oculomotor abnormalities, pyramidal signs parkinsonism, epilepsy, or cognitive impairment have been reported in some cases.
Identified in autosomal dominant pedigrees from multiple ethnic groups. Incomplete penetrance was reported in a pedigree.
A symptomatic mother-son pair from an autosomal dominant inheritance pedigree. LOF [9] p.T377M (i) Adolescence or adult-onset cerebellar ataxia; cognitive impairment in some patients. (ii) Hereditary spastic paraplegia.
A recurrently reported mutation identified in multiple ethnic groups. One single case identified from a cohort study of hereditary spastic paraplegia.
Identified in a case with autopsy-negative sudden unexplained death syndrome at first; one single case with Dravet syndrome was linked to the variant; a pair of siblings presented with cardiocerebral syndrome.
Identified in cases with Brugada syndrome at first; one case with autosomal dominant cerebellar ataxia was later reported.
One single case identified from a cohort study of hereditary spastic paraplegia. Two individuals observed in the cohort study of early-onset of persistent lone atrial fibrillation.
Recurrently observed from patients with Brugada syndrome or sudden unexplained death syndrome. GOF [11,42] p.P633S (p.P614S for the short isoform) Late onset cerebellar ataxia, decreased reflexes, and vibration sense. One single case from a cohort study of cerebellar ataxia. NSFC [14,36] To date, nearly 30 KCND3 variants have been associated with neurological or cardiac disorders (Table 3). A majority of KCND3 variants linked to neurological (e.g., SCA19/22) and cardiac (e.g., Brugada syndrome) disorders display loss-of-function and gain-offunction phenotypes, respectively, even though some of the cardiopathogenic gain-offunction KCND3 mutations were later associated with neurological pathogenicty as well. The p.R419H variant identified in our ataxic patient exerts a dominant gain-of-function effect on functional K + current level, which is probably attributed to a notable alteration of the steady-state voltage-dependent activation and inactivation of K V 4.3 channels. Moreover, our ECG analysis reveals that the patient carrying the p.R419H variant does not appear to display detectable cardiac arrhythmia. Previously, another gain-of-function KCND3 variant, p.L450F, was originally linked to the Brugada syndrome; however, this variant was later identified in a patient with cerebellar gait ataxia but no significant heart problems [11,14]. As far as cerebellar ataxia is concerned, the aforementioned observations support the idea that functional homeostasis of K V 4.3 plays an imperative role in the operation of cerebellar physiology, and that both loss-and gain-of-function phenotypes of K V 4.3 variant channels may considerably perturb neuronal excitability in the cerebellar circuit and therefore contribute to the pathogenesis of ataxia. Consistent with these notions, both loss-and gain-of-function variants in the KCNC3 gene encoding another voltage-gated K + channel (K V 3.3) have been associated with spinocerebellar ataxia type 13 [47]. Figure 5 summarizes the topographic localization of currently known disease-associated KCND3 variants within the K V 4.3 subunit. Interestingly, virtually all of the loss-of-function variants are located in the transmembrane region of the channel protein. In contrast, many of the gain-of-function variants, including the two ataxia-related gain-of-function variants p.R419H and p.L450F, are found in the cytoplasmic carboxyl-terminal region. It is unclear how the replacement of arginine with histidine at residue 419 may instigate such a significant gainof-function effect on voltage-dependent gating of K V 4.3 channel. The amino acid substitution at this evolutionary conserved K V 4.3 residue is unlikely to dramatically affect the secondary protein structure of the proximal carboxyl-terminal region. Nonetheless, our in silico analyses suggest that p.R419 may be in close proximity with two intracellular domains, the S4-S5 linker and the amino-terminal region ( Figure 2E), both essential for regulating voltage-dependent gating of K + channels. Detailed experimental analyses will be required in the future to determine whether the histidine substitution may have a direct impact on the potential interaction between p.R419 and its structural microenvironment in K V 4.3 channel.  Table 3 for genotype-phenotype relationship.
As depicted in Figure 1B, brain MRI revealed significant iron deposition in bilateral caudate nuclei and lentiform nuclei of the basal ganglia, and in bilateral dentate nuclei of the cerebellum. It is an open question regarding the mechanistic link between K V 4.3 current level and iron accumulation in neurons. Neuronal iron overload may result from enhanced postsynaptic iron uptake by the divalent metal transporter 1 (DMT1), and has been suggested to contribute to neurodegenerative diseases such as the Parkinson's disease [48]. In addition, activation of K + channels was shown to promote DMT1-mediated iron import into neuroblastoma cells [49]. We therefore speculate that the gain-of-function p.R419H variant may similarly potentiate DMT1-mediated iron uptake in specific regions in the brain. Since the iron-rich dentate nucleus serves as one of the largest deep cerebellar nuclei essential for the output signal from the cerebellum [50], dysregulation of iron homeostasis in bilateral dentate nuclei may lead to substantial anomaly of the cerebellar function. An analogous K V 4.3 p.R419H-induced enhancement of iron uptake may also take place in bilateral lentiform and caudate nuclei of the basal ganglia, resulting in the parkinsonism observed in the patient. To the best of our knowledge, the current study provides the first evidence suggesting a potential association between K V 4.3 gain-of-function and the susceptibility for brain iron accumulation. Taken as a whole, our findings highlight the wide variation in the phenotypical expression and pathophysiological outcome of disease-associated KCND3 variants.

Patient Evaluations and Ethics
This study was supported by the Neurogenetics Translational Center of Excellence, Department of Neurology, UPenn. The patient was evaluated by two neurologists with expertise in movement disorders and neurogenetics at UPenn. Molecular tests, neurocognitive evaluation, and neuroimaging studies were conducted as part of clinical care. Informed consent was provided by the patient.

Genetic Analyses
Genomic/mitochondrial DNA was extracted from white blood cells in the peripheral venous blood. Screening of a neurodegeneration with brain iron accumulation gene panel was implemented by Associated Regional and University Pathologists (ARUP) laboratories (Salt Lake City, UT, USA). Whole exome sequencing (WES), mitochondrial sequencing, and deletion testing were conducted by the XomeDxPlus test of GeneDx (BioReference Laboratories, Gaithersburg, MD, USA) to detect disease-relevant variant. The targeted exonic regions and flanking splice junctions of the genome were simultaneously sequenced with 100 bp paired-end reads by massively parallel sequencing on an Illumina HiSeq 2000 sequencing system (NextGen Healthcare, Irvine, CA, USA). A customized analysis tool (Xome Analyzer, GeneDx; BioReference Laboratories, Gaithersburg, MD, USA) was utilized to assemble and align the bi-directional sequence to reference genome sequences (GRCh37/UCSC hg19), as well as calling for sequence variants in the regions of interest throughout the genome. The potentially pathogenic variants originally identified were further confirmed by an appropriate method such as capillary sequencing. Sequence alterations were reported based on the Human Genome Variation Society (HGVS) nomenclature guideline. The identified variants were subject to further evaluation by searching in gnomAD and the 1000 Genome Browser (1000 Genomes).

cDNA Constructs
Amino-terminal Myc-tagged K V 4.3 (Myc-K V 4.3) was generated by subcloning human K V 4.3 cDNA into the pcDNA3.1-Myc vector (Invitrogen, Carlsbad, CA, USA). The K V 4.3 p.R419H variant was created by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Amino-terminal HA-tagged KChIP2 and KChIP3 (HA-KChIP2 and HA-KChIP3) were created by subcloning human KChIP2 and KChIP3 cDNAs, respectively, into the pcDNA3-HA vector (Invitrogen, Carlsbad, CA, USA). All the constructs were verified by DNA sequencing. For in vitro transcription, appropriate restriction enzymes were applied to linearize cDNAs, from which capped cRNAs were transcribed using the Ambion mMessage mMachine T7 kit (Thermo Scientific, Waltham, MA, USA).

Cell Culture and Transfection
Human embryonic kidney (HEK) 293T cells were grown in Gibco Dulbecco's modified Eagle's medium (DMEM) (Thermo Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Scientific, Waltham, MA, USA), 1 mM sodium pyruvate, 100 units/mL HyQ penicillin-streptomycin, and maintained at 37 • C in a humidified incubator with 95% air and 5% CO 2 . Cells were plated onto six-(for surface biotinylation) or 12-well plates (for total protein), or poly-D-lysine-coated coverslips in 24-well plates (for immunofluorescence) 24 h before transfection. Transient transfection was performed by the calcium phosphate method. Briefly, DNA/calcium phosphate precipitate was prepared by mixing one volume of DNA in 250 mM CaCl 2 with an equal-volume 2X HEPES-buffered saline (HBS) [(in mM) 280 NaCl, 50 HEPES, 1.5 Na 2 HPO 4 , pH 7.0]. The calcium phosphate precipitate was allowed to form for 20 min in the dark at room temperature prior to being added to the cultures. The quantities of cDNA used for different experiments are as follows: 400 ng/well for immunofluorescence, 800 ng/well for total protein, and 1600 ng/well for surface biotinylation. The DNA/calcium phosphate precipitates were added drop-wise to cells, which were subject to 37 • C incubation for 3-4 h. To terminate the transfection, the mixture solution was replaced with fresh medium pre-warmed in the 37 • C incubator. The cells were returned to the 5% CO 2 incubator at 37 • C until further processing. For co-expression experiments, cDNAs for individual K V 4.3 and the auxiliary subunit were mixed in equimolar ratio.

Immunoblotting
Transfected cells were lysed in an ice-cold lysis buffer [(in mM) 150 NaCl, 5 EDTA, 50 Tris-HCl pH 7.6, 1% Triton X-100] containing a complete protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). After adding the Laemmli sample buffer to the lysates, samples were sonicated on ice (three times for five seconds each) and heated at 70 • C for 5 min for further process. Samples were then separated by 7.5% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-Myc (clone 9E10), or rabbit anti-α-tubulin (Bethyal Laboratories, Montgomery, TX, USA) antibodies. Blots were exposed to horseradish-peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), or goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA), and revealed by an enhanced chemiluminescence detection system (Thermo Scientific, Waltham, MA, USA). Acquisition of chemiluminescent signals from immunoblots was achieved by using the UVP AutoChemi image system (Ultra-Violet Products, Upland, CA, USA). Data shown are representative of at least 3 independent experiments. Densitometric scans of immunoblots were quantified with ImageJ (National Institute of Health, Bethesda, MD, USA).
For surface biotinylation analyses, transfected cells were incubated in 1 mg/mL sulfo-NHS-LC-biotin (Thermo Scientific, Waltham, MA, USA) in ice-cold phosphate-buffered saline (PBS) [(in mM) 136 NaCl, 2.5 KCl, 1.5 KH 2 PO 4 , 6.5 Na 2 HPO 4 , pH 7.4] with 0.9 mM CaCl 2 and 0.5 mM MgCl 2 at 4 • C for 1 h on orbital shaker. After the biotin reagents were removed, the cells were rinsed with glycine-containing PBS, followed by once in Tris-buffered saline [(in mM) 20 Tris-HCl, 150 NaCl, pH 7.4] to terminate biotinylation. Cells were solubilized in the lysis buffer. Cell lysates were incubated overnight at 4 • C with streptavidin-agarose beads (Thermo Scientific, Waltham, MA, USA). Beads were washed four times in the lysis buffer, followed by heating in Laemmli sample buffer to elute biotin-streptavidin complexes.

Immunofluorescence
HEK293T cells were seeded on poly-D-lysine-coated coverslips in 24-well culture dishes. Forty-eight hours after transfection, the coverslips containing HEK293T cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Cells were permeabilized and blocked with a blocking buffer (5% normal goat serum in 20 mM phosphate buffer, pH 7.4, 0.1% (v/v) Triton X-100, and 450 mM NaCl) for 60 min at 4 • C. Appropriate dilutions of primary antibodies (1:200 for the mouse anti-Myc antibody; 1:200 for the rat anti-HA antibody) were appropriately applied in the blocking buffer overnight at 4 • C. Immunoreactivities were visualized with goat-anti-mouse antibodies conjugated to Alexa Fluor 488 (1:200; Invitrogen, Carlsbad, CA, USA), as well as goat-anti-rat antibodies conjugated to Alexa Fluor 633 (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Nuclei were labeled with DAPI. Finally, the coverslips were rinsed once in blocking buffer, twice in PBS, and twice in 0.1 M carbonate buffer, pH 9.2, before they were mounted on glass slides in a mounting medium (4% n-propyl gallate, 90% glycerol, 0.1 M carbonate, pH 9.2). A laser-scanning confocal microscope (Leica TCS SP8 STED; Wetzlar, Germany) was utilized to acquire fluorescence images.

Electrophysiological Analyses
Xenopus laevis oocytes (African Xenopus Facility, Knysna, South Africa) were used for functional studies. All animal procedures were in conformity with the animal protocol approved by the Institutional Animal Care and Use Committee of National Taiwan University. Frogs were anesthetized to dissect ovarian follicles, which were incubated in ND96 [(in mM): 96 NaCl, 2 KCl, 1.8 MgCl 2 , 1.8 CaCl 2 , and 5 HEPES, pH 7.2]. Stage V-VI oocytes were selected for cRNA injection, and the cRNAs for individual K V 4.3 and KChIP2 were mixed in equimolar ratio. For analyzing K V 4.3 WT/mutant heterotetramer, K V 4.3 WT, mutant and KChIP2 were mixed in a molar ratio of 1:1:2. Injected oocytes were stored in ND96 at 16 • C for 2-3 days before being used for functional analyses. OC-725C oocyte clamp (Warner Instruments, Hamden, CT, USA) was used to record K + currents through K V 4.3 channels utilizing two-electrode voltage-clamp technology. The recording bath contained Ringer solution [(in mM): 3 KCl, 115 NaCl, 1.8 CaCl 2 , 10 HEPES, and 0.4 niflumic acid, pH 7.4 with methanesulfonic acid]. Borosilicate electrodes (0.1-1 MΩ) filled with 3 M KCl were used for voltage recording and current injection. Data were acquired and digitized via Digidata 1440A using pCLAMP 10.2 (Molecular Devices, San Jose, CA, USA). Oocytes were held at −90 mV and leak currents arising from passive membrane properties were subtracted by using the −P/4 method provided in the pCLAMP system. Data were obtained, normalized and analyzed as reported previously [54].
To generate peak K + current-voltage curves for studying steady-state voltage-dependent activation of K V 4.3, the voltage protocol comprised 500 ms test pulses stepped from −60 mV to +60 mV, in 10 mV increments. The relative conductance (G/G max ) at a given test potential was calculated as G/G max = ∆I K /∆I K,+60 , where ∆I K is current increment determined from peak current difference between adjacent test pulses, and ∆I K,+60 is the ∆I K at the test potential +60 mV. A steady-state voltage-dependent activation curve was then generated by fitting the G/G max -voltage (G-V) curve with a Boltzmann equation: G/G max = 1/{1 + exp[(V 0.5a − V)/k a ]}, where V 0 . 5a is the half-activation voltage, and k a is the activation slope factor. To study the steady-state voltage-dependent inactivation of K V 4.3, oocytes were subject to 1 s prepulses stepping from −120 to +10 mV with 10 mV increments, followed by a +60 mV test pulse for 500 ms. Normalized peak currents at +60 mV (I/I max ) were then plotted against corresponding prepulse potentials. A steady-state voltage-dependent inactivation curve was then generated by fitting a I/I max voltage curve with a Boltzmann function: I/I max = 1/{1 + exp[(V 0.5i − V)/k i ]}, where V 0.5i is the half-inactivation voltage, and k i is the inactivation slope factor. Window current analyses were determined from the apex and the area of the triangular overlap area between activation and inactivation curves.

Statistical Analyses
Statistical analyses were performed with Origin 7.0 (Microcal Software, Northampton, MA, USA). Numerical values were presented as mean ± SEM. The significance of the difference between two means was tested using Student's t-test.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22158247/s1, Supplementary Video S1. The clinical presentation of the patient harboring KCND3 c.1256G>A (p.R419H) variant in this report. Neurological examination of the 69-yearold gentleman revealed cerebellar ataxia with mild parkinsonism, characterized by masked facial expression, perioral dyskinesias, mild intention tremor, bilateral dysrhythmokinesis, paratonia, lower greater than upper extremity bradykinesia, upright rigid posture, wide-based gait with short stride length, and mild tandem gait impairment. Supplementary Figure S1. Thumbnail image from Supplementary Video S1.