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 KCNC3R423H. 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.
Fig 2.
Expression of human KCNC3WT and KCNC3R423H 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 KCNC3WT, KCNC3R423H, 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, KCNC3WT, or KCNC3R423H 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 KCNC3R423H 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, KCNC3WT, or KCNC3R423H, demonstrating small, maldeveloped eyes in the mutant. (N-P) Scanning electron microscopy images of whole eyes from LacZ, KCNC3WT, or KCNC3R423H flies. Yellow square highlights region of eye shown at high resolution (Q-S).
Fig 3.
KCNC3R423H displays aberrant intracellular trafficking, glycosylation, and failure to express current in cell culture.
(A-C) Immunofluorescence of CHO cells expressing human KCNC3WT, KCNC3R420H, or KCNC3R423H 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 KCNC3WT, KCNC3R420H, KCNC3R423H, or KCNC3F448L. Fluorescence images of CHO cells transiently expressing (H-J) an ER marker, SIGMAR1CFP with KCNC3R423H-mRuby2; (K-M) an anterograde vesicle marker, JMYGFP with KCNC3R423H-mRuby2; and (N-P) a Golgi–endosome vesicle marker, PI4K2AGFP with KCNC3R423H-mRuby2. (N) Boxed area is magnified in (Q-S), with inset in S clearly demonstrating intravesicular retention of KCNC3R423H-mRuby2. Cropped immunoblot of human KCNC3WT, KCNC3R423H, or KCNC3R420H 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) KCNC3WT-Clover or (W) KCNC3R423H-Clover.
Fig 4.
KCNC3R423H causes dominant effects on KCNC3WT in Drosophila.
Eye images from gmr-Gal4 flies expressing (A) KCNC3R423H; (B) two copies of KCNC3R423H; and (C) KCNC3WT with KCNC3R423H. Wing images from dpp-Gal4 flies expressing (D) KCNC3R423H; (E) two copies of KCNC3R423H; and (F) KCNC3WT with KCNC3R423H. Red arrows and boxes (dashed lines) indicate areas of aberrations.
Fig 5.
KCNC3R423H causes dominant electrophysiological and trafficking effects on KCNC3WT.
(A) Representative currents evoked by a step from −70 mV to +70 mV in CHO cells expressing KCNC3WT-Clover or KCNC3R423H-Clover, and in those transfected with both constructs KCNC3WT-Clover:KCNC3R423H-mRuby2 in a 1:1 ratio. (B) Mean current densities recorded in CHO cells expressing either wild-type KCNC3WT-Clover (n = 7) or KCNC3R423H-Clover (n = 5) and in those expressing both KCNC3WT-Clover: KCNC3R423H-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 KCNC3WT-Clover (D) or KCNC3R423H-mRuby2 (G) individually, with no channel bleed-through (E,F). (H-S) Confocal fluorescence microscopy of cells co-expressing KCNC3WT-Clover and KCNC3R423H-mRuby2 at ratios of 1:1 to 6:1 (KCNC3WT:KCNC3R423H) showing co-localization and intracellular retention of both proteins, even at the highest concentration of KCNC3WT. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.
Fig 6.
Effects of Egfr on the KCNC3R423H Drosophila eye and wing phenotypes.
(A-C) gmr-Gal4–driven expression of controls LacZ, Egfr, and KCNC3R423H individually. (D) Co-expression of Egfr and KCNC3R423H shows rescue of the mutant phenotype. (E-G) dpp-Gal4–driven expression of KCNC3R423H and Egfr-RNAi, individually and co-expressed. Red arrows and boxes (dashed lines) indicate areas of aberrations.
Fig 7.
KCNC3R423H 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 KCNC3WT-mCerulean3:human EGFRCitrine (5:1 ratio), showing membrane localization for both proteins. (F-H) Representative cell co-expressing KCNC3R423H-mCerulean3: human EGFRCitrine (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 KCNC3R423H-mCerulean3:human EGFRCitrine (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 KCNC3R423H-mCerulean3:human EGFRCitrine (2:1 ratio) showing both plasma membrane trafficking and intracellular retention for EGFR. (O-Q) Confocal fluorescence images of cells co-expressing KCNC3R423H-mCerulean3:human EGFRCitrine (1:1 ratio) also demonstrating membrane and intracellular trafficking for EGFR with continued intracellular retention for KCNC3R423H. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.