Heteromeric Kv1 Potassium Channel Expression

AMINO ACID DETERMINANTS INVOLVED IN PROCESSING AND TRAFFICKING TO THE CELL SURFACE*

Jing Zhu, Itaru Watanabe, Barbara Gomez and William B. Thornhill {ddagger}

From the Department of Biological Sciences, Fordham University, Bronx, New York 10458

Received for publication, August 5, 2002 , and in revised form, April 16, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Kv1.4 and Kv1.1 potassium channels are expressed in brain as mature glycoproteins that are trans-Golgi glycosylated. When expressed in cell lines these homomers had very different trans-Golgi glycosylation efficiencies and cell surface expression levels with Kv1.4 > Kv1.1 for both parameters (Zhu, J., Watanabe, I., Gomez, B., and Thornhill, W. B. (2001) J. Biol. Chem. 276, 39419–39427). This previous study identified determinants in the outer pore region of Kv1.4 and Kv1.1 that positively and negatively, respectively, affected these events when expressed as homomers. Here we investigated which subunit exhibited positive or negative effects on these processes when expressed as heteromers. Kv1.4/Kv1.1 heteromers, by coexpression or expression as tandem-linked heteromers, were expressed on the cell surface at ~20-fold lower levels versus Kv1.4 homomers but they were trans-Golgi glycosylated. The lower Kv1.4/Kv1.1 expression level was not rescued by Kv{beta} 2.1 subunits. Thus Kv1.1 inhibited high cell surface expression and partially retained the heteromer in the endoplasmic reticulum, whereas Kv1.4 stimulated trans-Golgi glycosylation. The subunit determinants and cellular events responsible for these differences were investigated. In a Kv1.4/Kv1.1 heteromer, the Kv1.1 pore was a major negative determinant, and it inhibited high cell surface expression because it induced high partial endoplasmic reticulum retention and it decreased protein stability. Other Kv1.1 regions also inhibited high surface expression of heteromers. The Kv1.1 C terminus induced partial Golgi retention and contributed to a decreased protein stability, whereas the Kv1.1 N terminus contributed to only a decreased protein stability. Thus a neuron may regulate its cell surface K+ channel protein levels by different Kv1 subfamily homomeric and heteromeric combinations that affect intracellular retention characteristics and protein stability.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-gated potassium (K+) channels modulate cell excitability by repolarizing action potentials and shaping their waveforms (13). A large family of voltage-gated K+ channels have been identified by molecular cloning techniques that include the four subfamilies Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4) and many structural determinants within core K+ channel proteins that underlie their functional properties have been identified (4, 5). Tetramers composed of noncovalently associated homomeric or heteromeric subunits within a subfamily form a functional complex (4, 5).

Kv1.1 and Kv1.4 channels are components of the brain dendrotoxin-binding protein (6), are heavily glycosylated proteins (610) that form tetramers in many brain regions (6, 11, 12), and can be associated with cytoplasmic Kv {beta} subunits (12, 13). Oligomerization of Shaker or Kv1 channels to properly folded tetramers appears to take place in the endoplasmic reticulum (ER)1 and amino acid motifs involved in this include regions of the distal N terminus and the S1 and S2 transmembrane domains (14, 15). Other reports suggest that efficient cell surface expression of some Kv {alpha} subunits may be promoted by a cytoplasmic C terminus VXXSL motif (16), that different Kv {alpha} subunits may be differentially glycosylated (9, 10, 17), and that auxiliary Kv {beta} subunits promote the trafficking of some Kv {alpha} subunits (18). Within a Kv K+ channel subfamily most members function as homotetramers in expression systems, which indicated that a large diversity of functional channels could be generated by numerous heteromeric combinations. Less appreciated is the observation that differences in intracellular trafficking and retention of these homomeric and heteromeric channels could affect cell surface expression levels that could modify neuronal signaling characteristics (10, 17). Another study has identified regions on Kir-type K+ channels that appear to be possible ER export signals (19). The importance of proper membrane protein folding, oligomerization, and trafficking to the cell surface is highlighted by descriptions of ion channel trafficking defects associated with numerous disorders such as cystic fibrosis and a form of episodic ataxia (20).

Kv1.4/Kv1.1 heteromers and Kv1.4 homomers are both found in native brain (21) and are mature glycoproteins indicating they are heavily glycosylated in the trans-Golgi. In our previous work (Ref. 10, see also Ref. 22), Kv1.4 or Kv1.1 were expressed in CHO cells as homomers and we found that (a) Kv1.4 exhibited a high trans-Golgi glycosylation (TGG) conversion and Kv1.1 showed low conversion, and (b) Kv1.1 was expressed at only ~5% the cell surface level compared with Kv1.4, that is, Kv1.4 exhibited a ~20-fold higher surface level versus Kv1.1, as assayed by cell surface biotinylation and patch clamping, because Kv1.1 exhibited partial high ER retention. Kv1.1 homomers still expressed whole cell peak currents of ~1.5 nA per cell but this was much less than Kv1.4 homomers (10). We also mapped a trafficking determinant to the outer pore regions of these channels because exchanging these regions conferred upon the recipient channel the characteristics of the donor channel. Thus as homoteteramers, the pore of Kv1.4 was positive for high trans-Golgi glycosylation and high cell surface expression, whereas the pore of Kv1.1 was negative for these characteristics.

In our current study we investigated (a) the effects of expression of Kv1.4/Kv1.1 heteromers versus Kv1.4 homomers to determine which subunit, as well as which region of a subunit, contained dominant positive or dominant negative determinants for trans-Golgi glycosylation and cell surface expression levels, and (b) the cellular event(s) responsible for these differences, e.g. whether a change in the protein stability of the Kv1.4 heteromer, export from the ER to Golgi, and/or transport from the Golgi to cell surface were responsible for these cell surface level differences. CHO cells were used as a model system because in our previous work, as well as our current work, they gave similar results as neuronal-like CAD cells (10) and COS cells, and they do not endogenously express Kv1 subfamily proteins (23) nor Kv{beta}1 or Kv{beta}2 proteins (24).


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Plasmids, and Transfections—CHO pro5 cells and COS 7 cells, from American Type Culture Collection, Manassas, VA, were maintained in Dulbecco's modified Eagle's medium with 0.35 mM proline or {alpha}-minimal essential medium with 10% fetal bovine serum at 37 °C under 5% CO2. CAD cells from Dr. Wang were maintained in Dulbecco's modified Eagle's medium/F-12 with 10% fetal bovine serum at 37 °C under 5% CO2 as described previously (10). Rat brain Kv1.4 and Kv1.1 cDNAs (25) were modified using the PCR to have a 5' Kozak enhanced ribosomal binding sequence (CCACC) in front of the start methionine and no endogenous 5'-or3'-untranslated regions. Standard mutagenesis using PCR was used to construct different cDNAs as outlined in our previous work (10). Tandem-linked dimers were constructed by PCR by engineering unique restriction enzyme sites at the 5' and 3' ends, and the stop codon was eliminated from the 3' end of the first cDNA and the start methionine was eliminated from the 5' end of the second cDNA. These cDNAs were gel purified and ligated with an appropriate vector to produce a tandem-linked cDNA construct containing Kv1.4-Kv1.4 or Kv1.4-Kv1.1. These tandem-linked constructs would be expected to express at a defined stoichiometry of Kv1.44 and Kv1.42Kv1.12. Channel cDNAs in the eucaryotic expression vector pcDNA3 were sequenced to confirm construct integrity. LipofectAMINE Plus (Invitrogen) reagent was used for transient transfections following the manufacturer's method on cells plated in a 35-mm dish using 0.5 µg of plasmid for single transfection and cells were incubated for ~20 h post-transfection before they were processed. An incubation for ~20 h was chosen because we have shown that the cell surface expression levels for Kv1.4 and Kv1.1 were maximal at ~15 h (data not shown). For expression of Kv1.4 homomers we cotransfected with plasma membrane M2 muscarinic receptor cDNA to control for competition of reagents to produce mRNA in the nucleus and for components used for protein processing in the ER/Golgi and transport to the cell surface.

Crude Membrane Isolation and Glycosidase/Immunoblot Analysis of Kv1.4 —Crude total cell membranes (ER, Golgi, and plasma membrane) were isolated in ice-cold hypotonic media with protease inhibitors (5 mM Tris final, pH 8.0, 2 mM EDTA, 10 µM pepstatin A, 10 µM leupeptin, 1 mM 1,10-phenanthroline, and 0.2 mM phenylmethylsulfonyl fluoride) and stored at –80 °C as detailed previously (10). Glycosidase (Roche Diagnostics) treatments of solubilized crude membranes followed the manufacturer's protocol with the final concentration of glycosidases at 0.16 units/ml for Endo H or 13 units/ml for PNGase F for 20–24 h at 37 °C. Total cell membranes recovered from a well of a 6-well dish were used to run on 9% SDS gels with ~20 µg of membrane protein/gel lane (protein determined by Bio-Rad protein kit). Proteins were electrotransferred to nitrocellulose (Bio-Rad), the filter was blocked in phosphate-buffered saline with 5% nonfat milk, and then incubated overnight in Kv1.4 mouse monoclonal antibodies (to amino acids 13–37) at 1:1000 (Upstate Biotechnology). The Kv1.4 monoclonal antibody specificity has been described previously (26). Following washes, horseradish peroxidase-linked anti-mouse secondary antibodies were added and the bound antibodies were detected using enhanced chemiluminescence (ECL detection kit, Amersham Biosciences) and preflashed x-ray film (AR5, Eastman Kodak). Signals on immunoblots were quantified as described below. Nontransfected CHO, CAD, and COS cells do not express endogenous Kv1.4 or Kv1.1 proteins by immunoblotting (data not shown).

Biotinylation of Cell Surface Proteins and Immunoblot Analysis of Kv1.4 —Cell surface biotinylation used the hydrazide-LC-biotin (Pierce) reagent, which is specific for carbohydrates. The transfected cell procedure for surface carbohydrate oxidation and biotinylation, streptavidinagarose bead (Pierce) precipitation of solubilized biotinylated membrane glycoproteins, and the specific detection of cell surface Kv1.4 proteins by subsequent immunoblotting has been extensively described in our previous work (10). Immunoblotting of aliquots of the whole lysate for actin antibodies (clone AC-40, Sigma) was used to control for any cell density differences between wells. We also routinely checked for transfection efficiencies by cotransfecting with 0.1 µg of green fluorescence protein plasmid and performing immunoblots with green fluorescent protein antibodies (Clontech) (10). Filters were exposed to preflashed x-ray film (Kodak, AR5) for various lengths of time. A microtex 8700 scanmaker (dynamic range of 0–3.5) was used to scan a film image. Densitometry analysis was performed with the NIH Image 1.6 software that was calibrated with its own internal as well as an external standard (10). Values for cell surface expression levels of Kv1.4 homomers and Kv1.4 heteromers were normalized to Kv1.4 homomers that were taken as 100.0. We also estimated the total cell membrane (ER, Golgi, and plasma membrane) Kv1.4 protein levels of Kv1.4 homomers and Kv1.4 heteromers. Again these values were normalized to Kv1.4 homomers that were taken as 100.0. It appeared that only one or two Kv1.1 subunits in a Kv1.4/Kv1.1 heteromer were sufficient to inhibit surface expression (17). We predict that in our Kv1.4 with 4x Kv1.1 cotransfections the surface heteromers are a mixture of Kv1.43Kv1.11 and Kv1.42Kv1.12 (also see our data for tandem-linked Kv1.4-Kv1.1 versus Kv1.4-Kv1.4). Because our biotinylation/immunoblotting method measured surface Kv1.4 monomer levels, the surface protein value for Kv1.4 with 4x Kv1.1 was multiplied by 1.5 to correct for this and this corrected value is shown in the histograms in Fig. 1. Tandem-linked constructs of Kv1.4-Kv1.4 and Kv1.4-Kv1.1 would be expressed on the cell surface as Kv1.44 homomers and Kv1.42Kv1.12 heteromers, respectively. Cell surface Kv1.4-Kv1.4 denatured proteins on an immunoblot have two Kv1.4 antibody epitopes on each of their N termini whereas the Kv1.4-Kv1.1 protein has one. Thus the cell surface value obtained for Kv1.4-Kv1.1 was multiplied by two and this corrected value is shown in the histogram in Fig. 3C. The ECL detection system was linear over a ~1–20-fold range by an immunoblot assay on serially diluted total cell membranes from transfected cells (~100 to 5 µg of total membrane protein) (data not shown). Kv1 TGG (trans-Golgi glycosylation) percent = upper band signal/total band signal (upper and lower bands).



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FIG. 1.
In a Kv1.4/Kv1.1 heteromer, Kv1.1 inhibited cell surface expression levels, whereas Kv1.4 stimulated trans-Golgi glycosylation in three cell lines. CHO, neuronal-like CAD, and COS cells were cotransfected with different plasmids and used for immunoblot analysis using Kv1.4 antibodies of total cell membrane protein or cell surface proteins isolated by biotinylation of surface proteins. A, Kv1.4 protein profile in total cell membranes (ER, Golgi, and plasma membranes) from CHO cells transfected with Kv1.4 with increasing amounts of Kv1.1. tpTGG percent = upper band/total signal (upper + lower band). B, Kv1.4 protein profile on the cell surface of CHO cells transfected with Kv1.4/M2 muscarinic receptor or Kv1.4/Kv1.1. The lower panel is a longer time exposure of the upper one. spTGG percent was calculated from the lower panel and is displayed as a mean ± S.E., n = 3. C, group data for CHO Kv1.4 cell surface protein levels using the upper panel in B to avoid saturated bands. Values were normalized to Kv1.4, which was taken as 100.0 ± S.E., n = 3. D, group data for surface protein levels obtained as above for CHO cells transfected as Kv1.4 homomers or Kv1.4/Kv1.1 heteromers ± Kv{beta} 2.1 subunits. Values were normalized to Kv1.4 homomers, which was taken as 100.0 ± S.E., n = 3. Immunoblots are not shown. E and G, neuronal-like CAD cells were cotransfected with Kv1.4/M2 or Kv1.4/Kv1.1 and the Kv1.4 protein profile was determined for the total cell membrane protein (E) and the cell surface protein (G) as described above. F and H, COS cells were cotransfected as in E and G. I, group data for CAD and COS Kv1.4 cell surface proteins using the upper panels in G and H. Values were normalized to Kv1.4, which was taken as 100.0 ± S.E., n = 3.

 


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FIG. 3.
Tandem-linked Kv1.4–1.4 and Kv1.4-Kv1.1 constructs gave similar results as cotransfection experiments. CHO cells were transfected with tandem-linked Kv1.4-Kv1.4 or Kv1.4-Kv1.1 constructs and used for immunoblot analysis of total cell protein and cell surface protein and for patch clamping to estimate Gm/Cm levels. A, immunoblot analysis using Kv1.4 antibodies of total cell membranes (ER, Golgi, and plasma membrane) from cells transfected with 0.5 µg of plasmid. Endo H or PNGase F glycosidase were used to treat membrane proteins. tpTGG percent was calculated as described in the legend to Fig. 1. B, cell surface biotinylation/immunoblot analysis used Kv1.4 antibodies of cells transfected with Kv1.4-Kv1.4 or Kv1.4-Kv1.1 tandem-linked construct. spTGG percent was calculated as a mean ± S.E., n = 3, as in Fig. 1. The lower panels are longer time exposures of the upper panels. C, group data for cell surface protein levels by surface biotinylation from B. Values were normalized to Kv1.4-Kv1.4, which was taken as 100.0 ± S.E., n = 3. D, group data from whole cell current patch clamping (hold cell at –80 mV and depolarize to 50 mV for 80 ms) to estimate Gm/Cm. Values were normalized to Kv1.4-Kv1.4, which was taken as 100.0 ± S.E., n = 10.

 

Immunofluorescence Microscopy Analysis of Kv1.4 —Transfected COS cells on glass coverslips were incubated for 20 h following transfection. Cells were then washed, fixed in 3% paraformaldhyde for 10 min, and permeabilized (0.1% Triton X-100) and blocked in standard blocking solution for 1 h. Kv1.4 antibody (1:1000) was added overnight and following washes, the cells were incubated for 1 h with secondary antibody conjugated with a chromaphore (Alexa dyes, Molecular Probes). Cells were then washed, mounted on glass slides, and viewed with an Olympus BX50 microscope with a BX-FLA fluorescence attachment using the appropriate filter cube for the chromaphore. Photographs were taken using a x100 objective and an automatic camera setting that determined the optimal time exposure for the signal (short time exposures for strong signals and longer time exposures for weak signals). Therefore micrographs should be viewed for differences in the overall distribution pattern of the Kv1.4 antibody signal and not the intensity of the signal. Nontransfected cells, on a coverslip with transfected cells, showed no fluorescence from incubation in first and secondary antibodies (data not shown). In addition transfected cells showed no fluorescence when incubated with only secondary antibody (data not shown). Antibodies to localize the ER (BiP/Grp78, Stressgen; calnexin, Stressgen) and the Golgi (GM130, Transduction Laboratories; p230, Transduction Laboratories) in cells were also used in this study (data not shown).

Patch Clamping—Electrophysiological recordings and analysis of whole cell currents from transfected CHO cells have been described in our previous work (10). Cells were cotransfected with channel dimer cDNA constructs and 0.1 µg of green fluorescence protein construct to allow visualization of transfected cells by fluorescence microscopy. Cells were held at –80 mV and depolarized to 50 mV for 80 ms and maximal current levels were used to calculate maximum conductance/capacitance (Gm/Cm) values. Values were normalized to the Kv1.4-Kv1.4 dimer that was taken as 100.0 ± S.E. Gm/Cm values may under- or overestimate surface tetramer expression tetramer levels if channel constructs have different single channel parameters such as differences in single channel conductance and mean channel open time. Although Kv1.4 has a single channel conductance of 4 pS and fast inactivation and Kv1.1 has a single channel conductance of 10 pS and slow inactivation (25), we did not correct for these differences in the histograms of normalized Gm/Cm for Kv1.4-Kv1.4 versus the Kv1.4-Kv1.1 dimers.

Total Cell Protein Half-life Analysis of Kv1.4 Homomers and Kv1.4 Heteromers—Transfected CHO cells were incubated for 20 h following transfection and then incubated in [35S]methionine and [35S]cysteine (200 µCi/ml, Tran35S-label, Amersham Biosciences) in media without methionine or cysteine for 5 h. The media was changed and the cells were chased with media containing 5 mM methionine and cysteine. Cells were solubilized with a Triton X-100-solubilization buffer and Kv1.4 antibodies (1/200 dilution) were added overnight (9). Protein A/G-agarose (Invitrogen) was used for 3 h with rocking to precipitate Kv1.4 complexes and the beads were washed to remove nonspecific binding. The immunoprecipitate was then denatured in SDS sample buffer, run on a 9% gel, and the radiolabeled bands were detected by fluorography (Amplify, Amersham Biosciences) using preflashed x-ray film (AR5, Kodak). Bands were quantified by densitometric analysis as described above. Values were normalized to time 0 (time of chase with unlabeled media) and plotted as a function of time. A curve was fitted to a decaying single exponential time course and a protein half-life value was determined.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In a Kv1.4/Kv1.1 Heteromer, Kv1.1 Inhibited High Cell Surface Expression (Dominant Negative Effect), whereas Kv1.4 Stimulated High Trans-Golgi Glycosylation (Dominant Positive Effect)—Kv1.4 homomers in cell lines were detected on immunoblots as monomers consisting of two glycoprotein bands (Fig. 1A, lane 1) as shown previously (10), a high mannose-type glycoprotein p85 band (it was sensitive to glycosidase treatment by both Endo H which cleaves only N-linked high mannose-type sugars and PNGase F which cleaves all N-linked sugars), and a complex-type mature glycoprotein p110 band (it was insensitive to Endo H cleavage but sensitive to PNGase F), which suggested that it was trans-Golgi glycosylated. The site of N-glycosylation on Kv1.4 is on its extracellular loop between the S1 and S2 transmembrane domains at Asn-354 (Fig. 4A). Kv1.4 cDNA was cotransfected with increasing amounts of Kv1.1 cDNA and we performed immunoblots with Kv1.4 antibodies on total cell membranes (ER, Golgi, and plasma membrane). We found that the total protein trans-Golgi glycosylation (tpTGG) percent of Kv1.4 from total cell membranes was decreased from ~62 to ~1% as the Kv1.1 amount was increased (Fig. 1A). Cell surface biotinylation and immunoblotting methods were used to measure the cell surface protein level and the surface protein trans-Golgi glycosylation (spTGG) percent of Kv1.4 cotransfected with Kv1.1 compared with Kv1.4 cotransfected with M2 muscarinic receptor. Kv1.4 cotransfected with 4x M2 receptor will be referred to in the text as Kv1.4 homomers. The cell surface expression of Kv1.4 was dramatically inhibited when cotransfected with Kv1.1 and the levels were only ~20 and ~5% when cotransfected with 2 or 4x more Kv1.1 amounts, respectively, compared with Kv1.4 homomers (Fig. 1, B and C). In contrast to our results with total cell membrane immunoblots, cell surface Kv1.4 heteromers showed a high spTGG percent of ~88–92% that was slightly less than the ~97–98% value for Kv1.4 homomers (Fig. 1, A versus B). The spTGG percent was a more direct measure of trans-Golgi glycosylation than the tpTGG percent because only channels that were processed in the Golgi were assayed. Will Kv{beta} 2.1 subunits rescue the lower expression level of Kv1.4/Kv1.1 heteromers? It appeared that they did not. Kv{beta} 2.1 increased the surface level of Kv1.4 homomers and this percent increase was similar as seen in Kv1.4/Kv1.1 heteromers (Fig. 1D). We also performed cell surface biotinylation, as well as total protein determinations, of neuronal-like CAD cells and COS cells that were cotransfected with Kv1.4 with M2 or Kv1.4 with Kv1.1 (Fig. 1, E–I). Both cell lines showed similar results to what we recorded in transfected CHO cells, that is, Kv1.4 with Kv1.1 was expressed on the cell surface at ~5% the Kv1.4 homomers (Fig. 1, G, H, and I) and Kv1.4 showed significant spTGG although it was less than in CHO cells (Fig. 1, G and H).



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FIG. 4.
Schematic of Kv1.4, Kv1.1, and chimeras. A, a Kv1 monomer is shown with six transmembrane domains, its pore region as a hairpin loop between S5 and S6, and its N and C termini as cytoplasmic. The N-glycosylation site (*) on Kv1.4 or Kv1.1 is on the extracellular loop between S1 and S2. B, Kv1.4 and Kv1.1 schematics with numbers representing amino acid positions. Note that Kv1.4 and Kv1.1 have only 9 amino acid differences in their S5-pore-S6 regions and their S5 and S6 amino acids are identical (see C). For the Kv1.1 chimeras P1–P9, the light regions represent the a, c, and b regions of Kv1.4. For example, P1 has identical amino acids as the a, c, and b regions of Kv1.4 and this means its complete S5-pore-S6 region is identical to Kv1.4; P7 has only the Lys amino acid of Kv1.4 in the b region; and P9 is P1 with a VKESL C-terminal motif of Kv1.4 that was described previously (16). Note that the entire S5-pore-S6 amino acid regions of P1, P9, and P10–P15 are identical even though the shading is different (see C below). C, the amino acid sequence of the Kv1.4 S5-pore-S6 region compared with the Kv1.1 region. The dash lines in Kv1.1 represent identical amino acids as in Kv1.4. Numbers represent amino acid positions.

 

Immunofluorescence microscopy methods were used on fixed/permeabilized cells to compare the cellular localization of Kv1.4 homomers with Kv1.4/Kv1.1 heteromers. COS cells were used because they have consistent ER and Golgi staining and they have been employed by others for Kv1 localization studies (17). Antibodies specific for the ER (BiP or calnexin) or the Golgi (GM130 or p230) helped determine whether channels were predominantly localized to these organelles (data not shown). The parameter of interest in these micrographs was the similarities or differences in the Kv1.4 localization pattern among different conditions and not their signal intensity. A typical localization for Kv1.4 homomers in transfected COS cells is shown in Fig. 2A. The Kv1.4 homomeric pattern was characteristic of high cell surface expression (17), it showed a diffuse cell surface-like pattern with the cell perimeter clearly visible whereas the ER and Golgi were not prominently visible because of high homomer surface expression. In contrast, Kv1.4/Kv1.1 heteromers showed a high signal around the nucleus that spread out in a reticulate-like fashion that was indicative of high localization to the ER and there was much less diffuse-like surface staining (Fig. 2B). This pattern was similar to Kv1.1 homomers (10, 17). A similar pattern was seen in transfected CHO or CAD cells (data not shown). Thus our immunofluorescence data suggested that Kv1.4/Kv1.1 exhibited partial high ER retention as suggested previously (17).



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FIG. 2.
Immunofluorescence localization pattern of Kv1.4 as a homomer versus as a heteromer. AJ, immunofluorescence microscopy localization pattern using Kv1.4 antibodies in fixed/permeabilized COS cells transfected with Kv1.4 and different constructs. The plasmids used for cotransfections are listed below the pictures and P8–P15 constructs are schematically represented in Fig. 4B. In BD the nucleus is indicated by n. In C and D, the Golgi is indicated by a black line with a g, and two small intracellular foci are indicated by white lines with a f. A blow-up of the f area in D is shown in D1. The similarities and differences in the immunofluorescence localization pattern of Kv1.4 is the important parameter here and not signal intensity.

 

To further test these findings, cells were transfected with tandem-linked Kv1.4–1.4 homodimer or Kv1.4-Kv1.1 heterodimer constructs that would be expressed on the cell surface as Kv1.44 or Kv1.42Kv1.12 tetramers, respectively. The Kv1.4-Kv1.1 heteromer in total cell membrane immunoblots exhibited a lower tpTGG percent versus the Kv1.4-Kv1.4 homomer that showed ~52%, similar to our cotransfections (Fig. 3A, lanes 1 and 4). The Kv1.4-Kv1.4 homomer p210 band was Endo H-insensitive and PNGase F-sensitive, which suggested it was trans-Golgi glycosylated, whereas the Kv1.4-Kv1.4 p170 band and the major lower Kv1.4-Kv1.1 band were both sensitive to Endo H and PNGase F, which suggested that they were not trans-Golgi glycosylated (Fig. 3A, lanes 1–6). Kv1.4-Kv1.1 heteromers were expressed at only ~5% of the cell surface level compared with Kv1.4-Kv1.4 homomers (Fig. 3, B and C). Whole cell patch clamping also confirmed that Kv1.4-Kv1.1 heteromers exhibited a lower cell surface expression level versus Kv1.4-Kv1.4 homomers when assayed by Gm/Cm (Fig. 3D). In contrast to our results using total cell membranes, the cell surface pool of Kv1.4-Kv1.1 showed a significant spTGG of ~80% that was somewhat lower versus the Kv1.4-Kv1.4 homomer of ~97% (Fig. 3B).

These results suggested that in a Kv1.4/Kv1.1 heteromer, using both Kv1.4 and Kv1.1 cotransfections or Kv1.4-Kv1.1 tandem-linked transfections, Kv1.1 inhibited high cell surface expression levels, whereas Kv1.4 stimulated trans-Golgi glycosylation conversion of the surface pool. This inhibition was not rescued by Kv{beta} 2.1 subunits. This pattern of expression was found using three different cell lines, including the neuronal-like CAD line, which suggested that these effects were not cell line specific. It appeared that Kv1.1 induced the partial high ER retention of Kv1.4/Kv1.1 (dominant negative effect of Kv1.1 on surface levels) but that heteromers that were exported from the ER to the Golgi were glycosylated there similarly as Kv1.4 homomers (dominant positive effect of Kv1.4 on trans-Golgi glycosylation).

In a Kv1.4/Kv1.1 Heteromer, the Kv1.1 Pore Induced Partial High ER Retention—Our previous work showed that a Kv1.1 mutant homomer with the Kv1.4 pore exhibited high trans-Golgi glycosylation and high cell surface expression similar to Kv1.4 homomers (10). Will Kv1.4 cotransfected with Kv1.1 with the Kv1.4 pore exhibit a similar tpTGG as Kv1.4 homomers? First we tested the P1 mutant, which had the complete pore of Kv1.4, for its effect on tpTGG. Note that Kv1.4 and Kv1.1 have only nine amino acids differences in their S5-pore-S6 region and we term these areas of differences a, b, and c regions (Fig. 4, B and C). We reasoned that a high tpTGG percent of Kv1.4/P1 in total membranes (ER, Golgi, and plasma membrane) would suggest that the Kv1.1 mutation increased ER to Golgi export. Kv1.4/P1 exhibited a tpTGG of 55% that was similar to Kv1.4 homomers and this was in contrast to the little conversion of Kv1.4/Kv1.1 (Fig. 5A, lanes 1 and 4 versus 1 and 2). These results suggested that in a Kv1.4/Kv1.1 heteromer the Kv1.1 pore induced partial high ER retention and that substituting the pore of Kv1.4 into Kv1.1 helped relieve this and promoted ER to Golgi export.



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FIG. 5.
Kv1.4 homomers were expressed on the cell surface at higher levels than Kv1.4/Kv1.1 or Kv1.4/Kv1.1 mutant heteromers. CHO cells were cotransfected with Kv1.4 and different plasmids and used for immunoblot analysis of total cell membrane protein (ER, Golgi, and plasma membrane) and cell surface protein. A, immunoblot analysis using Kv1.4 antibodies of total protein (ER, Golgi, and plasma membrane) from cells transfected with Kv1.4 and different plasmids. tpTGG percent was calculated as described in the legend to Fig. 1. The lower panel is a longer time exposure of the upper panel. B and C, cell surface biotinylation/immunoblot analysis using Kv1.4 antibodies of cells cotransfected with Kv1.4 and other constructs. spTGG percent was calculated as a mean ± S.E., n = 3, using the lower panel. D, group data for cell surface protein levels for B and C. Values were normalized to Kv1.4/M2, which was taken as 100.0 ± S.E., n = 3, and conditions were all with 4x the second cDNA, e.g. Kv1.4/M2 is Kv1.4/4x M2. E, group data for total protein levels from cell membranes (ER, Golgi, and plasma membrane) from some of theconditions in A. The other total protein immunoblots for P10–P15 are not shown. Values were normalized to Kv1.4/M2, which was taken as 100.0 ± S.E., n = 3. F, surface protein in A was divided by total protein in B = surface protein/total protein ratio. G, dimer constructs expressed in CHO cells and group data from whole cell current patch clamping to estimate Gm/Cm levels. Values were normalized to the Kv1.4-Kv1.4 dimer, which was taken as 100.0 ± S.E., n = 8–12.

 

In a Kv1.4/Kv1.1 Heteromer, Tyr-379 and Ser-369 in the b and c Region of the Kv1.1 Pore Were Sufficient to Induce Partial High ER Retention—Which Kv1.1 a, b, or c subregion(s) or individual amino acid(s) were responsible for the partial high ER retention of Kv1.4/Kv1.1? Again we used tpTGG percent as a test for increased ER to Golgi export of a Kv1.4 heteromer containing Kv1.1 with different pore regions of Kv1.4 (P2–P8 mutants) (Fig. 4B). We found that Kv1.4/P1–P8 heteromers could be placed into three groups from their tpTGG percentage as compared with the 53% for Kv1.4 homomers. Group A (P1 = 55%, P4 = 42%, P5 = 46%, and P8 = 55%) was most similar to Kv1.4 homomers, group B (P2 = 21% and P6 = 31%) exhibited conversion but not as high as group A, and group C (P3 = 2% and P7 = 7%) had little conversion that was most similar to Kv1.4/Kv1.1 heteromers (Fig. 5A). P8 had only two amino acid substitutions from Kv1.4 (Fig. 4, B and C), Y379K in the outer pore b region and S369T in the inner pore region c, and this mutant supported a tpTGG of a Kv1.4 heteromer of 55% that was similar to Kv1.4 homomers. These results suggested that Ser-369 and Tyr-379 in Kv1.1 were responsible for the major effect of the pore of Kv1.1 on partial high ER retention of a Kv1.4/Kv1.1 heteromer and this also implied that the Kv1.4 amino acids Thr-523 and Lys-533, at the equivalent positions in Kv1.1 (Fig. 4C), may play an important role in its export from the ER to the Golgi.

In a Kv1.4/Kv1.1 Heteromer, the Kv1.1 Pore as Well as Other Regions Inhibited High Cell Surface Expression Levels—Will Kv1.4 cotransfected with a Kv1.1 mutant with the pore of Kv1.4 exhibit higher surface levels than Kv1.4/Kv1.1 heteromers? As predicted Kv1.4/P1 or Kv1.4/P8 exhibited a higher surface protein expression level than Kv1.4/Kv1.1 (15 versus 5% or ~3-fold higher) (Fig. 5D) but somewhat unexpectedly this value was much less than Kv1.4 homomers (Fig. 5D), a similar trend was obtained with the Kv1.4-P8 tandem dimer construct when maximum Gm/Cm was used to estimate surface expression levels (Fig. 5G). The latter finding was unexpected because these Kv1.4/P1 or Kv1.4/P8 heteromers exhibited similar ER to Golgi export as Kv1.4 homomers as described above. Thus Kv1.4/P1 and Kv1.4/P8 may be partially retained in the Golgi. It has been proposed that a cytoplasmic C terminus motif (VKESL) promotes cell surface expression of Kv1.4 homomers (16). To test whether a Kv1.4 heteromer required that Kv1.1 have both the pore and the VKESL motif of Kv1.4 to exhibit high surface expression a P9 mutant was constructed (Fig. 4B). However, Kv1.4/P9 still showed surface levels similar to Kv1.4/P1 or Kv1.4/P8 (Fig. 5, B and D). These results suggested that the Kv1.1 pore determinants, as well as other non-pore determinants, inhibited high cell surface expression and that placing a VKESL motif on the C terminus of Kv1.1 did not reverse this inhibition. We refer to Kv1.4/P1, Kv1.4/P8, or Kv1.4/P9 in the text as Kv1.4/group I heteromers and in Fig. 5D as group I.

In a Kv1.4/Kv1.1 Heteromer, Kv1.1 Determinants Inhibited High Cell Surface Expression Levels by Inducing a Decreased Protein Stability—Do Kv1.4/Kv1.1 or Kv1.4/group I heteromers have a decreased total protein stability versus Kv1.4 homomers? Kv1.4/Kv1.1 had a total protein (ER, Golgi, and plasma membrane) amount of only ~17% of Kv1.4 homomers and this was the lowest level recorded here (Fig. 5E). Kv1.4/group I total protein was ~28% of Kv1.4 homomers but ~1.6-fold higher than Kv1.4/Kv1.1 (Fig. 5E). The ~3-fold higher surface levels of Kv1.4/group I versus Kv1.4/Kv1.1 (Fig. 5D) appeared because of an increased protein stability and an increased ER to Golgi export. The decreased total protein levels of Kv1.4/Kv1.1 and Kv1.4/group I compared with Kv1.4 homomers implied they have a decreased protein stability and a decreased protein half-life. Indeed, we found that the total protein half-life of Kv1.4/P9 was only ~47% (2.5 h/5.3 h) of the level of Kv1.4 homomers (Fig. 6) and this suggested that the Kv1.1 pore region as well as other non-pore regions affected protein stability.



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FIG. 6.
Kv1.4 homomers have a longer total protein half-life compared with Kv1.4/P9 heteromers. CHO cells were cotransfected with Kv1.4/4x M2 or Kv1.4/4x P9 and incubated for 20 h. Cells were then incubated with [35S]methionine/cysteine, chased with unlabeled media for different times, and then channel complexes were immunoprecipitated as a function of time. The normalized data were plotted as a function of time and fitted to a single decaying exponential time course to derive protein half-life, mean ± S.E., n = 3.

 

Dividing the cell surface protein (Fig. 5D) by the total protein (Fig. 5E) for Kv1.4/Kv1.1 showed that this ratio was only ~0.33 of the level of Kv1.4 homomers and this was the lowest ratio in our study (Fig. 5F). In contrast, Kv1.4/group I had higher ratios than Kv1.4/Kv1.1 but these were still only ~0.49–0.61 the level of Kv1.4 homomers (Fig. 5F). A surface protein/total protein ratio that was much less than 1.00 implied that these heteromeric proteins may be partially retained in intracellular organelles compared with Kv1.4 homomers, whereas a ratio closer to 1.00 suggested that these proteins exhibited similar retention and trafficking characteristics as Kv1.4 homomers. Indeed, immunofluorescence localization of Kv1.4/group I in COS cells showed a unique pattern, somewhat between Kv1.4 homomers and Kv1.4/Kv1.1, with intracellular highly staining small foci or small plaques that we speculate may be induced from partial high Golgi retention (Fig. 2, C, D, and D1).

These results suggested that Kv1.4/Kv1.1 had the lowest cell surface level, compared with Kv1.4 homomers or other Kv1.4 heteromers, because it exhibited the highest ER retention and lowest protein stability because of the Kv1.1 pore. Kv1.4/group I had higher surface levels than Kv1.4/Kv1.1 because they had both an increased protein stability and an increased ER to Golgi transport because of the pore of Kv1.4 in each subunit of the tetramer. However, Kv1.4/group I still had lower surface levels than Kv1.4 homomers because it showed partial Golgi retention and a continued decreased protein stability.

In a Kv1.4/Kv1.1 Heteromer, the Kv1.1 Cytoplasmic C Terminus Inhibited High Cell Surface Expression Levels by Inducing Partial High Golgi Retention—Given the above results, we used additional chimeras of Kv1.1 that all contained the pore and cytoplasmic C terminus of Kv1.4 with increased amino acid sequences of the N-terminal region of Kv1.4 (Fig. 4B, P10–P13). Note that P13 is Kv1.4 with only the cytoplasmic N terminus of Kv1.1 and that the S5-pore-S6 amino acid sequences of P1, P9, and P10–P13 in Fig. 4B are identical to Kv1.4 even though in the schematic they have different shading (see Fig. 4C). Kv1.4/group II (P10–P13) had a cell surface level that was ~2.0-fold higher than Kv1.4/group I (30 versus 15%) (Fig. 5, C and D), a similar trend was obtained with the Kv1.4-P10 dimer construct when Gm/Cm was used to estimate surface expression levels (Fig. 5G). In contrast, Kv1.4/group II had a similar total protein level as Kv1.4/group I (Fig. 5E). These findings suggested that: 1) only the addition of the Kv1.4 C terminus on to P10–P13 was responsible for this increased surface expression because Kv1.4/P10 levels were similar to Kv1.4/P11–P13 levels, and 2) having the Kv1.4 C terminus on each subunit in the heteromer did not increase protein stability of Kv1.4/group II above that of Kv1.4/group I. These findings suggested that Kv1.4/group II had an increased Golgi to cell surface transport that increased their surface levels compared with Kv1.4/group I. This Kv1.4 C terminus effect was recorded only when a Kv1.1 mutant had both the pore and the C terminus of Kv1.4 because of the Kv1.1 pore inducing high ER retention in a heteromer (data not shown). Dividing the surface protein (Fig. 5D) by the total protein (Fig. 5E) showed that Kv1.4/P10–P13 all had ratios close to 1.00 and similar to Kv1.4 homomers (Fig. 5F). As we discussed above, a surface/total protein ratio close to 1.00 suggested that these heteromers may have similar immunofluorescence cell localization as Kv1.4 homomers. Indeed, Kv1.4/group II heteromers showed similar cell localizations to each other and were most similar to Kv1.4 homomers (Fig. 2, E–H). These results suggested that the Kv1.1 C terminus inhibited high surface levels of Kv1.4/Kv1.1 by inducing partial high Golgi retention of the heteromer.

In a Kv1.4/Kv1.1 Heteromer, the Kv1.1 Cytoplasmic N Terminus Inhibited High Cell Surface Expression Levels by Decreasing Protein Stability—Given the findings above, we used two Kv1.4 N terminus truncation mutants that had their T1 tetramerization domain but lacked the N-terminal amino acid sequences that the Kv1.4 antibody recognized (amino acids 13–37) (Fig. 4B, P14 and P15). Kv1.4/group III (P14 and P15) had a cell surface level that was ~1.8-fold higher than Kv1.4/group II (54 versus 30%) (Fig. 5, C and D), a similar trend was obtained with the Kv1.4-P15 dimer construct when Gm/Cm was used to estimate surface expression levels (Fig. 5G). Kv1.4/group III also had a ~2.0-fold increase (28 versus 59%) in total protein versus Kv1.4/group II, whereas this value was only ~59% the level of Kv1.4 homomers (Fig. 5E). This Kv1.4 N terminus effect was apparent only when a Kv1.1 mutant had both the pore and the N terminus of Kv1.4 (data not shown). These findings suggested that the increased surface level of Kv1.4/group III, versus Kv1.4/group II, was because of an increase in protein stability from the Kv1.4 N terminus. Dividing the surface protein (Fig. 5D) by the total protein (Fig. 5E) showed that both Kv1.4/P14 and Kv1.4/P15 had ratios close to 1.00 and similar to Kv1.4 homomers (Fig. 5F). Indeed, Kv1.4/group III members had similar cellular localization patterns to each other, as well as to Kv1.4/group II heteromers, and these patterns were most similar to Kv1.4 homomers (Fig. 2, I and J). These results suggested that the Kv1.1 cytoplasmic N terminus inhibited high surface levels of Kv1.4/Kv1.1 by decreasing the protein stability of the heteromer.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown that Kv1.4/Kv1.1 heteromers were expressed at only ~5% of the surface level of Kv1.4 homomers and that Kv{beta} 2.1 subunits did not rescue this lower expression level. In Kv1.4/Kv1.1 heteromers, Kv1.1 inhibited high cell surface expression whereas Kv1.4 stimulated high trans-Golgi glycosylation. These results were found in three different cell lines, including neuronal-like CAD cells, and this suggested that numerous cell lines were capable of reading the different intracellular processing and trafficking programs inherent on Kv1.4 and Kv1.1 proteins. A report has also suggested that subunit composition influences cell surface expression of some Kv1 channels (17) and we found similar results for the effects on Kv1.4/Kv1.1 surface levels. In addition, we mapped Kv1.1 regions, which included its pore, its C terminus, and its N terminus, that negatively affected surface levels of Kv1.4/Kv1.1 heteromers. The Kv1.1 pore was the most important negative determinant for surface expression of heteromers because it induced both a partial high ER retention and a decreased protein stability to the highest extent (Kv1.4/Kv1.1); the Kv1.1 C terminus induced partial Golgi retention and contributed to a decreased protein stability (Kv1.4/group I); and the Kv1.1 N terminus contributed to a decreased protein stability (Kv1.4/group II and Kv1.4/group III). Surface levels were decreased for all Kv1.4 heteromers to different degrees, and in all cases a decreased protein stability compared with Kv1.4 homomers was recorded, and thus Kv1.4 exhibited its highest cell surface expression as a homotetramer when each of its subunits were full-length.

Possible Mechanism(s) Involved in Partial High ER Retention, Partial Golgi Retention, and Protein Stability of Kv1.4/Kv1.1 Heteromers—What is a mechanism(s) for partial high ER retention of a Kv1.4/Kv1.1 heteromer? Shaker-like K+ channels in the ER appeared to be properly folded, tetramerized, and functional, that is, voltage-gated K+ currents were recorded from planar lipid bilayers fused with ER microsomes that contained newly synthesized Shaker-K+ channels (27). Kv1.1 homomers in the ER of transfected cell lines appeared to be folded properly and tetramerized, and there was no evidence for aggregation or misfolding (Ref. 22, and data not shown). Kv1.4/Kv1.1 heteromers that formed in the ER (28), as well as those found in native brain (21), would be predicted to be similar to this finding. Thus it appeared that ER retention of a Kv1.4/Kv1.1 heteromer was because of chaperone-mediated partial retention and not misfolding. However, high ER retention may contribute to the decreased protein stability of Kv1.4/Kv1.1. As we discussed in detail in our previous work (10) on expression of Kv1.1 homomers and high ER retention, this putative chaperone may be an ER transmembrane protein with a component in the lumen that associates transiently with a Kv1.4/Kv1.1 heteromer by interacting with its outer pore regions (see Ref. 10 for more discussion). It is also possible that the combined pore determinant region may conformationally affect another interacting/binding site that may be on the cytoplasmic side of the channel. In either scheme Kv1.4/Kv1.1 heteromers are a target of this putative ER retention-chaperone, because of the pore of Kv1.1, whereas Kv1.4 homomers are not and they exhibit high ER to Golgi export. In addition to affecting some of the intracellular transport of the Kv1 subfamily member, the putative chaperone may also be involved in partial ER retention of other K+ channels, such as endogenous leak K+ channels or inward rectifying K+ channels in tissue culture cells such as CHO cells, and even other ion channels.

What is a mechanism(s) for partial high Golgi retention of a Kv1.4/group I heteromer? Apparently, Kv1.4/group I heteromers were partially retained in the Golgi because they did not have the complete Kv1.4 C terminus in each of the four subunits of the tetramer. The VKESL motif (16), in the context of the Kv1.4 C terminus, or another uncharacterized region(s) of the C terminus, presumably was recognized by a chaperone-like protein and this interaction promoted more efficient transport from the Golgi to the cell surface. Other schemes are also possible. The chaperone-like protein may be a transmembrane protein in the Golgi whose cytoplasmic domain interacts with the VKESL motif on the C terminus of the channel or a cytoplasmic soluble protein may bind here and signal for increased transport to the cell surface. The chaperone-like protein may also promote Golgi transport to the cell surface of other K+ channels, or other ion channels or membrane proteins, and not be specific to only Kv1 subfamily members.

What is a mechanism(s) for the decreased protein levels of all Kv1.4 heteromers used in this study? Kv1.1 induced a decreased total protein (ER, Golgi, and plasma membrane) level in all Kv1.4/Kv1.1 heteromers tested (Kv1.4 protein levels were: Kv1.4 homomers > Kv1.4/group III > Kv1.4/group II or Kv1.4/group I > Kv1.4/Kv1.1). A decreased Kv1.4 protein level occurred because Kv1.1 promoted a decreased protein half-life of a heteromer. Regions of Kv1.1 responsible for this included its pore, C terminus, and N terminus. Although we only measured total protein half-life we suggest that the half-life of both intracellular and cell surface pools were decreased through both the proteasomal and lysosomal pathways. Although we argued that the surface protein/total protein ratio gave insight into underlying cellular mechanisms responsible for differences in surface expression level differences, it remained that Kv1.4/Kv1.1, Kv1.4/group I, Kv1.4/group II, and Kv1.4/group III all exhibited decreased cell surface levels of ~20-, ~6-, ~3-, and ~2-fold, respectively, compared with Kv1.4 homomers.

Possible Mechanisms That Affect the Cell Surface Expression Levels of K+ Channels—Both Kv1.4/Kv1.1 heteromers and Kv1.4 homomers are found in brain (24) and our data suggested that these different subunit compositions would give different channel cell surface levels in native tissue whether or not the tetramers were expressed with Kv{beta} subunits. A somewhat unusual but important role of Kv1.1 in different heteromeric combinations in brain may be to limit the cell surface expression level of a Kv1 heteromer. Differential gene expression and heteromeric formation of K+ channels have been appreciated as mechanisms to increase channel functional diversity in the brain (3). We have also presented data that the glycosylation extent affected the gating characteristics of some K+ channels and this may increase their functional diversity (29). Our data in this current report suggest that neurons may modulate their K+ channel cell surface protein levels, which can have profound effects on neuronal excitability and signaling (3), by differential gene expression that leads to different K+ channel homomeric and heteromeric combinations and different trafficking programs. In this scheme Kv1 homomers with different protein stabilities and internal organelle partial retention characteristics would exhibit a new profile for these parameters in a Kv1 heteromer that would either increase or decrease the steady-state level of the protein, which can affect cell surface expression levels.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant NS29633 (to W. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biological Sciences, Fordham University, Bronx, NY 10458. Tel.: 718-817-3688; Fax: 718-817-3645; E-mail: thornhill{at}fordham.edu.

1 The abbreviations used are: ER, endoplasmic reticulum; TGG, trans-Golgi glycosylation; tpTGG, total protein trans-Golgi glycosylation; spTGG, surface protein trans-Golgi glycosylation; CHO, Chinese hamster ovary; Gm/Cm, maximum conductance/capacitance. Back



    REFERENCES
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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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