A novel KCNA1 mutation in a patient with paroxysmal ...

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Molecular and Cellular Neuroscience 83 (2017) 6–12

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A novel KCNA1 mutation in a patient with paroxysmal ataxia, myokymia, painful contractures and metabolic dysfunctions Paola Imbrici a,⁎, Concetta Altamura a, Francesca Gualandi b, Giuseppe Felice Mangiatordi a, Marcella Neri b, Giovanni De Maria c, Alessandra Ferlini b, Alessandro Padovani d, Maria Cristina D'Adamo e, Orazio Nicolotti a, Mauro Pessia e,f, Diana Conte a, Massimiliano Filosto d, Jean-Francois Desaphy g a

Department of Pharmacy - Drug Sciences, University of Bari Aldo Moro, Bari, Italy Logistic Unit of Medical Genetics, Department of Medical Sciences, University-Hospital of Ferrara, Italy c Unit of Neurophysiopathology, ASST “Spedali Civili”, Brescia, Italy d Center for Neuromuscular Diseases and Neuropathies, Unit of Neurology, ASST “Spedali Civili”, and University of Brescia, Brescia, Italy e Faculty of Medicine, Department of Physiology and Biochemistry, University of Malta, MSD-2080 Msida, Malta f Department of Experimental Medicine, Section of Physiology & Biochemistry, University of Perugia School of Medicine, Perugia, Italy g Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Bari, Italy b

a r t i c l e

i n f o

Article history: Received 22 December 2016 Revised 5 April 2017 Accepted 25 June 2017 Available online 28 June 2017 Keywords: Episodic ataxia Kv1.1 channel Patch clamp Genetics Homology modeling

a b s t r a c t Episodic ataxia type 1 (EA1) is a human dominant neurological syndrome characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles that can be triggered by emotional stress and fatigue. This rare disease is caused by missense mutations in the KCNA1 gene coding for the neuronal voltage gated potassium channel Kv1.1, which contributes to nerve cell excitability in the cerebellum, hippocampus, cortex and peripheral nervous system. We identified a novel KCNA1 mutation, E283K, in an Italian proband presenting with paroxysmal ataxia and myokymia aggravated by painful contractures and metabolic dysfunctions. The E283K mutation is located in the S3–S4 extracellular linker belonging to the voltage sensor domain of Kv channels. In order to test whether the E283K mutation affects Kv1.1 biophysical properties we transfected HEK293 cells with WT or mutant cDNAs alone or in a 1:1 combination, and recorded relative potassium currents in the whole-cell configuration of patch-clamp. Mutant E283K channels display voltage-dependent activation shifted by 10 mV toward positive potentials and kinetics of activation slowed by ~2 fold compared to WT channels. Potassium currents resulting from heteromeric WT/E283K channels show voltage-dependent gating and kinetics of activation intermediate between WT and mutant homomeric channels. Based on homology modeling studies of the mutant E283K, we propose a molecular explanation for the reduced voltage sensitivity and slow channel opening. Overall, our results suggest that the replacement of a negatively charged residue with a positively charged lysine at position 283 in Kv1.1 causes a drop of potassium current that likely accounts for EA-1 symptoms in the heterozygous carrier. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Episodic ataxia type 1 is an autosomal dominant neurological disorder affecting the central and peripheral nervous systems (Graves et al., 2014; D'Adamo et al., 2015a). It occurs in early childhood and lasts throughout life with two main symptoms: myokymia (contractions of the muscles of the eyes, mouth and hands) and ataxia (abnormal movements of the head, legs and arms with loss of balance). Over the years, the phenotypic spectrum of the disease has widened and included other symptoms within the affected families such as neuromyotonia, ⁎ Corresponding author at: Department of Pharmacy - Drug Sciences, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy. E-mail address: [email protected] (P. Imbrici).

http://dx.doi.org/10.1016/j.mcn.2017.06.006 1044-7431/© 2017 Elsevier Inc. All rights reserved.

epilepsy, cognitive dysfunction, migraine, hyperthermia, hypomagnesemia and cataplexy (D'Adamo et al., 2015a, 2015b; Tomlinson et al., 2010; Brownstein et al., 2016; Parolin Schnekenberg et al., 2015; Imbrici et al., 2007). The attacks of ataxia are episodic, can last from minutes to hours, and are aggravated by physical and emotional stress (including fatigue and exercise), ischemia, and changes in temperature, through not yet clearly defined mechanisms. The frequency of attacks, severity and type of symptoms can vary between individuals. Acetazolamide and antiepileptic drugs represent the first line symptomatic pharmacological intervention in EA1, with variable efficacy (D'Adamo et al., 2015a; Imbrici et al., 2016). The disease is caused by loss-of-function missense mutations in KCNA1 gene encoding the voltage-gated potassium channel, Kv1.1 (D'Adamo et al., 2015a). This delayed rectifier K+ channel is composed of four homologous alpha subunits, each

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comprising six transmembrane segments, and can assemble as homomeric or heteromeric proteins with other members of the same subfamily. The S5–S6 segments and the H5 loop of each Kv1.1 α-subunit are part of the ion-conducting pore of the channel and provide the selectivity filter for K+. The S1–S4 segments form the voltage-sensor domain that is coupled, through the helical S4–S5 linker, to the potassium channel pore (Kuang et al., 2015). Electrophysiological recordings from heterologously expressed Kv1.1 mutant channels and from brain slices of an animal model of EA1 provided essential information for the understanding of the physiological role of Kv1.1 channels and the pathophysiological mechanisms underlying EA1 (D'Adamo et al., 2015a; Herson et al., 2003). In the central and peripheral nervous systems this channel contributes to neuronal excitability, firing properties and neurotransmitters release (Begum et al., 2016; Herson et al., 2003; Brunetti et al., 2012; Chen et al., 2005). In heterologous expression systems, EA1 mutations, located at highly conserved positions throughout the entire Kv1.1 primary sequence, were shown to cause mainly a marked reduction of surface expression with or without a dominant negative effect, a positive shift in the voltage dependent activation, altered kinetics or reduced single channel conductance (Imbrici et al., 2003; Imbrici et al., 2008; Zerr et al., 1998; Imbrici et al., 2011; D'Adamo et al., 2015a, 2015b). The consequent reduction of potassium efflux through the mutated Kv1.1 seems to be the main cause of the abnormal neuronal excitability responsible for the episodes of ataxia and neuromyotonia or myokymia (Tomlinson et al., 2010). Nevertheless, the correlation between the clinical manifestations of patients, that are variable among affected individuals, and the structural and functional consequences of specific EA1 mutations is not straightforward. The availability of the Xray solved structure of Kv1.2-Kv2.1 chimera (PDB code: 2R9R) was helpful to address some questions regarding the importance of various amino acid substitution for Kv1 channel function (Long et al., 2007), despite the role of some structural domains is still unclear. Here, we report the clinical and genetic study of an Italian family affected by EA1 and the functional and structural characterization of the novel mutation E283K in Kv1.1 channels, located in the S3–S4 linker, which results in changes in the voltage-dependence and kinetics of activation. 2. Material and methods 2.1. Genetic analysis DNA was isolated from the peripheral blood by standard methods. The coding regions and exon-intron boundaries of KCNA1 were PCR amplified and sequenced as previously described (Imbrici et al., 2008). 2.2. Mutagenesis and expression of hKv1.1WT and mutant channel The E238K mutation was introduced into the plasmid PMT2LF-hKv1.1 containing the full-length WT hKv1.1 cDNA using the QuickChange™ site-directed mutagenesis kit (Stratagene Cloning Systems, Santa Clara, CA, USA). The complete coding region of the cDNA was sequenced to exclude polymerase errors. HEK293 cells were transiently transfected with the hKv1.1WT or E238K (2.5 μg or 5 μg) and CD8 reporter plasmids (1 μg) using the calcium–phosphate precipitation method. Only cells decorated with anti-CD8 antibody-coated microbeads (Dynabeads M450, Invitrogen) were used for patch-clamp recordings. 2.3. Electrophysiology Standard whole-cell patch-clamp recordings were performed at room temperature (~20 °C) using an Axopatch 200B amplifier (Axon Instruments, Sunnyvale CA, USA) (Tricarico et al., 2013; Dinardo et al., 2012). The bath solution contained (mM): NaCl 142, KCl 2.8, MgCl2 1, CaCl2 1, HEPES 10, glucose 11, pH = 7.4 whereas the pipette solution contained (mM): NaCl 10, K-glutamate 132, MgCl2 2, CaCl2 0.9, EGTA

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1, HEPES 10, pH = 7.4 (Mele et al., 2012). Pipettes were pulled from borosilicate glass and had ~2.5 MΩ resistance. Outward currents were evoked by 200 ms depolarizing commands from a holding potential of − 80 mV to + 40 mV in 10 mV intervals, followed by a 150 ms voltage step at − 50 mV, where tail currents were measured. The voltage-dependence of channel activation was determined by plotting normalized tail currents as a function of the prepulse potential, and fitting data points with the Boltzmann function I = 1/1 + exp{−(V − Vh)/k}. Vh, the half-maximal activation potential and k, the slope factor, were calculated from fit. To measure activating kinetics, currents were elicited by 200 ms depolarizing pulses from a holding potential of − 80 mV to + 40 mV in 5 mV intervals. To measure deactivating kinetics, currents were elicited by 200 ms depolarizing pulse at + 20 mV followed by 200 ms depolarizing pulses from −80 to +20 mV in 5 mV intervals. Activating and deactivating kinetics were measured by fitting activating and deactivating current traces with a single exponential function. The resulting time constants were plotted as a function of voltage and fitted with the equation: τ = τVh exp.(V-Vh)/k, where τ Vh is the time constant at the mid-point activation voltage (Vh) of the channels, and k is the slope factor for the voltage-dependence of the time constants. To determine the C-type inactivation kinetics, a test pulse to +20 mV was delivered for 90 s to cells expressing Kv1.1 channels. The slow inactivation was estimated by fitting the time course of current decay with a double-exponential function and calculating the fast (τfast) and slow (τslow) time constants and the relevant amplitudes (A %). The recovery from C-type inactivation was determined by using a double-pulse protocol to +10 mV, separated by interpulse intervals of increasing duration (range: 0.1–17.1 s). The current amplitudes evoked by the second pulse (100 ms) were divided by the first pulse (20 s), normalized and plotted as a function of the interpulse interval. Data points were fitted with an exponential function from which the time constants were calculated. Currents were low-pass filtered at 2 kHz and digitized with sampling rates of 50 kHz using the Digidata 1440A AD/DA converter (Axon Instruments, Sunnyvale CA, USA). Data were analyzed by using Pclamp 10.3 (Axon Instruments, Sunnyvale CA, USA) and Kaleida Graph Software. Results are reported as mean ± SEM from n cells, and statistical analysis was performed using Student's t-test, with P b 0.05 or less considered as significant. 2.4. Homology modeling The sequence for Kv1.1 protein bearing mutation E283K was submitted to the I-TASSER server (Zhang, 2008; Roy et al., 2010; Yang et al., 2015). As a first step, I-TASSER identified the best structure templates by employing LOMETS (Wu and Zhang, 2007). In particular, the X-ray structure of the Kv1.2-Kv2.1 paddle chimera channel (PDB code: 2R9R) (Long et al., 2007) resulted the highest significance template. Next, several homology models were built by employing replica-exchanged Monte Carlo simulations. In particular, a large ensemble of structural conformations (decoys) were generated and clustered based on the structural similarity using SPICKER (Zhang and Skolnick, 2004). Finally, the model representative of the largest structure cluster was selected. Note that ITASSER estimates the quality of a model by measuring a confidence score (C-score) based on both significance of the template alignments and convergence parameters of the performed simulations. It ranges from −5 (low-quality model) to 2 (high quality model). Herein, the best Kv1.1E283K model returned a C-score equal to −1.1. All structures were visualized with PyMOL (https://www.pymol.org/). 3. Results 3.1. Case report A 45-year-old female patient complained of episodic lack of coordination involving head and limbs without loss of consciousness since

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childhood. Each episode was triggered by physical activity, strong emotions and some foods (bitter orange, chocolate, coffee, tea, salt-rich foods), and lasted few seconds to several minutes or a few hours. She also complained of postural instability when walking on rough terrain, involuntary, spontaneous and localized quivering of a few muscles or bundles within a muscle (myokymia), and painful muscle contractures involving face and limbs. An electromyographic study at rest showed continuous spontaneous activity in the form of bursts of triplets and multiplets with high frequency discharge. Neurological examination showed no abnormalities except for the presence of hand myokymia and mild deep sensory impairment in the lower limbs. Deep tendon reflexes and cerebellar tests were normal. There was neither nystagmus nor myotonia. The patient has never had epileptic episodes. She is suffering from glucose intolerance from about 16 years, high blood pressure for about 6 years treated with nebivolol, and obesity. She was treated with carbamazepine (400 mg a day) with a significant reduction in frequency, severity and duration of symptoms. Family history revealed the presence of episodic lack of coordination involving head and limbs, myokymia and painful muscle contractures in other family members (a 18-year-old son, paternal grandfather, father, one father's sister, a 53-year-old sister and her daughter) (Fig. 1A). A 61-year-old sister complained of myokymia and painful muscle contractures involving face and limbs but did not experience episodes of lack of coordination. Patient's father and sisters were suffering from diabetes mellitus. High blood pressure was described in the father and in the 53-year-old sister while no family history of obesity was present. No family member was available for genetic testing.

3.2. Genetic analysis The study of the KCNA1 gene in the proband showed the heterozygous variant c.847GNA causing the amino acid change p.Glu283Lys (E283K) (Fig. 1B). This variation was not described in the literature and its pathogenic significance was not known. In silico tools (Polyphen, SIFT) predict high probability of pathogenicity. The change is not present in control population databases (ExAc: http://exac.broadinstitute.org/).

3.3. Functional analysis of Kv1.1E238K channels The voltage-gated K+ channel Kv1.1 is formed by the tetrameric assembly of four pore-forming subunits, each containing six transmembrane segments (S1–S6). The mutation E283K is located in the S3–S4 linker belonging to the voltage sensor domain of Kv channels. Kv1.1 is unique within the Kv family to have a glutamate residue in position 283, whereas all the other members present a glutamine in the equivalent position (Fig. 1C). EA1 patients are heterozygous for this disease, so they presumably possess heterotetrameric channels composed of a mixture of WT and mutant subunits. To test the hypothesis that the E283K mutation altered Kv1.1 function and caused EA1 in the affected patient, we expressed equal amount of wild-type (2.5 μg or 5 μg) or E283K (2.5 μg or 5 μg) cDNAs alone or in 1:1 ratio (2.5 + 2.5 μg or 5 + 5 μg) in HEK293 cells. The current amplitude and biophysical properties of potassium currents elicited by E283K and WT + E283K channels were then compared with those obtained from WT currents. As shown in Fig. 2, E283K currents were smaller than those of WT channels whereas the co-expression of WT and E283K cDNAs gave rise to potassium currents that were below the calculated sum of those carried by WT and mutant subunits alone (Fig. 2A–D; Table 1), suggesting a weak dominant-negative effect. To determine whether possible modifications of the voltage dependent activation by E283K mutation might be of pathogenic relevance for EA1, tail current families were recorded at −50 mV after prepulse commands to several voltages (Fig. 3A, Supp. Fig. 1A) and data points were fitted to a Boltzmann function. Mutant E283K channels displayed voltage-dependent activation significantly shifted by 10 mV toward positive potentials compared to WT (Table 1). Potassium currents resulting from the co-transfection of WT and E283K showed voltage-dependent gating intermediate between WT and mutant homomeric channels, with a + 5 mV shift of V1/2 compared to WT. By contrast, the slope factor k calculated from the Boltzmann fit of tail currents was unaffected by the mutation (Fig. 3A, Table 1). To investigate whether the E283K mutation affected the kinetics of activation and deactivation of heteromeric WT + E283K channels, the activating and deactivating current traces of either WT, E283K or WT + E283K channels were fitted with a single exponential functions and

Fig. 1. (A) Pedigree of the family of the patient and sequence analysis of the KCNA1 gene mutation. (B) DNA sequence from the patient and a control individual. The sequencing profile shows the heterozygous single nucleotide change c.847GNA which leads to a glutamate (E) to lysine (K) substitution (p.E283K) in the protein Kv1.1. (C) Amino acid alignment of Kv channels.

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Fig. 2. (A–C) Representative current traces evoked by 200 ms depolarizing steps from a holding potential of −80 mV to 40 mV from Kv1.1WT (A), Kv1.1E283K (B), Kv1.1WT + Kv1.1E283K. (C) channels expressed in HEK 293 cells. (D) bar graph showing the average whole cell current density recorded at +20 mV from Kv1.1WT (2.5 μg and 5 μg), Kv1.1E283K (2.5 μg and 5 μg), Kv1.1WT + Kv1.1E283K (2.5 + 2.5 μg and 5 + 5 μg) (n = 10–20). Dashed line represents the calculated algebraic sum of WT + E283K currents. * p b 0.05, with respect to WT.

the calculated time constants were plotted as a function of membrane potential (Fig. 3B). This analysis revealed that E283K channels had significantly slower kinetics of activation compared with WT, whereas heteromeric WT + E283K channels showed an intermediate behaviour (Fig. 3B, Supp. Fig. 1B, Table 1). The kinetics of deactivation were similar to those of WT channels (Fig. 3B, Table 1). The Kv1.1 channels are characterized by a slow process of inactivation named C-type inactivation that develops upon tenths of seconds, thus increasing progressively during intense neuronal activity, and affecting both the firing rate and the shape of the action potentials (Aldrich et al., 1979). The analysis of the slow inactivation showed that the τfast and τslow for homomeric E283K and heteromeric WT + E283K channels was not statistically different from those of WT channels (Fig. 3C, Supp. Fig. 1C, Table 1). Accordingly, no differences were observed in the recovery from C-type inactivation of the three channel variants (Fig. 3D, Supp. Fig. 1C, Table 1). 4. Discussion 4.1. Clinical implications Here we characterized a novel mutation in Kv1.1 channel associated with EA1 in an Italian patient. Since earlier reports, N 30 different

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missense mutations in the KCNA1 gene have been classified as responsible for EA1 and the clinical manifestations associated with the disease have increased spanning from pure episodic ataxia with myokimia to more complex phenotypes (D'Adamo et al., 2015a, 2015b; Imbrici et al., 2003; Graves et al., 2014; van der Wijst et al., 2010). Interestingly, the clinical signs described here include typical ataxia and myokymia, painful muscle contractures, mild deep sensory impairment in the lower limbs and metabolic dysfunctions. Through functional studies of homomeric E283K and heteromeric WT + E283K mutant channels expressed in HEK 293 cells, we attempted to correlate the clinical phenotype with biophysical alterations induced by the mutation. E283K channels display smaller current amplitudes, slowing of activation kinetics and a 10 mV-positive shift in the voltage dependence of activation compared with WT channels. Upon co-expression with WT subunits, E283K shows a weak dominant-negative effect on WT subunits and resulting channels display intermediate gating properties. The shift of the voltage dependence of activation toward positive potentials and altered kinetics are recurrent channel dysfunctions among Kv1.1 mutant channels associated with typical EA1 symptoms (Imbrici et al., 2011; Zerr et al., 1998; Adelman et al., 1995; D'Adamo et al., 1999). Instead, more severe EA1 phenotypes with long-lasting ataxia are usually caused by Kv1.1 mutants producing very small currents and marked dominant-negative effects upon co-expression with WT (Adelman et al., 1995; Rea et al., 2002; D'Adamo et al., 2015b). In our proband, Kv1.1 E283K channels displayed milder, albeit significant, changes in the biophysical properties that result in a reduction in potassium current density within the physiological voltage range, likely sufficient to provoke characteristic loss of coordination, postural instability and hand myokymia. In the cerebellum, a crucial region for motor control, Kv1.1 and Kv1.2 subunits are co-expressed in the terminals of basket cells, which release GABA onto the Purkinje cells that are responsible for the final output of the cerebellum to the rest of the brain. Basket cells terminals of Kv1.1V408A/+ ataxic mice showed presynaptic spike broadening, increased Ca2+ influx and increased GABA release which led to reduced Purkinje cell firing (Herson et al., 2003; Begum et al., 2016). Similarly, the loss of E283K channels function in the proband, by increasing GABA release and consequently altering Purkinje cells firing, may trigger the episodic lack of motor coordination. Kv1.1 channels are also expressed at juxtaparanodal regions and at branch points of myelinated axons, where they dampen abnormal axonal excitability and allow proper neuromuscular transmission (Vacher et al., 2008). An electromyographic study in the Kv1.1V408A/+ ataxic mice revealed spontaneous bursting activity exacerbated by stress, fatigue and temperature (Brunetti et al., 2012). Consistently, the reduced K+ efflux through E283K mutant channels in this patient may likely cause hyperexcitability of the peripheral nerve endings and consequent bursts activity with high frequency discharge on EMG (myokymia). The proband and some family members also show painful face and limbs muscle contractures and distinctive glucose intolerance and hypertension. Unusual hypercontracted posture and marked muscle stiffness but without pain were already reported in a young patient affected by EA1 (Kinali et al., 2004). Kv1.1 channel has been reported to take part to the mechanosensitive K+ current that regulates the threshold of

Table 1 Biophysical parameters of Kv1.1WT, Kv1.1E283K and Kv1.1WT + Kv1.1E283K channels expressed in HEK 293 cells. Current density

Voltage dependence of activation

Kinetic of activation

Kinetic of deactivation

C-type inactivation [Amplitude %]

Recovery from inactivation

I (pA/pF)

V1/2 (mV)

τV1/2 (ms)

τV1/2 (ms)

τfast (s)

τ (s)

22.4 ± 0.5 (9) 30.8 ± 0.7 (9) 25.9 ± 0.6 (9)

3.9 ± 0.3 [51%] (9) 32.4 ± 2.4 [49%] (9) 4.2 ± 0.6 (6) 3.7 ± 0.5 [51%] (9) 31.8 ± 3.5 [49%] (9) 4.4 ± 0.9 (6) 3.0 ± 0.3 [55%] (9) 27.7 ± 2.7 [45%] (9) 6.1 ± 1.4 (6)

WT 114 ± 19 (20) E283K 76 ± 18 (20) WT + 163 ± 29 (20) E283K

k (mV)

−25.8 ± 0.4 (10) 7.4 ± 0.4 (10) 5.2 ± 0.2 (12) −16.5 ± 0.3⁎⁎ (10) 7.8 ± 0.2 (10) 11.7 ± 0.6⁎ (12) −21.4 ± 0.3 (10) 9.0 ± 0.3 (10) 6.2 ± 0.4 (12)

τslow (s)

To measure current density cells were transfected with 5 μg cDNAs of WT and E283K alone or in 1:1 combination (5 + 5 μg cDNAs of WT + E283K). Data are mean ± se. The number of cells is indicated in parenthesis. ⁎ p b 0.05. ⁎⁎ p b 0.01, with respect to WT.

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Fig. 3. (A) The current-voltage relationships for Kv1.1WT, Kv1.1E283K and Kv1.1WT + Kv1.1E283K channels were obtained by plotting the normalized peak tail currents measured at −50 mV as a function of the pre-pulse potentials and fitting data points with a Boltzmann function (n = 12 cells). (B) Activation and deactivation kinetics measured for the indicated channels. The time constants, resulting from the fit of the activating and deactivating current traces with a single exponential function, were plotted as a function of voltage (n = 9–12 cells). (C) Bar graphs showing the fast and slow time constants of the C-type inactivation for the indicated channels calculated by fitting current decay with a double-exponential function (n = 9 cells). (D) Recovery from C-type inactivation. The solid curves indicate the fit of the data points with an exponential function from which the time constants were calculated for the indicated channels (n = 6 cells).

mechanical sensitivity and the firing properties of fibers associated with mechanical perception (Baker et al., 2011; Hao et al., 2013; Ison and Allen, 2012). Thus, altered mechanosensation due to Kv1.1E283K defects might account for the painful sensation experienced by the patient when contracting face and limbs muscles. The involvement of Kv channel in vascular smooth muscle tone and blood glucose control is intriguing and has been recently documented although in a very few studies. In pancreatic β-cells, Kv1.1 channels, together with other potassium channels, can influence glucose-stimulated insulin release so that a reduction of Kv1.1 current would favor insulin release (Ma et al., 2011; Jacobson et al., 2007; Tricarico et al., 2003). An increase in blood insulin is often associated with insulin resistance, type 2 diabetes, weight gain, and hypertension (Ricci et al., 2017), that are pathological conditions experienced by the proband. Furthermore, heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter suggesting that Kv1 channels contribute to maintaining resting membrane potential and vascular tone (Plane et al., 2005; Fergus et al., 2003; Firth et al., 2011; Park et al., 2010). This would imply an increase in smooth muscle tone when Kv1-mediated currents are reduced. Overall these data could point to a link between glucose intolerance, alteration of arteries blood flow and Kv1 channel expression. Interestingly, diabetes and hypertension were observed also in the affected father and two sisters, suggesting possible co-segregation of these symptoms with ataxia. We may thus speculate that KCNA1 mutation may have contributed to the metabolic syndrome, at least unmasking or worsening it. However, as the role played by Kv1 channels in these pathological conditions is still poorly recognized, we would need more information to reasonably link the E283K mutation to the additional clinical symptoms observed in this patient and better understand the heterogeneity of the clinical manifestations associated with EA1.

4.2. Channel structure implications The E283K mutation is the first mutation associated with EA1 located in the S3–S4 extracellular linker belonging to the voltage sensor domain of Kv1.1 channel. This helix-turn-helix motif formed by the Cterminal half of S3 with the N-terminal half of S4 is proposed to move together during activation and to allow the conformational flexibility of S4 (Tabarean and Morris, 2002; Kuang et al., 2015). The S4 region presents regularly spaced arginine and lysine residues that make Kv channels voltage sensitive by driving S4 movement through the membrane bilayer upon depolarization. It has been proposed that S4 orientation and outward movement in the membrane is allowed by the formation of salt bridges between these positively charged residues and several negatively charged amino acids in the adjacent helices (S1, S2 and S3) (Papazian and Bezanilla, 1999; Tombola et al., 2005; Delemotte et al., 2012; Kuang et al., 2015; Moreau et al., 2015). From a molecular point of view, our results provide a direct evidence that the negatively charged glutamate E283 in the S3–S4 linker of Kv1.1 channel plays a specific role in voltage-sensitive gating since mutant channels (E283K) show voltage dependence shifted toward positive potentials (Vh = −16.5 mV). Kv1.2, that is 80% homologous to Kv1.1, presents a Q in the equivalent position of E283 in Kv1.1 (Fig. 1C) and requires stronger depolarization to activate (Vh = − 16 mV; D'Adamo et al., 1999). The visual inspection of the Kv1.2 crystal structure (PDB code: 3LUT) revealed a strong interaction between Q285 (corresponding to E283 in Kv1.1) and E276 (corresponding to E275 in Kv1.1, see alignment in Fig. 1C and Fig. 4). Interestingly, the superimposition of the homology model obtained for Kv1.1E283K and the Kv1.2 crystal structure suggests that a similar interaction might be established in the mutant. More specifically, K283 could establish

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Fig. 4. Tetrameric structure of the mutant Kv1.1E283K (homology model) showing the localization of the E283K mutation. Inset: Superimposition between the Kv1.2 crystal structure (PDB code: 3LUT) and the homology model of Kv1.1E283K channel. The crystal structure and the homology model are shown in yellow and cyan cartoon representation, respectively. Important residues are rendered as sticks.

an ionic interaction with E275 (Fig. 4). Although such interaction, at first sight, is not detectable in the homology model (distance between the oxygen of E275 oriented toward K283 and the nearest hydrogen of the positive charged residue equal to 6.17 Å), we can reasonably hypothesize that it can take place due to the conformational freedom of both the side chains that are not involved in other interactions. Thus, the replacement of a negatively with a positively charged residue in S4 might limit the flexibility of the S3–S4 linker and the consequent movement of S4, hence transforming the rapid voltage sensing Kv1.1 into a less sensitive mutant, more similar to Kv1.2 channels (Fig. 4). Other Kv1.1 mutations associated to EA1 affect the voltage sensitivity of the channel. The E325D mutation, for instance, in the S4–S5 linker of Kv1.1 channels induces a + 60 mV shift in activation voltage likely due to altered interaction with S4 (D'Adamo et al., 1999). Alternatively, the positive K283 can interact with the phospholipid head groups associated with the voltage sensor and pore domain limiting channel opening (Long et al., 2007; Zheng et al., 2011). Interestingly, our results are in agreement with previous reports showing that partial or full deletion of the S3– S4 linker in Shaker K+ channels shifted half-activation voltage to the right and slowed channel kinetics by reducing the mobility of S4 (Gonzalez et al., 2000; Tabarean and Morris, 2002). Besides providing a molecular explanation for the biophysical defect caused by the mutation, these structural information can be pivotal for the prospective development of more specific drugs to treat EA1, as reported for other channelopathies (Imbrici et al., 2016; Muraglia et al., 2007). 5. Conclusions In conclusion, we identified a novel KCNA1 mutation associated with a broader EA1 phenotype including ataxia, myokymia, painful contractures and metabolic complications. Our results demonstrate that the replacement of a negatively charged glutamate with a positively charged lysine in the 283 position of the S3–S4 linker may hinder voltage sensor movement, resulting in a slower activation and positive shift of voltage dependence. The resulting drop of potassium current likely causes EA1 symptoms in the heterozygous carrier. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mcn.2017.06.006.

Funding This work was supported by Telethon-Italy (grant number GGP14096 to D.C.), Association Française contre les Myopathies (grant number 19027 to J.-F. D.) and Fondi di Ricerca di Ateneo 2014 of University of Bari (CUP code H96J15001610005 to P.I. and J.-F. D).

Disclosure The authors report no disclosures relevant to the manuscript.

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