(Received for publication, June 14, 1996, and in revised form, October 24, 1996)
From the Department of Physiology, School of Medicine, and Molecular Biology Institute, University of California, Los Angeles, California 90095-1751
We have characterized the maturation of Shaker
K+ channel protein and the cellular site of assembly of
pore-forming and cytoplasmic
subunits in a transfected
mammalian cell line. Shaker protein is made as a partially
glycosylated, immature precursor that is converted to a fully
glycosylated, mature product. Shaker protein did not mature when
transport from the endoplasmic reticulum (ER) to the Golgi apparatus
was blocked. Consistent with this finding, only the immature form was
sensitive to digestion with endoglycosidase H. These results indicate
that the immature protein is core-glycosylated in the ER, whereas the
oligosaccharides of the mature protein have been further processed in
the Golgi compartment. After inhibiting ER-to-Golgi transport, the
oligomeric state of Shaker subunits was assessed by cross-linking in
intact cells or by solubilization and sucrose gradient sedimentation.
The results indicate that Shaker subunits assemble with each other in
the ER. When co-expressed, the Kv
2 subunit also associated with
Shaker in the ER. Assembly with the
2 subunit did not increase the
rate or extent of Shaker protein maturation. Our results indicate that
the biogenesis of Shaker K+ channels in vivo
involves core glycosylation and subunit assembly in the ER, followed by
efficient transfer to the Golgi apparatus where the oligosaccharides
are modified.
Ion channels are multisubunit membrane proteins involved in action potential propagation, neurotransmitter release, and excitation-contraction coupling in excitable tissues. To achieve their correct quaternary structures, ion channels undergo a complex series of events during biogenesis, including co- and posttranslational folding, modification, and subunit assembly. Although many multisubunit membrane proteins fold and assemble in the endoplasmic reticulum (ER)1 (1), there are notable exceptions among channel-forming proteins (2).
Voltage-gated K+ channels, which regulate the excitability
of nerve and muscle, contain four subunits that form the
ion-conducting pore (3-6). Each
subunit has a hydrophobic core
containing a pore-forming domain and six putative membrane-spanning
segments, flanked by cytoplasmic amino and carboxyl termini (7). The
subunits in a K+ channel may be identical or may be
different members of a subfamily of closely related proteins (8-12).
In neurons, the pore-forming
subunits may associate with
cytoplasmic
subunits in a one-to-one stoichiometry (13, 14). In
expression systems,
subunits have been shown to alter the
functional properties, stability, and cell surface expression of
K+ channels (15-18).
Recently, some of the molecular determinants that control assembly of
the Shaker channel and its mammalian homologues, the Kv1 subfamily,
have been identified. A conserved amino-terminal domain mediates the
recognition and assembly of subunits (19-24). An overlapping
region of the amino terminus is also important for the co-assembly of
and
subunits (25, 26). Much less is known about other aspects
of the biogenesis of K+ channels in cells.
We have characterized the maturation and cellular site of assembly of
the voltage-dependent Shaker K+ channel in a
transfected mammalian cell line. Our results demonstrate that
maturation of Shaker protein requires ER-to-Golgi transport, with a
corresponding change in the N-linked oligosaccharides from the high mannose type characteristic of the ER to the complex type
characteristic of the Golgi apparatus. In addition, we find that Shaker
channel subunits assemble with each other and with cytoplasmic
subunits in the ER, suggesting that only fully assembled channels are
transported to the Golgi. Unlike the effect of
subunits on some
mammalian
subunits (18), the
subunit does not act as a
chaperone to facilitate the maturation of Shaker channels.
Human embryonic kidney
cells (HEK 293T) (27), kindly provided by Dr. R. B. DuBridge, were
grown and transfected as described previously (28). For transfection
experiments, the Shaker B cDNA (29) and a mutant construct,
N259Q + N263Q, which eliminates N-linked glycosylation of
Shaker protein (30), were transferred into the vector pcDNA1/AMP
(Invitrogen). A subclone encoding the rat Kv2 protein in the pRGB4
vector was the generous gift of Dr. James S. Trimmer, SUNY, Stony Brook
(31). In co-transfection experiments, Shaker and Kv
2 DNAs were mixed
in a 3:1 mass ratio. A total of 2 µg of DNA was used per 35-mm well
of cells.
Forty-eight h after transfection with Shaker alone, HEK 293T cells were
incubated for 30 min in methionine- and cysteine-free DMEM (Mediatech),
pulsed for 3 h with 200 µCi/ml [35S]methionine and
cysteine (Tran35S-Label, ICN or EasyTag Express Protein
Labeling Mix [35S], DuPont NEN), and chased for 2 h
in complete, nonradioactive medium. A crude membrane fraction was
prepared and solubilized in the presence of protease inhibitors (30).
Shaker protein was immunoprecipitated with antisera directed against a
Shaker--galactosidase fusion protein (32).
Forty-eight h after co-transfection of Shaker and Kv2, HEK 293T
cells were pulsed for 10 min with 400 µCi/ml
[35S]methionine and -cysteine and chased for 2 h in
complete, nonradioactive DMEM supplemented with 5 mM
methionine (Sigma). The cell plates were chilled on
ice, and the cells were washed once with 1 ml per well of ice-cold
Dulbecco's phosphate-buffered saline (Mediatech) containing 5 mM NEM. Then, 0.75 ml of ice-cold lysis buffer (1% Triton
X-100, HEPES-NaOH, pH 7.4, plus the protease inhibitors previously
described (30) except that dithiothreitol was replaced by 5 mM NEM) was added, and the plates were rotated for 10 min at 4 °C. Cell lysates were centrifuged at 100,000 × g for 30 min at 4 °C to remove insoluble material. Shaker
and Kv
2 proteins were immunoprecipitated from the supernatant using
the anti-Shaker antibody described above or an anti-
antibody (14)
kindly provided by Dr. James S. Trimmer (SUNY, Stony Brook). The
anti-
antibody was used at a 1:200 dilution.
Immunoprecipitated protein was boiled for 3 min in endoglycosidase H (endo H) reaction buffer (50 mM sodium acetate, 10 mM EDTA, 0.06% sodium dodecyl sulfate, 10 mM 2-mercaptoethanol, pH 5.5). Samples were treated at 37 °C for 16 h with 20 milliunits/ml endo H (Boehringer Mannheim) in the presence of 0.36% Triton X-100, 0.5 mM phenylmethanesulfonate, 50 µg/ml antipain, 2 µg/ml aprotinin, and 0.7 µg/ml pepstatin. Enzyme treatment was terminated by adding Laemmli sample buffer and boiling for 3 min.
Low Temperature Incubation and Brefeldin A TreatmentFor incubation of cells at low temperatures, culture plates or flasks were sealed in plastic wrap and placed in a refrigerated incubator (15 °C), in an air-conditioned room (20 °C), or a standard incubator (27 °C). For treatment with both brefeldin A (BFA) and nocodazole, cells were incubated in methionine- and cysteine-free DMEM containing 20 µg/ml nocodazole (Sigma) in Me2SO (Sigma) for 40 min followed by continued starvation in the presence of both nocodazole and BFA (5 µg/ml in Me2SO, Sigma) for another 30 min. During subsequent pulse and chase, both drugs were present. Treatment with one drug alone or no drug followed the same incubation schedule except that the omitted drugs were substituted with an equal volume of Me2SO.
Cross-linking by Disulfide FormationSpecific disulfide bonds between Shaker subunits were oxidized in intact cells by exposing them to 1 mM iodine for 10 min as described previously (6). Shaker protein was then solubilized, immunoprecipitated, and boiled in sample buffer containing either 10% 2-mercaptoethanol (reducing conditions) or 16 mM iodoacetamide (nonreducing conditions).
Expression of a Covalent Tetramer of Shaker SubunitsA covalently linked Shaker tetramer construct in the vector pGEM-A, kindly provided by Dr. Fred J. Sigworth, Yale University (33), was expressed in Xenopus oocytes. The subclone was digested with NotI and cRNA was transcribed using the mMESSAGE mMACHINE kit (Ambion). RNA was co-injected with in vitro translation grade [35S]methionine (ICN) into oocytes as described previously (30).
Sucrose Gradient CentrifugationMetabolically labeled protein from HEK 293T cells or oocytes was solubilized in 50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA containing either 2% CHAPS (Sigma) or 2% Zwittergent 3-12 (Calbiochem-Novabiochem) plus protease inhibitors as described above. Solubilized protein (500-750 µl) was loaded on a 5-20% sucrose gradient (11 ml) containing either 2% CHAPS or 2% Zwittergent supplemented with 5 mM NEM and protease inhibitors except bestatin and antipain. Gradients were centrifuged at 36,000 rpm in an SW41 rotor for 20 h at 20 °C. Approximately 15 fractions (about 750-780 µl each) were collected from the bottom of each gradient and subjected to immunoprecipitation. Supernatants were saved for refractive index measurements, which were made with an Abbe-3L refractometer (Milton Roy) at 24 °C.
Electrophoresis and Immunoblot AnalysisProtein samples in Laemmli sample buffer were boiled for 3 min prior to electrophoresis. Proteins were fractionated by electrophoresis using standard Laemmli gels prepared with a 7.5 or 10% acrylamide separating phase and a 4% stacking phase. For analysis of cross-linked adducts of Shaker protein, gels were prepared with a 5% separating phase and a 3% stacking phase. Apparent molecular weights were determined using broad range standards (Bio-Rad) and high molecular weight standards (Pharmacia Biotech Inc.). After transfer of proteins to nitrocellulose, immunoblots were probed with an antibody specific for 14 amino acids at the carboxyl terminus of the Shaker protein (28).
Fluorography and DensitometryFollowing electrophoresis,
gels were stained in Coomassie Blue, destained, washed in
double-distilled water, and incubated in Fluoro-Hance (Research
Products International). Dried gels were exposed to X-Omat AR-5 film
(Eastman Kodak) at 80 °C. Fluorographs of gels were scanned and
analyzed using the Model GS-700 scanning densitometer and Molecular
Analyst Software version 1.4 (Bio-Rad). For low temperature and BFA
experiments, relative amounts of immature and mature Shaker protein
were quantified by peak integration analysis. For sucrose gradient
experiments, relative amounts of Shaker protein in gradient fractions
were quantified by volume analysis.
The glycosylation of Shaker K+ channel
protein has been characterized in several expression systems, including
an insect cell line (Sf9), Xenopus laevis oocytes, and a
mammalian cell line (HEK 293T) (28, 30). Asparagine-linked
glycosylation, which occurs at Asn-259 and Asn-263 in the first
extracellular loop of the protein, is not required for the assembly of
functional channels or their transport to the cell surface (30). In
oocytes and HEK 293T cells, glycosylation occurs in two stages, giving rise to a partially glycosylated, immature precursor that is
efficiently converted to a fully glycosylated, mature product (Ref. 28; see Fig. 3).
This differential glycosylation suggests that the immature precursor
has been core-glycosylated in the ER, whereas the mature product has
been further modified in the Golgi apparatus (34). To determine whether
maturation of Shaker protein requires transport from the ER to the
Golgi, movement of newly synthesized protein between these compartments
was arrested by two known inhibitors of intracellular transport in
mammalian cells, low temperature incubation, and brefeldin A (BFA)
treatment (35-39). The extent of maturation was then assayed by
comparing the relative amounts of the immature and mature forms of the
Shaker protein. At 37 °C, about 80% of the immature form is
converted to the mature form within 1.5 h, and conversion is
virtually complete within 2.5 to 3 h (Ref. 28; see Fig.
1D).
Incubation of mammalian cells at 15 °C prevents the transport of newly synthesized secretory and membrane proteins to the Golgi apparatus (35, 36). HEK 293T cells were metabolically labeled at 15 °C for 3 h and collected following a 2-h chase in nonradioactive medium at either 15 or 37 °C (Fig. 1A). Following pulse and chase at 15 °C, the extent of maturation was significantly reduced compared with that obtained after pulse and chase at 37 °C (see Fig. 1D). Of the total Shaker protein, nearly 90% was in the immature form after the 2-h chase at 15 °C. The blockade in maturation induced by incubation at 15 °C was reversible, however, as shown by the large increase in maturation following a 2-h chase at 37 °C (Fig. 1A).
In other mammalian cell lines, incubation at 20 °C does not block transport of membrane and secretory proteins to the Golgi compartment (35). However, similar to the results of incubation at 15 °C, we found that the extent of maturation was significantly but reversibly reduced following a 3-h pulse and a 2-h chase at 20 °C (Fig. 1B). These results suggest that ER-to-Golgi transport remains inhibited at 20 °C in HEK 293T cells. In contrast, incubation at 27 °C did not prevent maturation of the Shaker protein (Fig. 1C); the extent of maturation was similar to that seen after pulse and chase at 37 °C (Fig. 1D). Thus, the effects of low temperature incubation on the relative amounts of immature and mature Shaker protein suggest that ER-to-Golgi transport is required for maturation.
Alternatively, ER-to-Golgi transport was blocked with BFA in either the
presence or absence of nocodazole, a drug which makes BFA a more
specific inhibitor of protein transport (37-39). HEK 293T cells were
metabolically labeled during continued treatment with BFA, BFA and
nocodazole, nocodazole, or Me2SO, the drug vehicle (Fig.
2). Following treatment with BFA, either alone or in
combination with nocodazole, maturation of the Shaker protein was
completely blocked. In contrast, maturation was not inhibited by
treatment with nocodazole or Me2SO alone. Thus, these
results provide further evidence that ER-to-Golgi transport is required
for maturation of the Shaker protein.
Maturation of Shaker Protein Involves Modification in the Medial Golgi
To characterize the differential glycosylation of the
immature and mature forms of Shaker protein, wild-type protein was
expressed in HEK 293T cells and incubated in the presence (+) or
absence () of endoglycosidase H (endo H) (Fig. 3).
Conversion of a protein from an endo H-sensitive to an endo H-resistant
form indicates that the oligosaccharide moieties have been processed in
the medial Golgi compartment (40-42). The mature form of wild-type
Shaker protein (Fig. 3, 108 kDa) showed no significant change in
apparent molecular mass (107 kDa) following incubation with endo H,
indicating that it is endo H-resistant. The immature form (80 kDa),
however, decreased in apparent molecular mass by about 8 kDa
following incubation with endo H (72 kDa), indicating that it is
endo H-sensitive. The digested precursor comigrated with unglycosylated
protein (72 kDa) of the mutant construct, N259Q + N263Q, in
which the two asparagine residues that are normally modified by the
attachment of N-linked oligosaccharides have been replaced
by glutamine residues (30). The differential sensitivity of immature
and mature Shaker protein to endo H indicates that maturation involves
the conversion of high mannose oligosaccharides to a complex form in
the medial Golgi compartment.
To determine whether Shaker subunits assemble in the ER, ER-to-Golgi transport was inhibited, and the state of assembly of Shaker protein was examined in intact cells by cross-linking with iodine. Iodine, an oxidizing reagent, catalyzes the formation of a specific disulfide bond between Cys-96 in the amino terminus and Cys-505 in the carboxyl terminus of adjacent Shaker subunits (6). Although the subunits are not normally linked by disulfide bonds (28), functional assays under oxidizing conditions indicate that the formation of these bonds does not disrupt the native structure or function of the channel (6).
HEK 293T cells were metabolically labeled at 15 or 37 °C and treated
in the absence () or presence (+) of 1 mM iodine (Fig. 4). Under reducing conditions, only monomeric Shaker
protein was observed; after incubation at 15 °C, the immature form
was present (open arrow 1), whereas after incubation at
37 °C, primarily the mature form was present (filled arrow
1). Under nonreducing conditions, disulfide-bonded adducts of
Shaker protein corresponding to a dimer (arrows 2), trimer
(arrows 3), and two tetramers, one linear (arrows
4) and one circular (arrows 4c) were observed (6). After incubation at 15 °C, the adducts were composed of immature Shaker protein (open arrows), whereas after incubation at
37 °C, the adducts were primarily composed of mature protein
(filled arrows). The fact that adducts were observed after
blocking ER-to-Golgi transport by incubation at 15 °C suggests that
assembly of the subunits occurs in the ER. The intensities of the
tetrameric adducts indicate that the immature form of the Shaker
protein exists predominantly as fully assembled tetramers in the ER.
Blockade of ER-to-Golgi transport by BFA and nocodazole treatment gave
results similar to those obtained from low temperature incubation (data
not shown).
Sucrose Gradient Centrifugation Indicates that Shaker Subunits Assemble in the ER
The state of assembly of Shaker protein was
also assessed by sucrose gradient centrifugation (Fig.
5). The detergent CHAPS has been shown to solubilize
wild-type Shaker protein in an active, assembled form (43).
Furthermore, Shaker homologues expressed in vitro and
solubilized in CHAPS sediment with a velocity consistent with a
tetrameric structure (20). In contrast, Shaker homologues solubilized
in Zwittergent sediment as monomers (20). Following incubation at
37 °C (Fig. 5A) or treatment with Me2SO (Fig.
5C), Shaker protein solubilized in CHAPS sedimented with a
velocity consistent with a multimeric state, whereas Shaker protein
solubilized in Zwittergent sedimented with a velocity consistent with a
monomeric state. To determine whether Shaker protein assembles in the
ER, ER-to-Golgi transport was inhibited. Following incubation at
15 °C, immature Shaker protein solubilized in CHAPS sedimented to
the denser region of the gradient, consistent with a multimeric state of assembly (Fig. 5B). Following Zwittergent solubilization,
immature Shaker protein sedimented to a lighter region of the gradient, consistent with the monomeric state. Similar results were obtained after blocking ER-to-Golgi transport with BFA and nocodazole (Fig. 5D). Within experimental error, the sedimentation profiles
of the mature and ER resident, immature forms of the protein overlapped in gradients containing CHAPS or in gradients containing Zwittergent. Thus, the results of sucrose gradient centrifugation, like those obtained from disulfide cross-linking, demonstrate that assembly of
Shaker subunits occurs in the ER.
To determine whether the position of the Shaker protein on the CHAPS gradients corresponded to that of an authentic tetramer, we investigated the sedimentation properties of a covalently linked tetramer of Shaker subunits. This construct consists of four repeats of an inactivation-removed Shaker 29-4 monomer (44) connected by three 19-amino acid linkers (45) and has a predicted molecular mass of about 280 kDa (33). It provides an ideal marker for the hydrodynamic properties of the assembled channel (6). Similar to wild-type Shaker B protein expressed in HEK 293T cells, this covalent tetramer appears as an immature, core-glycosylated form in addition to the predominant, mature form (6).
The covalent tetramer was expressed and metabolically labeled in Xenopus oocytes, solubilized in CHAPS or Zwittergent, and subjected to sedimentation on sucrose gradients. As shown in Fig. 5E, covalent tetramer protein solubilized in CHAPS (filled circles) sedimented as one major peak to the denser half of the gradient. The position of this peak overlapped with that of the mature (Fig. 5, A and C) and immature (Fig. 5, B and D) forms of the protein in CHAPS. These results indicate that the multimers of mature and immature Shaker protein sediment with a velocity consistent with a tetrameric state following CHAPS solubilization. Thus, the results of sucrose gradient centrifugation confirm that Shaker subunits assemble into tetramers in the ER.
As expected, the covalent tetramer did not co-sediment with monomers after Zwittergent solubilization (open circles) but instead sedimented to the denser half of the gradient. This peak did not overlap with that seen in CHAPS, suggesting that Zwittergent weakens the interactions between the covalently linked subunits, which of course cannot be fully dissociated.
Cytoplasmic subunits have previously been shown to
associate with K+ channels, altering their functional
properties, cell surface expression, or stability (14-18). Endogenous
subunits, which are present in some cell lines, have not been
detected in HEK 293 cells, however (46). To determine whether
subunits assemble with Shaker subunits, the rat Kv
2 protein was
co-expressed with Shaker in HEK 293T cells. Kv
2 is homologous to the
Drosophila
subunit, Hyperkinetic, which is thought to
associate with Shaker subunits in vivo (16). Specific
association between the Shaker and Kv
2 proteins in HEK 293T cells
was demonstrated by reciprocal immunoprecipitation. Anti-Shaker
antibodies precipitated Kv
2 (~38 kDa), and anti-
antibodies
precipitated Shaker protein (Fig. 6). Interestingly, the
anti-
antibody precipitated both the immature and mature forms of
Shaker protein, suggesting that Shaker and
subunits associate in
the ER (Fig. 6, left panel). To test this idea further, the
Shaker and Kv
2 proteins were co-expressed and metabolically labeled
during a 10-min pulse in the presence of BFA and nocodazole. Following
blockade of ER-to-Golgi transport, both anti-
and anti-Shaker
antibodies co-precipitated the immature form of Shaker with Kv
2
(Fig. 6, middle panel), confirming that the
and
subunits assemble in the ER. Results from cells treated with
Me2SO alone were identical to those obtained from untreated cells (Fig. 6, right panel).
It has been previously reported that Kv2 acts as a chaperone for
mammalian K+ channel
subunits (18). To determine
whether this is a general property of Kv
2, we investigated the
effect of co-expression on the maturation of Shaker protein. The
presence of Kv
2 did not increase the rate or extent of Shaker
protein maturation (data not shown). In fact, co-expression slowed the
rate of Shaker maturation (data not shown). This is likely to be a
nonspecific effect, caused by competition for the cellular factors
needed to over-express two exogenous proteins simultaneously. Similar
effects have been seen upon co-expression of other
proteins.2
We have characterized three important features of Shaker
K+ channel biogenesis in a mammalian cell line. First,
maturation of the Shaker protein requires transport from the ER to the
Golgi apparatus. Second, the immature form of the Shaker protein
contains high mannose oligosaccharides characteristic of the ER,
whereas the mature form contains complex oligosaccharides
characteristic of modification in the Golgi apparatus. Third, Shaker
subunits assemble with each other and with subunits in the ER.
Thus, it is now possible to assign some of the steps in Shaker channel biogenesis to specific intracellular compartments.
Our conclusion that Shaker channels assemble in the ER is consistent with biochemical and electrophysiological analysis of K+ channel protein translated in vitro. Co-immunoprecipitation experiments suggest that Shaker homologues form heteromultimers within 15 min of the start of translation in vitro (47). Shaker protein translated in vitro in the presence of microsomal membranes can be reconstituted into lipid bilayers to produce functional channels (48), consistent with the idea that the core-glycosylated, ER form of the protein is not only assembled but functionally competent.
For many oligomeric proteins, assembly occurs in the ER and is required
for transport to the Golgi apparatus and subsequently to the cell
surface (1, 49). Among channel-forming proteins, however, there are
exceptions. Whereas subunits of the nicotinic acetylcholine receptor
appear to be transported out of the ER as assembled pentamers (50-52),
gap junction hemichannels and voltage-dependent Na+ channels assemble, at least in part, after leaving the
ER (53, 54). Connexin43, a subunit of gap junctions, assembles into hexameric connexons in a post-ER compartment, most likely the trans-Golgi network (53). Similarly, in rat brain neurons, association of the and
2 subunits of the Na+ channel occurs in
the Golgi apparatus late during biogenesis (54). In contrast, our data
indicate that both the pore-forming
and cytoplasmic
subunits of
voltage-dependent K+ channels assemble in the
ER.
Shi et al. (18) have recently reported that Kv2 increases
the glycosylation, stability, and cell surface expression of a
mammalian Shaker homologue, Kv1.2, expressed in a cell line. They
proposed that the
subunit acts as a chaperone to promote the proper
folding and assembly of pore-forming
subunits. In contrast,
co-expression of Kv
2 with Shaker does not increase the rate or
extent of Shaker maturation. Therefore, the chaperone action of the
subunit depends on the specific
subunit with which it is expressed.
One key difference between the Shaker and Kv1.2 proteins is their
efficiency of glycosylation in cell culture. When expressed in the
absence of Kv
2, Shaker undergoes efficient, virtually complete
glycosylation and transfer to the Golgi apparatus (28), whereas the
Kv1.2 protein is poorly glycosylated and is primarily found in the ER
(18). These results suggest that the Drosophila
subunit
is more robust than its mammalian counterpart, able to mature
efficiently in expression systems without the need for chaperone action
by a
subunit.
We have previously shown that maturation provides a consistent and reliable indication that the Shaker protein is in a native conformation (55).3 For wild-type Shaker protein expressed in HEK 293T cells, maturation is very efficient, occurring with an apparent t1/2 of 45 min and reaching virtual completion within 2.5 h (28). However, some site-directed mutations prevent maturation by disrupting specific structural interactions that are essential for the proper folding of the protein (55, 56). These mutations can be rescued by introducing specific, complementary, second-site mutations, which restore both maturation and function (55).3 Because we have now shown that maturation requires ER-to-Golgi transport, the non-maturing phenotype can be attributed to retention of the misfolded protein in the ER in a core-glycosylated state. Taken together, these results indicate that the Shaker protein is subject to quality control in the ER (57-59) and that only properly folded and assembled channels are transferred to the Golgi apparatus.
We thank Dr. Lily Y. Jan for antibodies, Dr.
Fred J. Sigworth for the covalent tetramer cDNA, Dr. James S. Trimmer for the Kv2 subclone and antibodies, Dr. Ernest M. Wright
for his support, and members of the Papazian laboratory for their
comments on the manuscript.