Human embryonic kidney-293 (HEK 293) cells were cultured as previous reported . Briefly, HEK 293 cells were maintained in minimum essential medium (MEM) (Gibco), 2 % w/v penicillin/streptomycin (Sigma), 10 % fetal bovine serum (FBS) v/v (Gibco), and incubated at 37 °C with 5 % CO2. Cells were grown to 80 % confluency on poly-L-lysine treated Corning dishes (Fisher Scientific) with fresh media exchanges every 48 h. African green monkey kidney fibroblast-like (COS-1) cells were cultured in a nearly identical way to the HEK 293 cells with the following changes: COS-1 cells were maintained in MEM (Gibco), 2 % w/v penicillin/streptomycin (Sigma), 10 % FBS v/v (Gibco) and incubated at 37 °C with 5 % CO2. Cells were grown to 50 % confluency on poly-L-lysine treated Corning dishes (Fisher Scientific) with fresh media exchanges every 48 h.
Construction of Kv1.3 channel mutants
Kv1.3 channels were expressed transiently in HEK 293 and COS-1 as previously described . pCDM8 was a kind gift from Dr. Brian Seed (Harvard University) . cDNA encoding human CD8 was amplified from pCDM8 and subcloned into the pcDNA3 vector as previously reported . The eGFP reporter was fused to the N-terminus of Kv1.3 immediately after the start codon of Kv1.3 (Kv1.3-eGFP) at the Florida State University Molecular Cloning Facility as previously described . Kv1.3 constructs containing the single point mutations (Kv1.3-eGFP E443A, Kv1.3-eGFP E445A, Kv1.3-eGFP E447A), double point mutations (Kv1.3-eGFP E443A-E445A, Kv1.3-eGFP E445A-E447A), triple point mutation (Kv1.3-eGFP E443A-E445A-E447) and the Kv1.3-eGFP C-terminal truncated protein (Kv1.3-eGFP ∆C) mutant were generated using the QuickChange® Mutagenesis kit (Stratagene) according to manufacturer’s specifications. The Kv1.3-eGFP ∆C cDNA has a stop codon at position 427 of Kv1.3 (Kv1.3-P427Stop). All constructs were sequenced to verify the mutation(s) and detect PCR errors.
cDNA transient transfection
cDNA was introduced into HEK 293 and COS-1 cells with a LipofectAMINE reagent (Invitrogen) five to seven days after passage as previously described . Briefly, HEK 293 and COS-1 cells were transfected for 4 h with 1.0 μg of each cDNA construct plus 4 μl of LipofectAMINE per 35 mm dish for electrophysiology and fluorescent microscopy. For Western blot analysis in HEK 293 and COS-1 cells, 6 μg of each cDNA construct plus 14 μl of LipofectAMINE per 60 mm dish was used. HEK 293 and COS-1 cells were either harvested for biochemical analysis, confocal microscopy, or used for electrophysiological recordings 36 h post-transfection, as previous studies have shown high surface expression levels at that time point . For all transient transfections the lipofectamine and appropriate cDNAs were complexed for 20 min at room temperature.
Thirty six hours post-transfection, HEK 293 cells were rinsed with bath solution (150 mM KCl, 10 mM HEPES, 1 mM EGTA, and 0.5 mM MgCl2, pH 7.4) and incubated with anti-hCD8 beads (Dynabeads®; Invitrogen) for 2 min to mark transfected cells [61, 62]. Co-expression with CD8 allowed visualization of cells taking up Kv1.3 cDNA by marking transfected cells with a red polypropylene-antibody-linked bead. Hoffman modulation contrast optics was used to visualize transfected HEK 293 cells at 40x magnification using an Axiovert 135 microscope (Zeiss). Transfected HEK 293 cells were rinsed two times with bath solution before beginning a recording session to remove any unbound beads. Patch pipettes were fabricated from #M15/10 borosilicate glass (Jencons) and were fire polished to approximately 1 μm (bubble number 5.0) . Pipette resistances between 8 and 14 MΩ were used to produce high-resistance giga seals by applying a small amount of suction to the pipette lumen in contact with the target cell. HEK 293 cells were voltage-clamped at a holding potential (Vh) of -90 mV and patches were generally depolarized to a command voltage (Vc) of +40 mV for pulse durations of 1000 milliseconds. Pulses were typically delivered at intervals of 60 s to prevent cumulative inactivation of the Kv1.3 channel . Macroscopic currents were recorded in the cell-attached configuration using an AxoPatch200B integrating patch-clamp amplifier (Molecular Devices). The analog output was filtered between 2-5 kHz and digitally sampled every 0.5 - 4 milliseconds. Data acquisition was carried out using pClamp 10 software (Molecular Devices). Data were subsequently analyzed using software from Microcal Origin (Northampton) and QuattroPro (Borland International). The inactivation of the macroscopic current (τinact) was fitted to the sum of two exponentials (y = y0 + A1exp(x/t1) + A2exp(x/t2)) by minimizing the sums of squares, where y0 is the Y offset, t1 and t2 are the inactivation time constants, x is the time, and A1 and A2 are the amplitudes. The two inactivation time constants were mathematically combined by multiplying each by its weight (A) and summing. The deactivation of the macroscopic current (τdeact) was fitted similarly, but to only a single exponential (y = y0 + A1 exp(x/t1)). The voltage-dependent activation of Kv1.3 was determined by fitting current-conductance relationships with a Boltzmann expression. The Boltzmann equation that was used for fitting was: y = [(A1-A2)/(1 + exp(X -Xo)/dx) + A2] [44, 66].
Kv1.3-eGFP and C-terminal mutant localization studies
BSC40 cells at 50 % confluency were co-transfected with cDNAs encoding the membranous ER resident protein Sec61β fused with the mCherry reporter and various mutants of Kv1.3-eGFP using Fugene HD (Promega). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % FBS, 1x penicillin and streptomycin, and L-glutamine at 37 °C in the presence of 5 % CO2. 24 h post-transfection, cells were fixed for 15 min at 37 °C in 4 % paraformaldehyde v/v, washed three times with Tris buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.6) and permeabilized with 0.5 % v/v Triton X-100 for 10 min at room temperature. Immunolabelling was carried out using mouse anti-Golgin 97 primary antibodies (Life Technologies) and anti-mouse secondary antibodies conjugated to Alexa 647 (Jackson ImmunoResearch). Images were acquired using a swept-field confocal microscope (Nikon Ti-E) equipped with a Roper CoolSnap HQ2 charge-coupled device (CCD) camera using a Nikon 60x, 1.4 numerical aperture Planapo oil objective lens. Acquisition parameters were controlled by Nikon Elements software. Line scan profiles of the pixel intensity versus their location in the respective images for all fluorophores were generated using ImageJ. Line scans were drawn in order to maximize the amount of cellular content that could be measured in a given scan.
ER microsome extraction
Intact HEK 293 ER microsome isolates were prepared as previously reported with the following modifications . HEK 293 cells were cultured and transfected as stated above for biochemical analysis. Cell cultures were harvested by first washing two times with hypo-osmotic lysing buffer (2 mM KCl, 25 mM Tris, 5 μg/ml aprotinin (Sigma), 1 μg/ml pepstatin A (Sigma), 0.2 mM phenylmethanesulfonylfluoride (PMSF) (Sigma), and 1 μg/ml leupeptin (Sigma), pH 7.4). Once washed, cells were collected and resuspended in 5 mM HEPES pH 7.4 and stored at -20 °C. Frozen cell suspensions were thawed in a 18 °C water bath, centrifuged at 700 g for 7 min at 4 °C, resuspended in 5 mM HEPES pH 7.4, and homogenized with a Dounce homogenizer (7 strokes). Homogenates were cleared of any intact cells by centrifugation at 700 g for 7 min at 4 °C. 200 μg of the cleared cell homogenate samples were taken for Western blot analysis and immediately mixed with SDS-PAGE sample buffer (250 mM Tris, 10 % w/v SDS, 30 % v/v glycerol, 5 % v/v 2-mercaptoethanol, 0.02 % w/v bromophenol blue, pH 6.8) to a final concentration of 10 μg/μl and heated to 95 °C for 5 min. The remaining lysate was centrifuged at 18,000 g for 20 min at 4 °C. The supernatant was isolated and spun at 100,000 g for 45 min at 4 °C. The microsome enriched pellet was resuspended in SDS-PAGE sample buffer to a final concentration of 10 μg/μl and heated to 95 °C for 5 min. Protein concentrations were determined using a ND-1000 spectrophotometer (NanoDrop) and absorbance values at 280 nm were recorded. Control Western blots were performed to determine the efficiency of ER microsome isolation by probing for the luminal ER resident protein BiP (anti-BiP; Sigma), the luminal Golgi resident protein GCP60 (anti-GCP60; Origene), the nuclear and perinuclear heat shock protein of 90 kDa (HSP90) (anti-HSP90; Origene) and the cytosolic protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (anti-GAPDH; Origene). Host-specific secondary antibodies conjugated with an alkaline phosphatase reporter (Sigma) were added after three washes in TBST for 1 h at room temperature. All Western blots were developed using the SIGMAFAST™ BCIP®/NBT tablets (Sigma). All antibodies were diluted in 5 % non-fat dry milk w/v in TBST to the following working dilutions: BiP (1:500), GCP60 (1:500), GAPDH (1:500), HSP90 (1:500) and all secondary antibodies (1:2500).
Western blot and quantitative densitometry
Western blots were performed using standard biochemical techniques. 150-200 μg of total protein from the ER microsome and the corresponding homogenate samples were separated on independent 8 % SDS-PAGE gels. After visual separation of a pre-stained protein standard (BioRad), gels were electro-transferred to nitrocellulose (Pall Life Sciences) at 4 °C in Towbin transfer buffer (25 mM Tris-Base, 192 mM glycine, 10 % v/v methanol, pH 8.3). Membranes were first blocked with 5 % non-fat dry milk w/v in tween TBS (TBST) (0.05 % Tween-20 v/v; (Sigma)) for 1 h at room temperature. Blocked membranes were incubated overnight at 4 °C with Kv1.3 antisera, FSU120, generated against the intracellular C-terminal domain  and an anti-actin primary antibody (Millipore). Host-specific secondary antibodies conjugated with an alkaline phosphatase reporter (Sigma) were added after three washes in TBST for 1 h at room temperature. All antibodies were diluted in 5 % non-fat dry milk w/v in TBST to the following working dilutions: FSU120 (1:1000), anti-actin (1:2500),
Densitometry measurements were performed using the ImageJ software package as previously reported  with the following modifications. Western blots (n = 3 for both the homogenate and ER microsome enriched fraction) were digitized on an Epson Perfection 4490 Photo scanner and band intensities of Kv1.3-eGFP proteins located at ~100 kDa were recorded (the combined mass of a Kv1.3 monomer and eGFP). The band intensities recorded from homogenate and ER microsome enriched fractions expressing the channel or mutant proteins were rationalized by comparing the amount of Kv1.3-eGFP protein in the ER microsome enriched fraction to the total amount of Kv1.3-eGFP protein in the cleared homogenate sample. The anti-actin band was used as a visual loading marker to ensure all lanes had equal amounts of Kv1.3-eGFP proteins. All data are presented as mean ± standard error of the mean.
siRNA silencing of Sec24
Knock down of all SEC24 isoforms (a-d) was performed using a siRNA silencing system (Invitrogen). COS-1 cells were transfected using predesigned stealth RNA duplex oligoribonucleotides (purchased as sets of three siRNAs per isoform from Invitrogen) and the appropriate controls as recommended by the manufacturer (Invitrogen). The supplier siRNA label codes were as follows: SEC24AHSS145804, SEC24AHSS145805, and SEC24AHSS145806 (for SEC24A); SEC24BHSS115967, SEC24BHSS115968, and SEC24BHSS173629 (for SEC24B); SEC24CHSS114388, SEC24CHSS114389, and SEC24CHSS114390 (for SEC24C); SEC24DHSS114919, SEC24DHSS114920, and SEC24DHSS190682 (for SEC24D). COS-1 cells were transfected using the RNAiMAX™ reagent (Invitrogen) according to manufacturer’s specifications to achieve a final siRNA concentration of 50 nM on cells. Negative controls (stealth RNAi negative control duplexes; Invitrogen) were included in each experiment as recommended by the manufacturer. 24 h following SEC24 silencing, COS-1 cells were tested via Western blot for Sec24 knockdown using primary antibodies specific to each Sec24 isoform (Cell Signaling Technologies; Sec24a: #9678S, Sec24b: #7427S, Sec24c: #8531S, Sec24d: 9610S). Western blots were performed using standard biochemical techniques as outlined above. All primary Sec24 antibody dilutions were at 1:333 and secondary antibody dilutions were at 1:2500 in 5 % non-fat dry milk w/v in TBST.
Kv1.3-eGFP localization studies after Sec24 knock down
COS-1 cells were grown to 50 % confluency on poly-L-lysine coated glass cover slips (Thermo Scientific). Following siRNA treatment, COS-1 cells were further co-transfected with Kv1.3-eGFP and cDNAs encoding for either the vesicular stomatitis virus glycoprotein tagged with a cerulean fluorophore (VSVG-Cerulean) (Addgene; plasmid 11913) , or the membranous ER resident protein Sec61β tagged with the mCherry fluorophore (Sec61β-mCherry) (Addgene; 49155)  using the Lipofectamine™ 2000 reagent (Invitrogen) as described above. Following a 36 h transfection, cells were washed 3 times with phosphate buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4⋅2H20, 2 mM KH2PO4, pH 7.4) and cellular nuclei were stained using 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI) according to manufacturer’s recommendations (Invitrogen) then mounted onto clear microscope slides (Electron Microscopy Sciences) using the ProLong® Anti-Fade kit (Invitrogen) according to manufacturer’s specifications. Images were acquired from a Leica TCS SP2 AOBS confocal microscope equipped with the Chameleon TI:Sapphire multi-photon laser. All acquisition parameters were controlled within the Leica acquisition suite. Images were acquired using an Olympus 63X 1.4 numerical aperture oil immersion objective with a detector pinhole diameter set to 1 airy unit. Detector gain was kept under 800 using the Leica control panel to maintain equal exposure and detection of all emission spectra. 4 individual scans were acquired and averaged to create the final image. All Images were acquired in a 2048 × 2048 pixel array in LEI file format, and then were converted to a 16 bit TIFF file format using the Leica acquisition software. Line scans were drawn in the same fashion as described above.
In electrophysiological studies, biophysical properties were compared across mutant Kv1.3-eGFP channels using an analysis of variance (ANOVA; one-way) with the channel as the independent variable and the biophysical property (i.e. peak current amplitude, voltage at half-activation, rate of inactivation, or rate of deactivation) as the dependent variable. Post-hoc multiple comparison tests were performed using a Bonferoni test. Current-voltage relationships were compared using a two-way ANOVA with the channel and voltage as independent variables and current as the dependent variable. Comparison of biophysical properties between wildtype and eGFP tagged Kv1.3 channels were made by Student’s t-test. For all electrophysiology experiments, the level of statistical significance was determined at the 95 % percent confidence level or greater and is noted by the p-value.
For Western blot densitometry studies, pairwise one-way ANOVAs were performed to determine if the mutated Kv1.3-eGFP protein was retained in the ER compared to the Kv1.3-eGFP protein. To adjust for the possibility of Type-1 errors, a Bonferoni correction was applied and yielded a new p-value of 0.008.
Kv1.3 recombinant expression, purification, and reconstitution
Mouse Kv1.3 cDNA was cloned from the Kv1.3-pcDNA3 expression vector described above in the ‘Construction of Kv1.3 channel mutants’ section by enzymatic digestion at the unique Nco and Kpn restriction sites of the wild type Kv1.3-pcDNA3 vector. Kv1.3 cDNA was ligated in the pFastbac HT expression vector (Invitrogen) between the Nco and Kpn restriction sites. Cloning at these sites provided the addition of a TEV cleavable histidine tag (His6) downstream of the polyhedrin promoter using standard molecular techniques. The final recombinant expression vector was sequence verified to detect the presences of any PCR errors. The Kv1.3 pFastbac recombinant expression vector was transformed into competent DH10bac cells (Invitrogen) according to manufacturer’s recommendations. Two rounds of blue/white selection were performed and the Kv1.3 bacmid DNA was isolated using the Qiagen endotoxin free MaxiPrep kit (Qiagen). Baculovirus generation and amplification was performed by incubating 1 μg of Kv1.3 bacmid DNA that was complexed with 6 μl of the Cellfectin reagent (Invitrogen) and further transfected into Sf9 cells seeded at 1 x 105 cells/mL in Graces modified media (Gibco) supplemented with 50 μg/ml gentamycin (Sigma) and 5 % FBS (Gibco). Sf9 cells were cultured at 27 °C with 5 % CO2 and constant shaking at 150 rpm. After 72 h the culture media was removed and used for further rounds of viral amplification as recommended by the manufacturer. Control infections were performed in parallel and did not receive bacmid DNA but only the Cellfectin reagent.
Kv1.3 proteins were expressed in Hi5 insect cells at a multiplicity of infection (MOI) of one after three consecutive rounds of virus amplification. Hi5 cells were cultured at 27 °C with 5 % CO2 and constant shaking at 150 rpm. Viral titers were greater than 1 x 109 plaque forming units (pfu) pfu/mL. Hi5 cells were cultured in ESF-921 serum free media (Expression Systems) for 72 h post infection. Infected Hi5 cells were harvested by centrifugation at 700 g and pellets were stored at -80 °C until purification.
Kv1.3 proteins were purified as previously reported using a modified protocol . Briefly, infected Hi5 cells were resuspended in Kv1.3 resuspension buffer (25 mM Tris, 150 mM NaCl, 150 mM KCl, one EDTA-free protease inhibitor cocktail (PIC) tablet (Roche) per 50 mL of buffer, pH 7.4). Resuspended Hi5 cells were lysed in a Microfluidizer (Microfluidic Corporation) and lysates were pre-cleared by centrifugation at 60,000 g. Lysate supernatants were cleared of any remaining membrane fragments by ultra centrifugation at 230,000 g. Both membrane-containing pellets were combined and resuspended in Kv1.3 solubilization buffer (resuspension buffer with 4 mM n-dodecyl phosphatidylcholine (Fos-12) (Affymetrix)). Kv1.3 proteins were solubilized overnight at room temperature on a rotating carousel. Insoluble material was removed by centrifugation at 60,000 g and the remaining supernatant was loaded onto a 25 ml column packed with Chelating Sepharose™ fast flow resin (GE Healthcare) charged with cobalt ions (Sigma) using an ÄKTA fast protein liquid chromatography (FPLC) machine (GE Healthcare). The column was equilibrated with 2 column volumes of purification buffer A (resuspension buffer containing 1 mM Fos-12) and Kv1.3 proteins were eluted with purification buffer B (purification buffer A with 500 mM imidazole (Sigma)). Eluted Kv1.3 proteins were concentrated to 5 mg/ml, loaded onto a Superose 6 10/300 GL column (GE Healthcare), and size excluded after the column was equilibrated with 2 column volumes of Kv1.3 purification buffer A using an ÄKTA FPLC. Eluted Kv1.3 proteins were analyzed for purity on a 12 % SDS-PAGE gel and fractions containing Kv1.3 protein were concentrated using a 100,000 kDa molecular weight cut off (MWCO) spin concentrator (Corning) to 1 mg/ml as determined by absorbance at 280 nm. Purified Kv1.3 proteins were stored at -80 °C until further use.
Kv1.3 proteins were reconstituted into membranes using a lipid composition of 95 %:5 % 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC): 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) w/w with a lipid to protein ratio of 1:1 mol:mol by detergent dialysis. Kv1.3 was dialyzed against detergent-free dialysis buffer (25 mM Tris, 150 mM NaCl, 150 mM KCl, 0.01 % NaN3 w/v, pH 7.5) in a 100,000 kDa MWCO slide-a-lyzer cassette (Thermo). Kv1.3 proteins were dialyzed for 1 week at room temperature in the presence of SM2 bio-beads (Bio-Rad) (1 g of washed and equilibrated bio-beads per liter of detergent-free dialysis buffer) with buffer changes every 24 h. Membrane reconstituted Kv1.3 proteins (termed Kv1.3 proteoliposomes) were recovered by centrifugation at 230,000 g. Kv1.3 proteoliposomes were stored at -80 °C until further use. Control liposomes were formed in the identical fashion as Kv1.3 proteoliposomes but do not contain any Kv1.3 protein. Both Kv1.3 proteoliposomes and control liposomes were examined by negative stain electron microscopy (EM) to assess their morphology and intactness using standard EM procedures . Samples were deposited on continuous carbon coated copper mesh grids that were rendered hydrophilic by glow discharge using a Solarus Model 950 Advanced Plasma System (Gatan) in a 75/25 % Ar/O mixture and then stained with 2 % uranyl acetate. Micrographs were collected on a CM-120 (Phillips) operating at 120 keV at room temperature with a nominal pixel size of 2.33 angstroms per pixel (Å/pix) equipped with a Tem-Cam F224 slow scan CCD camera (Tietz).
Recombinantly expressed Kv1.3 protein molecular weight characterization
To determine the oligomeric state of purified Kv1.3 proteins, Kv1.3 proteins solubilized in Fos-12 were exchanged into amphipols (A8-35) (Anatrace) following a modified protocol . Briefly Kv1.3 proteins were incubated with a 5-fold excess of A8-35 (w/w) for 4 h at 4 °C. The mixture was then diluted to a 0.8-fold reduction of the Fos-12 critical micellular concentration (CMC) using Kv1.3 resuspension buffer and dialyzed overnight in a 100,000 MWCO slide-a-lyzer (Thermo) at 4 °C against detergent-free dialysis buffer in the presences of SM2 bio-beads (BioRad). Unbound A8-35 was removed by size exclusion chromatography using a 10/300 Superose 6 column (GE Healthcare). Kv1.3 proteins in Fos-12 and A8-35 were analyzed on a 4-16 % native gel (Invitrogen) according to manufacturer’s recommendations. Kv1.3 proteins in A8-35 were subjected to dephosphorylation using calf intestinal phosphatase (CIP) (New England Biolabs) according to manufacturer’s recommendations using a ratio of 1 CIP unit to 1 μg of Kv1.3 protein. Dephosphorylated proteins were analyzed on 8 % SDS-PAGE.
Negative stain EM was used to visualize purified Kv1.3 tetramers using standard EM procedures . Samples were prepared in the same manner as mentioned above. Micrographs were collected using a Titan Krios EM (FEI) at 120 keV at room temperature equipped with a DE20 direct electron detector camera (Direct Electron) with a nominal pixel size of 1.26 Å/pix in an automated fashion using the Leginon software . EM micrographs were uploaded to the Appion processing suite . Kv1.3 tetramers were picked in a semi-automatic fashion using the template picker FindEM . Two dimensional (2D) class averages were generated using the maximum likelihood alignment algorithm within the Xmipp package .
Sec24a341 protein expression and purification
The Sec24a expression vector lacking the first 341 residues (Sec24a341) was a generous gift from Dr. Jonathan Goldberg (Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center) . Sec24a341 proteins were expressed in the same manner as Kv1.3 proteins using the baculovirus expression system according to manufacturer’s recommendations.
Sec24a341 proteins were purified by resuspending infected Hi5 insect cells in Sec24a341 lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM MgOAc, 5 mM 2-mercaptoethanol, one PIC tablet per 50 ml of buffer, 2 mL PopCulture reagent per 50 ml of buffer (EMD Millipore), pH 7.4) and incubated for 1 h at room temperature on a rotating carousel. Insoluble material was removed by centrifugation at 60,000 g. Cleared lysate supernatants were incubated with 3 ml of the HisPur™ Colbalt resin per 50 ml lysate supernatant and incubated at room temperature for 30 min. Loaded resin was collected by centrifugation at 2000 g and washed twice with Sec24a341 purification buffer A (25 mM Tris, 150 mM NaCl, 1 mM MgOAc, 5 mM 2-mercaptoethanol, pH 7.4). Sec24a341 proteins were eluted with Sec24a341 purification buffer B (purification buffer A with 500 mM imidazole). Eluents were analyzed on an 8 % SDS-PAGE gel and fractions containing Sec24a341 proteins were concentrated in a 30,000 kDa MWCO spin concentrator (Corning) to 10 mg/ml. Concentrated Sec24a341 proteins were loaded onto a FPLC Superose 6 10/300 GL size exclusion column (GE Healthcare) equilibrated with 2 column volumes of Sec24a341 purification buffer A using an ÄKTA FPLC. Sec24a341 proteins were analyzed on an 8 % SDS-PAGE gel for purity and fractions containing Sec24a341 were concentrated in a 30,000 kDa MWCO spin concentrator (Corning) to 1 mg/ml. Sec24a341 proteins were stored at -80 °C until further use.
Kv1.3-Sec24a341 floatation assay
The Kv1.3-Sec24a in vitro association was detected using a modified membrane floatation assay. 15 μg of Sec24a341 protein was incubated with 15 μg of Kv1.3 proteoliposomes or 15 μg of Kv1.3 in detergent micelles in a total volume of 265 μl in floatation assay buffer (25 mM Tris, 150 mM NaCl, 160 mM KOAc, 1 mM MgOAc, pH 7.5) and 30 % sucrose w/v. Each reaction was incubated at 32 °C for 1 h. Reactions were first overlaid with 250 μl of floatation assay buffer containing 25 % sucrose w/v then followed by an additional overlay of 50 μl floatation assay buffer lacking sucrose. Each condition was centrifuged at 200,000 g for 1 h at 4 °C. Each layer was carefully removed and 50 μl samples were taken and mixed with 50 μl SDS-PAGE sample buffer. Control reactions were performed in parallel that contained Sec24a341, Sec24a341 in the presence of control liposomes, Kv1.3 micelles, Kv1.3 micelles in the presence of control liposomes, Sec24a341 and Kv1.3 micelles in the presence of control liposomes, and control liposomes alone. Samples were analyzed on an 8 % SDS-PAGE gel and were electro-transferred to nitrocellulose (Pall Life Sciences) at 4 °C in Towbin transfer buffer. Membranes were first blocked with 5 % non-fat dry milk w/v in TBST for 1 h at room temperature. Blocked membranes were incubated overnight at 4 °C with FSU120 and an anti-Sec24a primary antibody (Abcam). Host-specific secondary antibodies conjugated with an alkaline phosphatase reporter (Sigma) were added after three washes in TBST for 1 h at room temperature. Western blots were developed using the SIGMAFAST™ BCIP®/NBT tablets (Sigma). Antibodies were diluted in 5 % non-fat dry milk w/v in TBST to the following working dilutions: FSU120 (1:500), anti-Sec24 (1:250), secondary antibody (1:2500).
Protein sequence alignments
All protein sequences were aligned using the Clustal Omega© program as instructed . Accession numbers are as follows: Shaker; CAA29917, mouse Kv1.1; NP_034725, mouse Kv1.2; NP_032443, mouse Kv1.3; AAI37668, mouse Kv1.4; NP_067250, mouse Kv1.5; AAG40241, human Sec24a; O95486.2, human Sec24b; O95487.2, human Sec24c; P53992.3, human Sec24d; O94855.2.
Availability of supporting data
The data collected for the manuscript are presented within the article and Additional files 1, 2, 3, 4, 5, 6 and 7.