Human genotyping and MRI

Patient DNA was isolated from blood (QIAamp Blood kit) or saliva (Oragene) following written informed consent and approval from the Institutional Review Board of the University of Florida, Gainesville, Florida (IRB project 484–2007). Written informed consent for minors was obtained from the next of kin. This study was performed in accordance with the Declaration of Helsinki. All patient-derived sequencing was performed with specific primers (S1 Table) at the DNA Sequencing Core, University of Florida. Midline sagittal T1-weighted MR images of patients were collected at multiple institutions.

Plasmid constructs

The human KCNC3^WT cDNA was provided by Dr. James L. Rae (Mayo Foundation, Rochester, Minnesota). Individual mutants (KCNC3^R420H, KCNC3^R423H, or KCNC3^F448L) were generated by polymerase chain reaction (PCR) with QuikChange Mutagenesis (Agilent Technologies) (S1 Table). The cDNA encoding human KCNC3^WT and KCNC3^R423H was amplified with primers appropriate for translation in flies, subcloned into the fly expression vector pUAST, then injected into yw embryos (Rainbow Transgenic Flies). All constructs were verified by sequencing. PCR-amplified human KCNC3^WT cDNA and all mutant coding sequences with an enhanced Kozak site were subcloned into modified green fluorescent protein (GFP) and red fluorescent protein (RFP) vectors, pcDNA3-Clover and pcDNA3-mRuby2 (Addgene #40259, #40260, provided by Michael Lin) [17], or Cerulean3-N1 [18] (Addgene #54742, provided by Michael Davidson, S1 Table). Human perbB1-Citrine (EGFR^Citrine) was purchased from Addgene (#40266), courtesy Martin Offterdinger. GFP-JMY-Full length (GFP-junction-mediating and regulatory protein [JMY^GFP]) and GFP-PI4K2A wild-type (PI4K2A^GFP) were provided by Theresia E.B. Stradal (Helmholtz Centre for Infection Research, Braunschweig, Germany) and Tamas Balla (National Institute of Child Health and Human Development, National Institutes of Health), respectively. N-Cadherin-EGFP (Addgene plasmid # 18870) was provided by Valeri Vasioukhin.

Drosophila genetics and imaging

Fly stocks were obtained from the Bloomington Drosophila Stock Center (flystocks.bio.indiana.edu, S2 Table). All crosses were at 25°C unless indicated otherwise. Adult wings (n = 6) and frozen adult eyes (n = 7) were processed and imaged, as previously described [19].

Cell culture and transfection

Flp-In-CHO (Chinese hamster ovary, Thermo Fisher Scientific) and human glioblastoma U87 cells (ATCC, HTB-14) were cultured in Ham’s F-12 or DMEM (Dulbecco’s modified Eagle’s medium) (Corning), respectively, with 10% fetal bovine serum (FBS), 25mM glucose, 4mM glutamine, and pen/strep (penicillin-streptomycin) at 37°C, 5% CO₂. Transient transfections were carried out using Lipofectamine LTX with Plus reagent (Life Technologies). Total DNA was kept constant by adding control pcDNA3.1 plasmid.

Immunoblot analysis

Protein from individual 2-day-old flies or CHO or U87 cells was analyzed, as previously described, [19] with the modification of resolution with 3–8% Tris-acetate gels (Life Technologies). Co-immunoprecipitation experiments were performed with Dynabeads Protein G (Thermo Fisher Scientific) using α-EGFR (Abcam) for binding and α-EGFR (EMD Millipore) and α-KCNC3 (Alomone Labs) for detection. Full-length blots are presented in S1 Fig.

Electrophysiology

CHO cells were grown in Iscove’s modified Dulbecco’s medium (Invitrogen), with 10% FBS, 100 units/ml pen/strep, 5% HT supplement (Thermo Fisher Scientific) in 5% CO₂ at 37°C. Seeded cells were transfected 24h later using Lipofectamine reagent (Invitrogen) with the mRuby2- or Clover-tagged KCNC3^WT or KCNC3^R423H constructs. On the recording day, patch electrodes were pulled from 1.5mm outside diameter borosilicate glass capillaries (World Precision Instruments). The resistance of a typical electrode was 2-3M for whole-cell recording when filled with intracellular solution (in mM) of 97.5 potassium gluconate, 32.5 KCl, 10 HEPES, 5 EGTA, and pH7.2, with KOH. The bath solution consisted of (in mM) 140 NaCl, 5.4 KCl, 1.3 CaCl₂, 25 HEPES, 33 glucose, and pH 7.4, with NaOH. Series resistance was 2-4M and was compensated by 80–85%. The data were acquired at 10 kHz and filtered at 5 kHz, using pClamp8 software (Molecular Devices).

Immunofluorescence

Eye-imaginal discs of third instar fly larvae were immunostained using a standard protocol [20] with bovine serum albumin (BSA) for blocking. Rat α-elav (7E8A10, 1:200), developed by Gerald M. Rubin, and mouse α-chaoptin (24B10, 1:200), developed by S. Benzer and N. Colley, were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa). CHO cells were transiently transfected with pBKCMV-KCNC3^WT, pBKCMV-KCNC3^R420H, and pBKCMV-KCNC3^R423H plasmids, grown on poly-D-lysine–coated coverslips, fixed (4% formaldehyde, 20 min), permeabilized (0.1% Triton X-100, 10 min) and blocked with 10% BSA. The samples were stained overnight with α-KCNC3 (1:500, Alomone Labs) at 4°C, washed with PBS, and subjected to secondary-antibody (Alexa Fluor 594, anti-rabbit, Molecular Probes) for 1h with samples without the primary processed simultaneously. CHO cells transiently transfected with Clover-, mCerulean3-, or mRuby2-tagged KCNC3^WT, KCNC3^F448L, KCNC3^R420H, and KCNC3^R423H vectors were imaged 48h post-transfection in Live Cell Imaging Solution (Molecular Probes) using a Nikon A1 confocal or inverted Olympus IX-HLSH100 microscope. Images were processed using NIS-Elements (Nikon) and Adobe Photoshop CS6.

Statistical analysis

Statistical analysis with SEM values are presented using an unpaired, two-tailed t-test for significance (***, p<0.001). All analyses were performed using Microsoft Excel and GraphPad Prism.

Causative inheritance, phenotype and MR imaging associated with the KCNC3^R423H allele

Four multigenerational pedigrees segregating KCNC3^R423H provided definitive genetic proof of causation through complete fidelity in allele segregation of sequenced, affected, and unaffected family members (Fig 1A–1D). Case 423–2 illustrates de novo inheritance in patient II-2. Midline T1-weighted sagittal MR images demonstrate pronounced cerebellar hypoplasia. A 35-year-old control (Fig 1E) is compared to a 42-year-old affected family member (423–1, II-3, Fig 1F). T1-weighted MR images of a 10-month-old (423–1, III-1, Fig 1G; inset; age-matched control) and a 21-month-old (423–2, II-2, Fig 1H) demonstrate severe cerebellar hypoplasia, consistent with neurodevelopmental onset. Patient 423–2, II-2 presented with a seizure at 7 months and was diagnosed by genotyping and head CT. Review of available patient histories confirms infantile onset.

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Fig 1. Pedigrees of the probands’ families and MRI.

Four pedigrees (A) 423–1, (B) 423–2, (C) 423–3, and (D) 423–4, illustrating the inheritance pattern of KCNC3^R423H. De novo inheritance in patient II-2 is illustrated in 423–2. Midline T1-weighted sagittal magnetic resonance images (MRIs) of (E) a 35-year-old control; (F) patient 423–1, II-3 at age 42 years; (G) patient 423–1, III-1 at age 10 months (inset shows age-matched control); (H) de novo patient 423–2, II-2 at age 21 months. Midline T1-weighted sagittal MRIs of (I,J) patient 423–3, III-2 at age 7 and 17 years, respectively; and (K,L) patient 423–4, III-4 at age 16 and 26 years, respectively, demonstrating the lack of progressive cerebellar hypoplasia and/or atrophy.

https://doi.org/10.1371/journal.pone.0173565.g001

The MR images for individuals 423–3, III-2 at 7 and 17 years (Fig 1I and 1J) and 423–4, III-4 at 16 and 26 years (Fig 1K and 1L) similarly illustrate severe cerebellar hypoplasia. Strikingly, these two individuals display minimal age-dependent atrophy in 10-year serial scans (compare Fig 1I to 1J; 1K to 1L). This contrasts to the KCNC3^R420H allele where patients demonstrate marked progression of cerebellar atrophy over time with concomitant symptom progression [8]. Furthermore, individual III-2 (423–3, Fig 1C, 1I and 1J) was wheelchair-bound during early adolescence, transitioning to a walker, then to a cane, and ambulating independently by late teens, although not completely normally. Patient III-4 (423–4, Fig 1D, 1K and 1L) was initially diagnosed with cerebral palsy and moderate cognitive impairment, but progressed to unassisted ambulation and/or running and normal-range cognition by age 26 years. A motor evaluation in 2013 yielded a SARA (Scale for the Assessment and Rating of Ataxia) score [21] of 13, which remained unchanged at12-month follow-up. Both patients therefore display non-progressive cerebellar hypoplasia, along with displaying many other activity-dependent improvements in motor and cognitive milestones. Phenotypic data and ataxic parameters (S3 and S4 Tables), demonstrate uniformity in clinical features within and across families. These kindreds establish several novel aspects not typical of other SCAs [4, 22–24]: a neurodevelopmental pattern with infantile onset, non-progression of cerebellar atrophy, and clinical symptoms accompanied by cognitive and motor improvement suggestive of compensatory neural mechanisms despite severe cerebellar hypoplasia.

Expression of human KCNC3^R423H in Drosophila melanogaster wing and eye

Developmental consequences of R423H expression were examined in strains of Drosophila melanogaster expressing human KCNC3^WT and KCNC3^R423H controlled by the yeast UAS/Gal4 expression system [25]. All driver and responder fly strains are summarized in S2 Table. Expression of KCNC3^WT and KCNC3^R423H in transgenic flies was verified by immunoblot (Fig 2A) from individual 2-day-old flies controlled by the ubiquitous daughterless (da)-Gal4 driver [26]. We expressed LacZ, KCNC3^WT, or KCNC3^R423H in the developing wing (Fig 2B–2G), along the anteroposterior boundaries extending to the anterior compartment using the decapentaplegic (dpp)-Gal4 driver. Gal4/KCNC3^R423H expression caused complete loss of the anterior cross vein (ACV) and partial loss of vein L3 (Fig 2G). The flies were kept at 29°C to achieve maximal Gal4-transgene expression with minimal effects on viability and fertility [27]. KCNC3^R423H expression in the developing wing pouch, using the A9-Gal4 driver [28], resulted in severely altered wing morphology and vein patterning (Fig 2H–2J).

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Fig 2. Expression of human KCNC3^WT and KCNC3^R423H in the Drosophila wing and eye.

(A) Cropped immunoblot using antibodies directed against mammalian KCNC3 and Drosophila tubulin illustrates da-Gal4 expression of the indicated transgenes (human KCNC3^WT, KCNC3^R423H, or β-galactosidase (LacZ)) under control of the ubiquitous da-Gal4 driver (full-length blot shown in S1 Fig). (B-D) Wing images from adult flies maintained at 29°C, where the dpp-Gal4 drives expression of LacZ, KCNC3^WT, or KCNC3^R423H in the anterioposterior border of the wing. (E-G) Magnified images showing loss of the anterior crossvein (ACV [E]; red arrow [G]) and disruption of the longitudinal vein L3 as a consequence of KCNC3^R423H expression (dotted brackets [G]). (H-J) Wing images from adult flies where A9-Gal4 drives expression in the wing compartment. (K-M) Eye images from adult flies where gmr-Gal4 drives expression of LacZ, KCNC3^WT, or KCNC3^R423H, demonstrating small, maldeveloped eyes in the mutant. (N-P) Scanning electron microscopy images of whole eyes from LacZ, KCNC3^WT, or KCNC3^R423H flies. Yellow square highlights region of eye shown at high resolution (Q-S).

https://doi.org/10.1371/journal.pone.0173565.g002

To test the mutant allele in a developing neuronal lineage, we utilized the eye-specific driver gmr-Gal4 [29] for expression throughout eye development. KCNC3^R423H expression displayed marked eye dysmorphology and a profound reduction in size (Fig 2M), disrupted ommatidial organization, fused ommatidia, and malformed eye bristles (Fig 2P and 2S) compared to those of a normal eye phenotype in KCNC3^WT and a control LacZ (Fig 2K, 2L, 2N, 2O, 2Q and 2R). Sagittal sections of the KCNC3^WT eyes illustrate regular arrangement of each lens at the apex of the elongated ommatidium, in contrast with the disrupted pattern observed in the KCNC3^R423H eyes (S2A and S2B Fig). In addition to the adult eye, we stained the eye imaginal discs from wandering third instar larvae for the pan-neuronal marker elav [30]. Control KCNC3^WT larval imaginal discs revealed expected organized ommatidia (S2C Fig), whereas KCNC3^R423H larvae showed disorganized and fused ommatidia with maldeveloped smaller ommatidial clusters (S2D Fig). Detection of chaoptin, a photoreceptor cell- and axon-specific membrane protein required for cell morphogenesis [31], illustrates normal organization of the axon bundle from the ommatidia to the brain in the KCNC3^WT larvae (S2E Fig), whereas KCNC3^R423H larvae displayed disorganized and diminished axonal projections (S2F Fig).

KCNC3^R423H intracellular location and biophysics

We recently reported that the KCNC3^R420H allele [7, 8, 32] displayed altered post-translational modifications with aberrant retention in the Golgi [19]. CHO cells, with no detectable endogenous KCNC3, transiently transfected with human KCNC3^WT (Fig 3A), show normal plasma membrane localization by immunofluorescence. In contrast, cells expressing KCNC3^R423H (Fig 3C; compare to KCNC3^R420H in Fig 3B), demonstrate aberrant trafficking with strong perinuclear staining analogous to the KCNC3^R420H allele [19]. As illustrated with other representative cells (Fig 3B and 3C insets) aberrant trafficking is consistently observed with both mutants. To corroborate the aberrant trafficking, we then generated C-terminal Clover-tagged vectors [17] harboring. Confocal microscopy illustrates normal plasma membrane localization (Fig 3D) for KCNC3^WT, in contrast to absent plasma membrane localization and clear perinuclear retention for each mutant (Fig 3E and 3F). This aberrant trafficking is found in all cells from multiple independent transfection experiments. For further comparison, we also expressed the Clover-tagged SCA13 mutation from a French pedigree, KCNC3^F448L, which displays normal plasma membrane trafficking (Fig 3G), although associated with altered biophysical properties [7].

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Fig 3. KCNC3^R423H displays aberrant intracellular trafficking, glycosylation, and failure to express current in cell culture.

(A-C) Immunofluorescence of CHO cells expressing human KCNC3^WT, KCNC3^R420H, or KCNC3^R423H using a KCNC3 antibody. Insets illustrate other representative cells from each transfection. (D-G) Confocal fluorescence images of CHO cells transiently expressing Clover-tagged human KCNC3^WT, KCNC3^R420H, KCNC3^R423H, or KCNC3^F448L. Fluorescence images of CHO cells transiently expressing (H-J) an ER marker, SIGMAR1^CFP with KCNC3^R423H-mRuby2; (K-M) an anterograde vesicle marker, JMY^GFP with KCNC3^R423H-mRuby2; and (N-P) a Golgi–endosome vesicle marker, PI4K2A^GFP with KCNC3^R423H-mRuby2. (N) Boxed area is magnified in (Q-S), with inset in S clearly demonstrating intravesicular retention of KCNC3^R423H-mRuby2. Cropped immunoblot of human KCNC3^WT, KCNC3^R423H, or KCNC3^R420H expressed in (T) CHO cells or (U) human U87 glioblastoma cells illustrating aberrant glycosylation for both causative mutant alleles (full-length blot shown in S1 Fig). Representative currents evoked by commands to potentials between −80 mV and +70 mV, recorded in CHO cells expressing (V) KCNC3^WT-Clover or (W) KCNC3^R423H-Clover.

https://doi.org/10.1371/journal.pone.0173565.g003

To demonstrate that protein overexpression was not responsible for aberrant localization, we show that over about an 8-fold range of transfected plasmid concentrations, with vectors harboring human KCNC3^WT and KCNC3^R423H C-terminally tagged with mCerulean3 [18], KCNC3^{R423H-mCerulean3} remains intracellularly localized, with no detectable plasma membrane trafficking, in contrast to normal localization with equivalent concentrations of the KCNC3^WT allele (S3 Fig). This finding clearly demonstrates that channel mis-trafficking is not attributable to protein expression levels.

To determine the intracellular localization of the mutant channel, we used fluorescently tagged markers of endoplasmic reticulum (ER), Golgi, and intracellular vesicles. Sigma receptor 1 (SIGMAR1) is a highly conserved, transmembrane chaperone protein located in the ER membrane [33]. On co-expression of the cyan fluorescent protein (CFP) tagged SIGMAR1 (SIGMAR1^CFP) with KCNC3^R423H-mRuby2 (Fig 3H–3J), we observed no co-localization, which rules out aberrant retention in the ER. JMY is linked to cytosolic actin assembly and acts as a DNA damage-induced transcriptional co-activator of p53 [34, 35]. Recently, Schlüter et al. [36] also demonstrated JMY’s role in vesicular trafficking at the trans-Golgi network through actin-dependent elongation and/or tubulation of anterograde vesicles. Co-expression of JMY^GFP with KCNC3^R423H-mRuby2 resulted in co-localization (Fig 3K–3M) in vesicular structures, implying the retention of KCNC3^R423H in trafficking vesicles. PI4K2A, a membrane-bound phosphatidylinositol-4 kinase, localizes to the trans-Golgi network (TGN) and early endosomes [37, 38] and is responsible for phosphorylation of phosphatidylinositol (PI) to phosphatidylinositol 4-phosphate (PI4P), a critical lipid in endocytosis, Golgi function, protein sorting, and membrane trafficking [39]. Co-expression of KCNC3^R423H-mRuby2 with PI4K2A^GFP clearly demonstrates that the mis-trafficked mutant channel accumulates in the intravesicular space of PI4K2A^GFP–positive vesicles (Fig 3N–3S). These results suggest that KCNC3^R423H is retained in the interior of anterograde or endosomal vesicles rather than being incorporated into the vesicular membrane.

As an integral membrane protein, N-glycans are added to the nascent KCNC3 protein in the ER with trimming of the oligosaccharide precursor, a critical quality-control measure for proper glycoprotein folding and vesicular trafficking through the Golgi en route to the plasma membrane. The expression of human KCNC3^R423H in CHO (Fig 3T) or U87 cells (Fig 3U) demonstrates altered glycosylation patterns consistent with our previous studies on KCNC3^R420H [19].

As a voltage-dependent potassium channel, KCNC3/Kv3.3 displays high activation thresholds with fast activation and deactivation kinetics, conveying the property of sustained trains of high-frequency action potentials in neurons expressing them [40]. To assess the functional consequences of the KCNC3^R423H mutation, we performed electrophysiology in CHO cells transiently expressing the human KCNC3^WT or KCNC3^R423H channels. For KCNC3^WT, Fig 3V illustrates the slow inactivating outward current evoked during application of depolarizing voltage steps (increments of 10 mV from −80 mV to +70 mV). In contrast, cells expressing the KCNC3^R423H channels demonstrate the complete absence of current conductance by the mutant channel over the same voltage range (Fig 3W), consistent with aberrant glycosylation and intracellular retention.

KCNC3^R423H has a dominant effect on KCNC3^WT

The consequences of an increased dosage of the KCNC3^R423H allele were determined by creating flies harboring 2× copies of KCNC3^R423H, resulting in a more severe eye phenotype entirely lacking ommatidia (Fig 4B compared to Fig 4A). Moreover, simultaneous expression of exogenous KCNC3^WT with KCNC3^R423H did not overcome the eye phenotype (Fig 4C compared to Fig 4A), which implicates the dominant nature of the R423H allele. Similarly, KCNC3^R423H self-cross, driven by dpp-Gal4 in the wing, led to disruption of veins L2, L3, and ACV in the wing (Fig 4E compared to Fig 4D), whereas co-expression of KCNC3^WT with KCNC3^R423H resulted in a phenotype similar to KCNC3^R423H alone (Fig 4F compared to Fig 4D). Studies in the eye and wing thus support the strong dominant negative effects exerted by KCNC3^R423H.

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Fig 4. KCNC3^R423H causes dominant effects on KCNC3^WT in Drosophila.

Eye images from gmr-Gal4 flies expressing (A) KCNC3^R423H; (B) two copies of KCNC3^R423H; and (C) KCNC3^WT with KCNC3^R423H. Wing images from dpp-Gal4 flies expressing (D) KCNC3^R423H; (E) two copies of KCNC3^R423H; and (F) KCNC3^WT with KCNC3^R423H. Red arrows and boxes (dashed lines) indicate areas of aberrations.

https://doi.org/10.1371/journal.pone.0173565.g004

KCNC3 functions as a tetrameric voltage-gated potassium channel [41, 42]. This disease presents as an autosomal dominant phenotype, [7, 32] presumably resulting from the formation of a heterotetramer composed of WT and mutant monomers. We have determined the probabilities of heterotetramer interactions based on WT:423 ratios of 1:1 to 6:1 (S4A and S4B Fig). Biophysical studies of CHO cells co-expressing KCNC3^WT: KCNC3^R423H at a ratio of 1:1 demonstrate a significant reduction in current amplitude and mean current densities compared to those of cells expressing KCNC3^WT alone (Fig 5A, 5B and 5C), thereby showing a dominant electrophysiological effect. Studies using other model systems have implied that the dominant phenotypic effects manifested by KCNC3^R423H and KCNC3^R420H mutations are based on alterations in channel electrophysiology [43]. These results are not supported by our data [19] (Fig 3A–3S), which demonstrate that neither mutation is appreciably trafficked to the plasma membrane. To address the mechanism underlying the dominant phenotypic effects of KCNC3^R423H, we also used differentially C-terminally labeled mutant and WT channels to explore intracellular trafficking and tetrameric protein association. KCNC3^WT-Clover was co-expressed with KCNC3^R423H-mRuby2 in cells at ratios from 1:1 to 6:1 and visualized by confocal microscopy (Fig 5D–5S). As expected, KCNC3^WT-Clover alone traffics normally to the plasma membrane (Fig 5D), whereas KCNC3^R423H-mRuby2 alone displays complete retention in intracellular vesicles (Fig 5G). Co-expression with increasing ratios of KCNC3^WT: KCNC3^R423H demonstrates complete co-localization (Fig 5H–5S) suggestive of tetrameric co-assembly, along with complete retention of the KCNC3^WT protein in the same intracellular vesicles even at a ratio of 6:1 (Fig 5Q–5S).

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Fig 5. KCNC3^R423H causes dominant electrophysiological and trafficking effects on KCNC3^WT.

(A) Representative currents evoked by a step from −70 mV to +70 mV in CHO cells expressing KCNC3^WT-Clover or KCNC3^R423H-Clover, and in those transfected with both constructs KCNC3^WT-Clover:KCNC3^R423H-mRuby2 in a 1:1 ratio. (B) Mean current densities recorded in CHO cells expressing either wild-type KCNC3^WT-Clover (n = 7) or KCNC3^R423H-Clover (n = 5) and in those expressing both KCNC3^WT-Clover: KCNC3^R423H-mRuby2 (1:1) constructs (n = 6). Current density was calculated by dividing the peak current evoked by a step from −70 to +70 mV by cell capacitance. Values are shown as mean±SEM, and significance was tested using a one-way ANOVA. (C) Current-voltage relations for cells in the three conditions shown in (A) and (B). Confocal fluorescence microscopy of cells expressing KCNC3^WT-Clover (D) or KCNC3^R423H-mRuby2 (G) individually, with no channel bleed-through (E,F). (H-S) Confocal fluorescence microscopy of cells co-expressing KCNC3^WT-Clover and KCNC3^R423H-mRuby2 at ratios of 1:1 to 6:1 (KCNC3^WT:KCNC3^R423H) showing co-localization and intracellular retention of both proteins, even at the highest concentration of KCNC3^WT. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.

https://doi.org/10.1371/journal.pone.0173565.g005

dEgfr rescues the Drosophila KCNC3^R423H eye phenotype

To identify the Drosophila pathway(s) affected by KCNC3^R423H, we evaluated modifiers of the mutant phenotypes upon co-expression with a series of eye and wing specific determinants, including dEgfr, Ras, Rolled (mitogen-activated protein kinase [MAPK]) and Notch [44–46] (S2 Table). Effects of Egfr overexpression (UAS-Egfr.B) [47] were evaluated on a gmr-Gal4 (Fig 6A and 6B) or gmr-Gal4, UAS-KCNC3^R423H background (Fig 6C and 6D). Elevated expression of dEgfr with the UAS-Egfr.B allele resulted in a striking rescue of the KCNC3^R423H eye phenotype (Fig 6D), which supports a link between the mutant voltage-gated potassium channel and Egfr or its downstream signaling pathway. To further demonstrate interactions between KCNC3^R423H and Egfr in eye development, we expressed two dEgfr RNAi strains in the context of KCNC3^R423H at 25°C (S5A–S5F Fig). Due to lower Gal4 expression at 25°C, the KCNC3^R423H allele displayed no overt eye pathology (S5D Fig), with the dEgfr RNAi strains displaying only minor effects on ommatidia organization (S5B and S5C Fig). However, co-expression of KCNC3^R423H and dEgfr RNAi transgenes resulted in a reduction in eye size (S5E and S5F Fig) reminiscent of the KCNC3^R423H phenotype at 29°C (Fig 2M).

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Fig 6. Effects of Egfr on the KCNC3^R423H Drosophila eye and wing phenotypes.

(A-C) gmr-Gal4–driven expression of controls LacZ, Egfr, and KCNC3^R423H individually. (D) Co-expression of Egfr and KCNC3^R423H shows rescue of the mutant phenotype. (E-G) dpp-Gal4–driven expression of KCNC3^R423H and Egfr-RNAi, individually and co-expressed. Red arrows and boxes (dashed lines) indicate areas of aberrations.

https://doi.org/10.1371/journal.pone.0173565.g006

Egfr signaling in Drosophila involves a linear pathway with equivalent mammalian orthologs as summarized in S6 Fig. We thus evaluated overexpression of downstream mediators Ras (S7B and S7F Fig) and Rolled (MAPK, S7C and S7G Fig), which displayed little effect on the KCNC3^R423H eye phenotype. These data imply that the mutant channel may be impairing Egfr signaling at the level of the receptor, consistent with KCNC3^R423H dominant effects (Figs 4 and 5). During the third instar larval stage, cells arrest in the eye imaginal disc and constrict at the disc’s posterior edge to form the morphogenetic furrow (MF) [48, 49]. The MF then travels anteriorly, where Notch signaling establishes specification of the initial R8 photoreceptor neurons as the founding cells of the developing ommatidia. Signaling from the Egfr pathway leads to the recruitment of the remaining photoreceptor cells in a pairwise manner (R2/5, R3/4, R1/6, and finally R7), along with non-neuronal cone and pigment cells [46]. Therefore, we also evaluated the effects of Notch overexpression and found no effect on the KCNC3^R423H eye phenotype (S7D and S7H Fig), further supporting specificity for the interaction of the mutant potassium channel allele with Egfr. The connection with EGFR was further corroborated with experiments in the wing demonstrating that co-expression of Egfr-RNAi and KCNC3^R423H causes more significant abnormalities in wing vein development than either strain alone (Fig 6E–6G). These results thus provide evidence that the developmental effects of KCNC3^R423H expression may be mediated through disruption of EGFR signaling.

KCNC3^R423H allele contributes to aberrant trafficking of EGFR

To test whether the potassium channel directly interacts with EGFR in mammalian cells, we transiently transfected U87 cells, known to express EGFR [50], with either human KCNC3^WT or KCNC3^R423H.Cellular lysates were subjected to immunoprecipitation with EGFR-specific antibodies. As expected, U87 cells expressed EGFR (Fig 7A, left panel), while immunoblot analyses of the identical immunoprecipitation lysates showed no detectable bands in either the KCNC3^WT or KCNC3^R423H transfected cells (Fig 7A, right panel), despite clear overexpression of KCNC3 (Fig 7B).

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Fig 7. KCNC3^R423H causes aberrant EGFR trafficking resulting in intracellular retention.

(A) Immunoblot with α-EGFR illustrating positive immunoprecipitation (IP) of EGFR in the eluent from beads bound to anti-EGFR antibody (left panel), with absence of KCNC3 in the same complex (α-KCNC3, right panel). (B) Immunoblot (α-KCNC3) showing presence of KCNC3 in the lysate. (C-E) Confocal fluorescence images of cells co-expressing KCNC3^{WT-mCerulean3}:human EGFR^Citrine (5:1 ratio), showing membrane localization for both proteins. (F-H) Representative cell co-expressing KCNC3^{R423H-mCerulean3}: human EGFR^Citrine (5:1 ratio) showing that both proteins do not reach the plasma membrane and are retained in intracellular vesicles. Insets provide additional examples. (I-K) Representative cell co-expressing KCNC3^{R423H-mCerulean3}:human EGFR^Citrine (4:1 ratio) also showing aberrant trafficking for both proteins. Insets magnify the co-localization of the two proteins in vesicles. (L-N) Representative cell co-expressing KCNC3^{R423H-mCerulean3}:human EGFR^Citrine (2:1 ratio) showing both plasma membrane trafficking and intracellular retention for EGFR. (O-Q) Confocal fluorescence images of cells co-expressing KCNC3^{R423H-mCerulean3}:human EGFR^Citrine (1:1 ratio) also demonstrating membrane and intracellular trafficking for EGFR with continued intracellular retention for KCNC3^R423H. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.

https://doi.org/10.1371/journal.pone.0173565.g007

With no apparent direct protein-protein interaction, we examined the potential influence of aberrant KCNC3^R423H cellular trafficking on EGFR transit through the ER/Golgi to the plasma membrane. Cells were co-transfected with KCNC3^{WT-mCerulean3} or KCNC3^{R423H-mCerulean3} and with varying concentrations of a Citrine-tagged human EGFR [51] construct, using KCNC3:EGFR molar ratios from 5:1 to 1:1 (Fig 7C–7Q). Representative confocal Z-stacks of cells expressing both KCNC3^WT and EGFR (Fig 7C–7E) illustrate that, despite an excess of KCNC3^WT (5:1), both proteins trafficked normally to the plasma membrane. Conversely, co-expression of KCNC3^R423H and EGFR at ratios of 5:1 (Fig 7F–7H) and 4:1 (Fig 7I–7K) reproducibly led to aberrant trafficking of EGFR with sequestration in intracellular vesicles that co-register with KCNC3^R423H (Fig 7H and 7K). As the molar ratio of KCNC3^R423H:EGFR is decreased to 2:1 (Fig 7L–7N), EGFR is found both intracellularly sequestered and at the plasma membrane. At a ratio of 1:1, EGFR traffics normally to the plasma membrane, while in some cells the receptor continues to intracellularly co-register with KCNC3^R423H (Fig 7O–7Q). The titration was repeated with KCNC3^R423H tagged with a different fluorescent protein, mRuby2, co-expressed with EGFR^Citrine at ratios of 3:1 (S8 Fig). These results further illustrate that complete normal trafficking of the receptor to the plasma membrane is achieved only with reduction of the molar ratio of KCNC3^R423H:EGFR to 0.7:1. To determine whether the mutant channel could cause aberrant trafficking of other membrane proteins, we co-expressed KCNC3^WT or KCNC3^R423H with N-cadherin (Cadherin-2 [CDH2]) fused with eGFP [52] at molar ratios of 3.5:1 and 2.5:1, with no effects on plasma membrane localization for cadherin (S9 Fig). Together with the rescue of the Drosophila eye phenotype (Fig 6E and 6G), these observations strongly implicate a specific link between this causative mutant allele and EGFR.