N-glycosylation promotes the cell surface expression of Kv1.3 potassium channels

N-glycosylation promotes the cell surface expression ofKv1.3 potassium channelsJing Zhu, Jenny Yan* and William B. Thornhill

Department of Biological Sciences and Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Bronx, NY, USA

Keywords

cell surface expression; monosaccharide

supplementation; N-glycosylation; potassium

channel; trafficking

Correspondence

W. B. Thornhill, Department of Biological

Sciences, Fordham University, Bronx, NY

10458, USA

Fax: +718 817 3645

Tel: +718 817 3688

E-mail: [email protected]

*Present address

College of Arts and Science, New York

University, New York, NY 10012, USA

(Received 28 February 2012, revised 27

April 2012, accepted 15 May 2012)

doi:10.1111/j.1742-4658.2012.08642.x

The voltage-gated potassium channel Kv1.3 plays an essential role in mod-

ulating membrane excitability in many cell types. Kv1.3 is a heavily gly-

cosylated membrane protein. Two successive N-glycosylation consensus

sites, N228NS and N229ST, are present on the S1–S2 linker of rat Kv1.3.

Our data suggest that Kv1.3 contains only one N-glycan and it is predomi-

nantly attached to N229 in the S1–S2 extracellular linker. Preventing

N-glycosylation of Kv1.3 significantly decreased its surface protein level

and surface conductance density level, which were � 49% and � 46%

respectively of the level of wild type. Supplementation of N-acetylglucos-

amine (GlcNAc), L-fucose or N-acetylneuraminic acid to the culture med-

ium promoted Kv1.3 surface protein expression, whereas supplementation

of D-glucose, D-mannose or D-galactose did not. Among the three effective

monosaccharides ⁄derivatives, adding GlcNAc appeared to reduce sialic

acid content and increase the degree of branching in the N-glycan of

Kv1.3, suggesting that the N-glycan structure and composition had chan-

ged. Furthermore, the cell surface half-life of the Kv1.3 surface protein was

increased upon GlcNAc supplementation, indicating that it had decreased

internalization. The GlcNAc effect appears to apply mainly to membrane

proteins containing complex type N-glycans. Thus, N-glycosylation pro-

motes Kv1.3 cell surface expression; supplementation of GlcNAc increased

Kv1.3 surface protein level and decreased its internalization, presumably

by a combined effect of decreased branch size and increased branching of

the N-glycan.

Introduction

The voltage-gated potassium (Kv) channel Kv1.3 is a

key regulator of various cellular functions in many cell

types. In neurons, Kv1.3 helps set the resting membrane

potential, modulates action potential firing and influ-

ences neurotransmission [1–3]. In T blood cells, Kv1.3

is the major Kv channel regulating the resting mem-

brane potential and calcium signaling [4,5]. Blockage of

Kv1.3 channels prevented T cell activation and attenu-

ated the immune response [6–8]. Kv1.3 has also been

localized to the inner mitochondrial membrane of

lymphocytes, mediating apoptosis [9]. In addition,

Kv1.3 expression was correlated with the proliferation

and apoptosis of colon, prostate and breast cancers

[10–12]. Kv1.3 is becoming a molecular target for treat-

ment of T-cell-mediated autoimmune diseases as well as

different cancers. Thus, understanding the mechanisms

regarding the cellular regulation of Kv1.3 expression

and function is of biological and clinical importance.

Kv1.3 is a heavily glycosylated tetrameric protein [13].

The topology of a subunit reveals six transmembrane

Abbreviations

CAD, cath-a-differentiated; CHO, Chinese hamster ovary; Endo H, endo-b-N-acetylglucosaminidase H; GlcNAc, N-acetylglucosamine;

GLUT, glucose transporter; Kv, voltage-gated potassium; Neu5Ac, N-acetylneuraminic acid; NXT ⁄ NXS, N-glycosylation consensus site;

PNGase F, peptide N-glycosidase F; TbR, transforming growth factor b receptor.

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 1

domains, S1–S6, linkers that connect the domains, the

pore region, and cytoplasmic N- and C-termini. An

N-glycan(s) is attached to the S1–S2 extracellular lin-

ker of the channel protein. N-glycosylation is a com-

mon and highly diverse co- or post-translational

modification to proteins. N-glycosylation begins in the

rough endoplasmic reticulum with the addition of an

N-glycan precursor (Glc3Man9GlcNAc2) to the aspara-

gines of the N-glycosylation consensus sequence, or

sequon, NXS ⁄T (where X represents any amino acid

but proline) on the nascent polypeptide. The occu-

pancy rate of an extracellular sequon is � 67% based

on data from 749 well characterized glycoproteins [14].

The N-glycans are then trimmed and modified when

transported through the Golgi apparatus. N-glycans

have diverse compositions and structures due to the

inherent inefficiency of pathway enzymes and avail-

ability of N-glycan biosynthesis substrates. N-glycosyl-

ation can affect not only protein folding, stability,

trafficking and localization, but also functional proper-

ties such as ligand binding, signal recognition and elec-

trophysiological parameters of a variety of proteins

[15–21]. Furthermore, defects in glycosylation of pro-

teins appear to be involved in a group of congenital

multisystemic diseases, also called congenital disorders

of glycosylation, which often cause severe psychomo-

tor retardation [22].

The aim of this paper is to investigate the surface

expression of Kv1.3 by (a) identifying the N-glyco-

sylation site(s), (b) assessing the effect of N-glyco-

sylation on surface protein level and conductance

density level, and (c) examining the effects of mono-

saccharide ⁄derivative concentration on its surface

protein level, protein banding pattern and protein

internalization.

Results

Kv1.3 channels have heterogeneous

N-glycosylation

To assess the N-glycosylation state of Kv1.3

(Fig. 1A,B), the cDNA of rat Kv1.3 was transiently

transfected into Chinese hamster ovary (CHO) cells.

CHO cells are a fibroblast-like line, which do not

express endogenous Kv channels and are an ideal system

for exogenous Kv channel expression [23]. Immunoblot

analysis revealed that Kv1.3 consists of two major

forms, a diffuse upper band with a molecular mass of

� 80 kDa and a sharp lower band with a molecular

mass of � 60 kDa (Fig. 1C, lane 1). The predicted

molecular mass from the amino acid sequence of rat

Kv1.3 is � 58 kDa. The apparent molecular mass of

Kv1.3 protein bands is greater than their predicted

mass, suggesting that these molecules are post-transla-

tionally modified. Glycosidase gel shift analysis was

employed to verify the presence of N-glycans. Endo-b-N-acetylglucosaminidase H (Endo H) removes only

high-mannose type and some hybrid type glycans while

peptide-N-glycosidase F (PNGase F) removes high-

mannose, hybrid and complex type N-glycans. The

� 80 kDa bands of Kv1.3 were not sensitive to Endo

H but were sensitive to PNGase F, suggesting that these

bands contained complex type N-glycans, whereas the

� 60 kDa band of Kv1.3 was sensitive to both Endo H

and PNGase F, indicating that this band contained

high-mannose or hybrid type N-glycans (Fig. 1D, lanes

1–3). Therefore, Kv1.3 has heterogeneous N-glycosyla-

tion.

Kv1.3 has only one N-glycan attached to

its S1–S2 linker

Two successive N-glycosylation consensus sites, NNS

and NST, are present on the S1–S2 linker of rat Kv1.3

(Fig. 1B). To determine whether one or both sites are

N-glycosylated, single mutants N228Q and N229Q,

with one site mutated, and the double mutant

N228Q ⁄N229Q, with both sites mutated, were con-

structed and transiently transfected into CHO cells.

Immunoblot analysis showed that there was little or

no difference in the total protein profiles between wild

type Kv1.3 and single N-linked mutants – both formed

two bands, a diffuse upper band at � 80 kDa and a

sharp lower band at � 60 kDa. However, the double

mutant only formed one band, and its gel mobility

was significantly increased compared with the

� 60 kDa band of wild type and single mutants (Rf

� 51.9% versus � 51.4%) (Fig. 1C). This finding

implies that both single mutants are N-glycosylated,

N228Q to asparagine at position 229 and N229Q to

asparagine at position 228 (Fig. 1B). Glycosidase treat-

ment showed that the � 80 kDa bands of single

mutants were sensitive to PNGase F but resistant to

Endo H, suggesting they contained complex type

N-glycans, whereas the � 60 kDa band was sensitive

to both enzymes, suggesting it had a high-mannose or

hybrid type N-glycan (Fig. 1D, lanes 4–9). The double

mutant was insensitive to both glycosidases, indicating

that it did not contain any N-glycans (data not

shown). Therefore, Kv1.3 and two single mutants

appear to have a similar molecular mass and a similar

N-glycosylation pattern. Since each single mutant con-

tains one N-glycan, Kv1.3 should also contain one

N-glycan; otherwise, the wild type would be expected

to show higher molecular mass than the single

An N-glycan promotes Kv1.3 surface expression J. Zhu et al.

2 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

mutants. Our results suggest that either N-glycosyla-

tion site on rat Kv1.3 could be glycosylated but only

one site was occupied at a time.

To determine which of the two sites is occupied in

wild type, the surface protein profile of Kv1.3 and sin-

gle mutants was examined using cell surface biotinyla-

tion ⁄ immunoblotting and was analyzed by

densitometry. The diffuse upper band of Kv1.3 and

the single mutants have three bands, denoted as bands

1, 2 and 3. Kv1.3 and N228Q have mainly bands 1

and 2, whereas N229Q has mainly bands 1 and 3. The

percentage of the upper band is � 91%, � 94% and

� 61% for Kv1.3, N228Q and N229Q, respectively

(Fig. 1E). It appears that the protein banding pattern

of Kv1.3 is more similar to N228Q than N229Q, which

suggests that most of the N-glycans were attached to

asparagines at position 229. Based on the upper band

percentage, we estimated that � 91% of wild type

Kv1.3 has N-glycans attached to N229 and � 9% of

wild type Kv1.3 has N-glycans attached to N228.

Thus, although rat Kv1.3 has two extracellular N-gly-

cosylation consensus sites, at N228 and N229, it

appears that the N229 site is preferred in the wild type.

In addition, rat brain Kv1.3 also appears to have only

a single N-glycan by glycosidase treatment and immu-

noblotting (data not shown).

N-glycosylation promotes cell surface expression

of Kv1.3

We next asked whether N-glycosylation affects Kv1.3

cell surface expression. Kv1.3 and the double mutant

N228Q ⁄N229Q, which does not contain N-glycosyla-

tion consensus sites, were transiently expressed in

CHO cells. The surface protein levels were analyzed by

cell surface biotinylation ⁄ immunoblotting and the

surface conductance density levels, obtained by maxi-

mum conductance divided by capacitance (Gm ⁄Cm),

were estimated by whole-cell patch clamping. Our data

showed that the surface protein level of N228Q ⁄N229Q

was � 49% the level of Kv1.3 (Fig. 2A), suggesting

that preventing N-glycosylation significantly decreased

the surface protein level of Kv1.3. Furthermore, the

surface conductance density level of N228Q ⁄N229Q

was � 46% of the level of Kv1.3 (Fig. 2B,C),

suggesting that preventing N-glycosylation significantly

N C

228

229

Pore

S1 S2 S3 S4 S5 S6

LPEFRDEKDYPASTSQDSFEAAGNSTSGSRAGASSFSDP

LPEFRDEKDYPASPSQDVFEAANNSTSGAPSGASSFSDP

LPEFRDEKDYPASPSQDVFEAANNSTSGASSGASSFSDP

mKv1.3

hKv1.3

r Kv1.3228229

231232

227

Kv1.3 N228Q N229Q

Endo HPNGase F

+ + ++ ++

Kv1.3 Ab60 kDa--

80 kDa-

1 2 3 4 5 6 7 8 9

Kv1.3

N228Q

N229Q

N228Q

/

N229Q

Kv1.3 Ab

Rf (%) 51.38+0.06

51.88+0.05

51.43+0.04

51.35+0.06

60 kDa--

80 kDa-

1 2 3 4

123

--LowerUpper

123

Kv1.3

N228Q

N229Q

90.9+1.4

60.5+3.6

94.0+1.8Upper %

123Lower

Upper

60 kDa--80 kDa-

A

B

D

C

E

Fig. 1. Identification of N-glycosylation sites on the S1–S2 linker of rat Kv1.3. (A) Schematic of a rat Kv1.3 monomer which shows six trans-

membrane domains (S1–S6), linkers and the pore region between S5 and S6. Two consensus sites for N-glycosylation (NXT ⁄ S) are present

in the S1–S2 linker. (B) Sequence alignment of Kv1.3 S1–S2 linkers from human, mouse and rat. Numbers denotes the N-glycosylation site.

(C) Total protein profiles of Kv1.3 and N-glycosylation mutants in transfected CHO cells. A representative immunoblot is shown. The relative

mobility (Rf) of the lower band (� 60 kDa band) of Kv1.3 constructs is displayed, ±SE, n = 3. The Rf value of N228Q ⁄ N229Q is significantly

different from the Rf values of other constructs (P < 0.05, ANOVA). (D) Immunoblot analysis of glycosidase-treated Kv1.3 and N-glycosyla-

tion mutants: +, glycosidase treatment. Upper panel is a shorter time exposure of the lower panel. (E) Surface protein profiles of Kv1.3 con-

structs. A representative immunoblot is shown, n = 3. Densitometry analysis revealed that the upper band (� 80 kDa band) of Kv1.3

constructs consists of three bands, denoted as bands 1, 2 and 3. The percentage of the upper band is calculated by dividing the upper band

by the sum of the upper band and the lower band.

J. Zhu et al. An N-glycan promotes Kv1.3 surface expression


decreased the surface conductance density level of

Kv1.3. Thus, these results suggest that N-glycosylation

promotes cell surface expression of Kv1.3, although it

is not required for the functional expression of Kv1.3.

To assess whether the effect of N-glycosylation on

Kv1.3 is cell line specific, Kv1.3 and N228Q ⁄N229Q

were transiently expressed in cath-a-differentiated

(CAD) cells. CAD cells are a mouse CNS cell line and

exhibit biochemical and morphological characteristics

of primary neurons but do not express endogenous

Kv1 channels [24]. Immunoblot analysis suggests that

the surface protein level of N228Q ⁄N229Q was � 66%

of the level of Kv1.3 (Fig. 2D). It appears that pre-

venting N-glycosylation decreased the surface protein

level of Kv1.3 in two different cell lines, although to

differing degrees. This finding could be due to some

differences in the intracellular glyco-processing

machinery between the two lines.

Supplementation of GlcNAc, L-fucose and

Neu5Ac increases Kv1.3 cell surface expression

whereas D-mannose, D-galactose and D-glucose

have no effect

Three types of N-glycans (high-mannose, hybrid and

complex) are produced during Golgi processing of gly-

coproteins in mammalian cells, and the component

monosaccharides and their derivatives include d-man-

nose, d-glucose, N-acetylglucosamine (GlcNAc),

l-fucose, d-galactose and N-acetylneuraminic acid

(Neu5Ac), the most common member of sialic acid

(Fig. 3A). N-glycans differ not only in the nature of

their constituents but also in the length of their chains

and in the number of branches. To investigate whether

the monosaccharide ⁄derivative level affects Kv1.3 sur-

face expression, CHO cells were exposed to increased

concentrations of different monosaccharides or deriva-

tives. Kv1.3 transfected cells without monosaccharide

supplemented were used as a control. The surface pro-

tein level of Kv1.3 was measured by cell surface bioti-

nylation and immunoblotting. Our data showed that

supplementation of d-mannose, d-glucose and d-galac-

tose had little or no effect on cell surface expression

of Kv1.3 (Fig. 3B,C,F), whereas supplementation of

GlcNAc, l-fucose and Neu5Ac caused a significant

increase in Kv1.3 surface levels. GlcNAc enhanced

Kv1.3 surface expression in a dose dependent manner

– the surface protein level of Kv1.3 increased 2, 2.5

or 3 times when GlcNAc was at 25, 50 or 100 mm

(Fig. 3D). l-Fucose also increased the Kv1.3 surface

protein level but only when its concentration reached

50 mm (Fig. 3E). Supplementation of Neu5Ac

increased Kv1.3 surface protein level when its concen-

tration reached 10 mm (Fig. 3G). Thus, addition of

GlcNAc, l-fucose or Neu5Ac to the culture medium

significantly increased the cell surface expression of

Kv1.3, presumably through altering N-glycosylation.

We speculate that the altered N-glycosylation might

further affect protein internalization or stability

resulting in a higher surface expression level of

Kv1.3.

GlcNAc supplementation reduced the branch size

of complex type N-glycans of Kv1.3, whereas

L-fucose and Neu5Ac did not

To assess whether the increased surface level of Kv1.3

upon supplementation of monosaccharides was due to

a change in N-glycan structure, the surface protein

profile of Kv1.3 was analyzed by immunoblotting and

densitometry. The gel mobility of the � 80 kDa bands

of Kv1.3 was increased significantly when GlcNAc was

Sur

face

pro

tein

(nor

mal

ized

)

Sur

face

pro

tein

(nor

mal

ized

)

0

50

100

*

Kv1.3N228Q/

N229Q

CHO transfected

Kv1.3

0

5

10

15

20

25

Gm

/Cm

(nS

/pF)

*

CHO transfected

N228Q/N229Q

Kv1.3

N228Q/N229Q

10 nA

10 ms

0

50

100

*

Kv1.3N228Q/

N229Q

CAD transfected

A

C D

B

Fig. 2. Effect of N-glycosylation on the cell surface expression of

Kv1.3. (A) Bar graphs of surface protein levels of Kv1.3 and

N228Q ⁄ N229Q in transfected CHO cells. The mean value of

N228Q ⁄ N229Q was normalized to the mean value of Kv1.3, which

was taken to be 100. Error bars represent ±SE of triplicate values.

An asterisk indicates significant difference from control (P < 0.05,

unpaired t test). (B) Bar graphs of conductance densities of Kv1.3

and N228Q ⁄ N229Q in transfected CHO cells. Gm ⁄ Cm, maximum

conductance divided by capacitance. Error bars represent ± SE of

11–13 values. An asterisk indicates a significant difference from

control (P < 0.05, unpaired t-test). (C) Whole cell current traces of

Kv1.3 and N228Q ⁄ N229Q. (D) Bar graphs of surface protein levels

of Kv1.3 and N228Q ⁄ N229Q in transfected CAD cells. The mean

value of N228Q ⁄ N229Q was normalized to the mean value of

Kv1.3, which was taken to be 100. Error bars represent ± SE of

triplicate values. An asterisk indicates significant difference from

control (P < 0.05, unpaired t-test).



used, whereas the gel mobility of the � 60 kDa band

of Kv1.3 did not change with the treatment (Fig. 4A).

Glycosidase treatment showed that the upper bands

of Kv1.3 ⁄GlcNAc were sensitive to PNGase F but

resistant to Endo H, suggesting that the bands

contained complex type N-glycans, whereas the

� 60 kDa band of Kv1.3 ⁄GlcNAc was sensitive to

both PNGase F and Endo H, indicating that it con-

tained a high-mannose or hybrid type N-glycan

(Fig. 4B). As shown in Fig. 1, the � 80 kDa bands of

Kv1.3 contain complex type N-glycans and the

� 60 kDa band of Kv1.3 contains a high-mannose or

hybrid type N-glycan. These results indicate that the

type of N-glycosylation did not change upon addition

of GlcNAc; the difference in gel mobility of the

� 80 kDa bands in Kv1.3 and Kv1.3 ⁄GlcNAc sug-

gests that there was a change in structure and ⁄orcomposition of the complex type N-glycan. Previous stu-

dies showed that the gel mobility of the Na,K-ATPase

b1 subunit was increased when the length of the

branches or the number of branches decreased in com-

plex type N-glycans [25]. The increased gel mobility of

the � 80 kDa bands of Kv1.3 suggests that the branch

size and ⁄or branching of the complex type N-glycan

must be decreased.

Mature mammalian N-glycans are commonly termi-

nated with sialic acids [26]. To assess whether supple-

mentation of GlcNAc altered sialic acid content,

0

25

50

75

100

125

150

D-galactose (mM)

0

50

200

150

250

100

0 10 205 15Neu5Ac (mM)

*

050100150200250300350

GlcNAc (mM)

**

*

D-glucose (mM)

0

25

50

75

100

125

150

L-fucose (mM)

0255075100125150175 *

*

0

25

50

75

100

125

150

A

B C

D E

F G

0 50 1007525

0 50 1007525

0 50 1007525

0 50 1007525

0 50 1007525

D-mannose (mM)

Sur

face

Kv1

.3 p

rote

in(n

orm

aliz

ed)

Sur

face

Kv1

.3 p

rote

in(n

orm

aliz

ed)

Sur

face

Kv1

.3 p

rote

in(n

orm

aliz

ed)

Sur

face

Kv1

.3 p

rote

in(n

orm

aliz

ed)

Sur

face

Kv1

.3 p

rote

in(n

orm

aliz

ed)

Sur

face

Kv1

.3 p

rote

in(n

orm

aliz

ed)

GlcNAc

Galactose Neu5Ac

Glucose

Fucose

Mannose

Asn Asn Asn Asn

ER Golgi

GlycanPrecursor

High-MannoseGlycan

Hybrid typeGlycan

Complex typeGlycan

Fig. 3. Effect of the monosaccha-

rides ⁄ derivatives on the surface protein

level of Kv1.3. (A) Schematics of N-glycan

precursor, high-mannose, hybrid and com-

plex type N-glycans. (B)–(G) Surface protein

levels of Kv1.3 in transfected CHO cells

maintained in the culture medium supple-

mented with different concentrations of

monosaccharides ⁄ derivatives, including

D-mannose (B), D-glucose (C), GlcNAc (D),

L-fucose (E), D-galactose (F) and Neu5Ac (G).

Mean values of Kv1.3 in the presence of

monosaccharides ⁄ derivatives were normal-

ized to the mean value of Kv1.3 in the

absence of monosaccharides ⁄ derivatives,

which was set to 100. Error bars represent

± SE of triplicate values. *differences are

significant (P < 0.05, ANOVA).



surface Kv1.3 was treated with sialidase, which

removes terminal sialic acid residues. The gel mobility

of both upper and lower bands of Kv1.3 was

increased after sialidase treatment, indicating that

both of them contained sialic acids (Fig. 4C). Since

the lower band was also sensitive to Endo H

(Fig. 1D), it appeared to contain a hybrid type N-gly-

can. In contrast, after supplementation of GlcNAc,

the upper bands of Kv1.3 were resistant to sialidase,

indicating that they did not contain detectable sialic

acids, whereas the lower band of Kv1.3 was sensitive

to sialidase, indicating that it contained sialic acids

(Fig. 4D). It appears that supplementation of GlcNAc

decreased sialic acid content in the complex type

N-glycan of Kv1.3. These results suggest that the length

of branches in complex type N-glycans decreased when

GlcNAc was added, but whether or not the number of

branches also changed was not clear. Thus, the

increased surface protein level of Kv1.3 upon supple-

mentation of GlcNAc appears to be due to decreased

branch size and ⁄or altered branching of complex type

N-glycans.

The addition of l-fucose or Neu5Ac caused little or

no change in gel mobility of the � 80 kDa bands of

Kv1.3 (Fig. 4E,F), indicating no dramatic changes in

the branch size and ⁄or branching of the complex type

N-glycans; however, whether or not the carbohydrate

composition changed was unclear. Therefore, the

increase of Kv1.3 surface expression upon supplemen-

tation of l-fucose or Neu5Ac might be due to different

mechanisms compared with GlcNAc. We speculate

that increasing the substrate concentration would

improve the efficiency of fucosylation or sialylation of

N-glycans and therefore stabilize the structure of

Kv1.3.

Supplementation of GlcNAc stabilized the cell

surface Kv1.3 population

We next asked whether GlcNAc promotes the surface

protein level of Kv1.3 by affecting its internalization.

To address this question, Kv1.3 transfected cells were

maintained in culture medium with or without GlcNAc

added, and then surface biotinylated and chased for

various times. The cell surface signals of Kv1.3 were

analyzed using a two-phase exponential decay equa-

tion. Kv1.3 had half-lives of 0.6 and 9.2 h (Fig. 5A),

whereas Kv1.3 ⁄GlcNAc had half-lives of 1.0 and

14.9 h (Fig. 5B). It appears that surface Kv1.3 was a

mixture of two components, each with its own inter-

nalization half-life, which suggests that two internali-

zation temporal pathways might exist. The half-lives

of both components of Kv1.3 were increased upon

supplementation of GlcNAc, indicating that they

exhibited decreased internalization. These results sug-

gest that supplementation of GlcNAc eventually sta-

bilized the Kv1.3 surface population and inhibited its

internalization, presumably due to decreased branch

size and ⁄or altered branching in complex type N-gly-

cans of Kv1.3.

Sialidase

60 kDa--80 kDa-- Surface

Kv1.3

+

60 kDa--70 kDa--

Sialidase

GlcNAc (25 mM)

SurfaceKv1.3

+

Neu5Ac (10 mM)– +

SurfaceKv1.321

12

80 kDa-

L-fucose (50 mM)– +

21SurfaceKv1.3

12

80 kDa-

GlcNAc (25 mM)– +

12

21 Surface

Kv1.380 kDa-

A

B

C D

E F

GlcNAc (25 mM)– +

SurfaceKv1.360 kDa--

60 kDa band

80 kDa-

Over-exposed

Endo HPNGase F

++

GlcNAc (25 mM)

60 kDa--80 kDa-- Total

Kv1.3

Fig. 4. Effect of GlcNAc, L-fucose and Neu5Ac on the protein band-

ing pattern of Kv1.3. (A) Surface protein banding patterns of Kv1.3

in CHO cells with GlcNAc supplemented: ), control, without

GlcNAc added; +, GlcNAc added. A densitometry analysis of Kv1.3

proteins is displayed in the lower panel showing the position of

two major bands. The right panel is the longer time exposure of

the left panel. (B) Immunoblot analysis of glycosidase-treated Kv1.3

with GlcNAc supplemented: +, glycosidase treatment. (C) Immuno-

blot analysis of sialidase-treated Kv1.3: +, sialidase treatment. (D)

Immunoblot analysis of sialidase-treated Kv1.3 with GlcNAc supple-

mented: +, sialidase treatment. (E), (F) Surface protein banding pat-

terns of Kv1.3 in CHO cells with L-fucose (E) and Neu5Ac (F)

supplemented: ), control, without monosaccharide added; +,

monosaccharide added. A densitometry analysis of Kv1.3 proteins

is displayed in the lower panel showing the position of two major

bands.



Supplementation of GlcNAc affects only

glycoproteins and has no effect on

non-glycosylated proteins

To test whether the effect of GlcNAc supplementation

is specific to Kv1.3, glucose transporter 1 (GLUT1),

which is a heavily glycosylated membrane protein

endogenously expressed in CHO cells, was also used.

The GLUT1 on the cell surface was detected as a

broad protein band of � 70–80 kDa on SDS gels.

With GlcNAc supplementation, the gel mobility of

GLUT1 was increased significantly to � 45–75 kDa,

suggesting that there was a decrease in N-glycan

branch size and ⁄or a change in branching. GLUT1

contains one N-glycan (Fig. 6A). The deglycosylated

GLUT1 had a molecular mass of � 40 kDa on SDS

gels [27], although the predicted molecular mass of

GLUT1 was 54 kDa, suggesting that GLUT1 had

other post-translational modifications in addition to

N-glycosylation. Furthermore, the surface protein level

of GLUT1 was increased � 1.5-fold upon supplemen-

tation of GlcNAc (Fig. 6B). These results are similar

to those observed with Kv1.3. Thus, addition of

GlcNAc in cell culture also has an effect on endoge-

nous GLUT1.

To determine whether supplementation of GlcNAc

affects non-glycoproteins, Kv2.1 and N228Q ⁄N229Q

were transiently expressed in CHO cells. Kv2.1, a

potassium channel from the Kv2 subfamily, does not

contain an N-glycosylation site that is glycosylated,

and N228Q ⁄N229Q is the Kv1.3 mutant with both

N-glycosylation sites mutated. Our data showed that

the surface protein level and the protein banding pat-

tern of Kv2.1 or Kv1.3N228Q ⁄N229Q were similar in

the presence or absence of GlcNAc in the culture med-

ium (Fig. 6C–F). Actin is a non-glycosylated cytoplas-

mic protein endogenously expressed in CHO cells. Our

data showed that addition of GlcNAc in the culture

medium did not affect the total protein level or the

banding pattern of actin (Fig. 6G). Thus, supplementa-

tion of GlcNAc appears to affect mainly glycoproteins.

These results further suggest that supplementation of

GlcNAc does not affect protein synthesis, but it affects

N-glycan Golgi processing or endocytosis.

Discussion

Mouse and rat Kv1.3 have two successive N-glycosyla-

tion consensus sites, NNS and NST, on their S1–S2

linker, while human Kv1.3 has only one, NST. The

S1–S2 linker of Kv1.3 from the three species has the

same length, and mouse and rat share 97.4% sequence

identity whereas human shares 84.6% with the rodents.

Our results showed that either of the two sites on rat

Kv1.3 could be glycosylated if one is mutated, but that

only one site is occupied at a time. We are not aware

of any examples in the literature of neighboring N-gly-

can consensus site asparagines on membrane proteins

being glycosylated. Fly Kv1.1 (Shaker) has two N-gly-

cosylation sites at positions 259 and 263 on its S1–S2

linker, which are three amino acids apart; both sites

are occupied with N-glycans [28]. Rat Kv1.2 has one

N-glycosylation site at the position 207 on its S1–S2

linker. A Kv1.2 construct was engineered to contain

two additional sites at positions 199 and 203, for a

total of three NST sites, each being three amino acids

apart; all of them were N-glycosylated [20]. Human

Kv4.1 has two N-glycosylation sites at positions 352

and 355 on its S5-P loop, which are two amino acids

Chase time (h)

Chase time (h)

Nor

mal

ized

sig

nal

Nor

mal

ized

sig

nal

HL1: 0.6 hHL2: 9.2 h

Kv1.3 surface protein half-life (HL)

25

75

50

100

A

B

0

25

75

50

100

0

86420 10

86420 10

HL1: 1.0 hHL2: 14.9 h

Kv1.3/GlcNAc surface protein half-life (HL)

Fig. 5. The surface protein half-life of Kv1.3. Cell surface protein

half-lives of Kv1.3 in transiently transfected CHO cells in the

absence (A) and presence (B) of GlcNAc were estimated by surface

biotinylation ⁄ immunoblotting and chasing in nonbiotinylation media

as described in Experimental procedures. Cell surface signals of

Kv1.3 were normalized to time zero of chase, which was taken as

100. Symbols (squares and circles) represent the normalized

mean ± SE of triplicate values at each time point. Lines represent

the two-phase exponential decay curves.



apart. Neither site was N-glycosylated [29]. Presumably

these two sites are too close to the predicted S5 trans-

membrane domain – they are five and eight amino

acids respectively away from the S5. In general, an N-

glycosylation site that is occupied is supposed to be 10

amino acids from a transmembrane domain [30]. Our

data showed that two successive N-glycosylation sites,

with no amino acids in between, could not be occupied

at the same time. We speculate that this finding is due

to steric hindrance from the close proximity of these

two sites preventing two N-glycans being added.

While both sites (NNS and NST) on the S1–S2 lin-

ker were capable of being N-glycosylated, the N229

site (NST) was preferred over the N228 site (NNS) in

wild type Kv1.3. It appears that 91% of Kv1.3 has

an N-glycan attached to N229 and 9% of Kv1.3 has

an N-glycan attached to N228. This finding might be

due to the sequon or the position of the site. The

serine-containing sequon (NXS) is less frequently

glycosylated and more affected by the nature of the X

amino acid compared with the threonine-containing se-

quon (NXT) [31,32]. Furthermore, the position of

N229 (NST) on the S1–S2 linker of rat Kv1.3 is equiv-

alent to the position of N227 (NST) in human. Our

previous study showed that the N-glycosylation con-

sensus site of Kv1 channels appears to be located in a

similar relative position in the S1–S2 linker and the

strict relative position of N-glycosylation consensus

sites on these linkers may be correlated with the func-

tional effect of N-glycans on some Kv1 potassium

channels [33]. Thus Kv1.3 from either rat or human

has only one N-glycan attached its S1–S2 linker.

The effect of N-glycosylation on surface expression

varies between different glycoproteins even within the

Kv1 subfamily. Removal of the N-glycosylation con-

sensus site had little effect on Kv1.1 protein levels, but

it considerably reduced surface protein levels of Kv1.2

and Kv1.4, which were � 60% and � 15% of the wild

0

50

150

100

*S

urfa

ce p

rote

in(n

orm

aliz

ed)

Sur

face

pro

tein

(nor

mal

ized

)

Sur

face

pro

tein

(nor

mal

ized

)

GLUT1

GLUT1/

GlcNAc

GLUT1 (1 N-site)Surface protein

51 kDa--

75 kDa--

45 kDa--

Kv2.1

Kv2.1/

GlcNAc

0

50

100

Kv2.1 (0 N-site)Surface protein

110 kDa--

GlcNAc

Actin Ab

+

Actin (0 N-site)CHO Lysates

45 kDa--

–

0

100

50

N228Q

/N22

9Q

N228Q

/N22

9Q/

GlcNAc

N228Q/N229Q (0 N-site)Surface protein

58 kDa--

A

C

D

E

FB

G

Fig. 6. Supplementation of GlcNAc increases surface protein levels of other membrane glycoproteins but it does not affect non-glycopro-

teins. (A) Surface protein profiles of endogenously expressed GLUT1 in CHO cells with or without monosaccharides added. A representative

immunoblot is shown. (B) Bar graphs of GLUT1 surface protein levels. (C) Surface protein profiles of Kv2.1 (contains no N-glycans) in trans-

fected CHO cells with or without monosaccharides added. A representative immunoblot is shown. (D) Bar graphs of Kv2.1 surface protein

levels. (E) Surface protein profiles of N228Q ⁄ N229Q (contains no N-glycan) in transfected CHO cells with or without monosaccharides

added. A representative immunoblot is shown. (F) Bar graphs of N228Q ⁄ N229Q surface protein levels. The mean values of proteins tested

were normalized to the mean value of the control, which was set to 100. Error bars represent ± SE of triplicate values. *differences are

significant (P < 0.05, unpaired t-test). (G) Total protein profiles of actin (cytoplasmic protein) in CHO cells: +, monosaccharide added. A

representative immunoblot is shown, n = 3.



type, respectively [17,20]. Our data showed that

N228Q ⁄N229Q was � 49% of the level of Kv1.3 in

CHO cells. The differential effect of N-glycosylation

on closely related Kv channels presumably reflects the

complexity of the N-glycan structures.

Increasing the GlcNAc level has been shown to

enhance cell surface expression of some receptors and

transporters including transforming growth factor breceptor (TbR) and glucose transporter 4 (GLUT4)

[34]. The TbR surface levels were increased 1.5- and

2-fold in Mgat5) ⁄ ) tumor cells supplemented for 48 h

with 25 and 50 mm GlcNAc, respectively, quantified

by 125I-TGF-b1 binding and galectin-3 immunoprecipi-

tation; GLUT4 surface levels were increased 2-fold in

HEK293T cells supplemented for 48 h with 30 mm

GlcNAc, quantified by immunostaining and confocal

microscopy [34]. We found that the surface levels of

Kv1.3 were increased 2- and 2.5-fold in CHO cells sup-

plemented for 24 h with 25 and 50 mm GlcNAc,

respectively, quantified by cell surface biotinylation

and immunoblotting. It appears that GlcNAc supple-

mentation had similar effects on the surface expression

of Kv1.3, TbR and GLUT4, although to differing

degrees. These differences could be from varied

GlcNAc concentrations, incubation times, cell lines, as

well as techniques used to measure surface protein

levels. Furthermore, the internalization of Kv1.3 was

slowed upon GlcNAc supplementation, suggesting that

the increased surface protein level was due to

decreased endocytotic processing.

Supplementation of GlcNAc increased the degree of

N-glycan branching of TbR I and TbR II in Mgat5) ⁄ )

tumor cells and GLUT4 in HEK293T cells, which in

turn enhanced surface protein expression [34]. GlcNAc

supplementation also increased the number of

branched N-glycans in CHO cells [34], and therefore it

is predicted that the degree of N-glycan branching of

Kv1.3 in CHO cells was also increased. The branching

or branch size of an N-glycan was directly correlated

with the protein gel mobility. A previous report

showed that the Na,K-ATPase b1 subunit exhibited

increased gel mobility when the degree of N-glycan

branching was reduced with swainsonine treatment

and decreased gel mobility when the degree of N-gly-

can branching was increased using small interfering

RNA against the stop-branching enzyme, presumably

due to altered steric hindrance [25]. Our results showed

that the gel mobility of Kv1.3 was increased upon

GlcNAc supplementation. Similar results were

obtained with GLUT1 (Fig. 5), TbR I and TbR II

[34]; they all showed increased gel mobility when

GlcNAc was added. Since the degree of N-glycan

branching is generally increased with GlcNAc supple-

mentation, we speculate that the size of branches must

be decreased. Indeed, the sialic acid content of the

N-glycan in Kv1.3 was reduced when GlcNAc was

added. The increased gel mobility of Kv1.3 upon Glc-

NAc supplementation appeared to result from a com-

bined effect of decreased branch size and increased

branching.

Externally applied GlcNAc goes into the salvage

pathways in cells to form UDP-GlcNAc, which is

transported into the Golgi apparatus as a substrate in

the synthesis of N-glycoconjugates [35–37]. GlcNAc is

linked by b1,2 to the terminal mannose residue of the

Man (a1,3) branch or at the free Man (a1,6) arm

yielding the precursor for mono- or bi-antennary struc-

tures, or by b1,4 at the Man (a1,3) or b1,6 at the Man

(a1,6) branches yielding the precursor for tri- or tetra-

antennary structures [38]. The N-acetyl-d-glucos-

aminyltransferases I, II, IV and V, encoded by the

genes Mgat1, 2, 4a ⁄b and 5, act sequentially to cata-

lyze these reactions, respectively. The concentration of

Golgi UDP-GlcNAc is proportional to the degree of

GlcNAc branching in N-glycans [39]. The branched

N-glycan was crosslinked with extracellular galectins, a

family of N-acetyllactosamine binding animal lectins,

to form a molecular lattice which restricted endocyto-

sis of surface glycoproteins. b1,6 GlcNAc branching

by Mgat5 is preferentially extended by poly-N-acetyl-

lactosamine, further enhancing avidity for galectins

[38]. The desialylation of N-glycans would expose

underlying N-acetyllactosamine and enhance the

galectin binding.

The enhancement of surface proteins by GlcNAc

supplementation appeared to be dependent on complex

type N-glycans. The unglycosylated proteins, e.g.

Kv2.1 and Kv1.3N228Q ⁄N229Q (Fig. 5), proteins with

hybrid type N-glycans, e.g. the � 60 kDa band of

Kv1.3 (Fig. 4), and proteins with high-mannose type

N-glycans, e.g. in Lec1 [34], were not affected. There-

fore, GlcNAc supplementation might have a global

effect on membrane proteins containing complex type

N-glycans. However, in certain cells that highly express

Mgat5, this effect might be reduced.

In mammalian tissues, l-fucose is usually linked by

a1,2 to terminal d-galactose, by a1,3 or a1,4 to subter-

minal GlcNAc residues of the antennae and by a1,6 to

the innermost GlcNAc [40,41]. It has been shown that

the a1,6-linked fucose modification promoted protein

stability and cell surface expression of polysialic-acid-

carrying glycoproteins [42]. The a1,6-linked fucosyla-

tion was found to reduce the conformational flexibility

of the antennae of a bi-antennary N-glycan, which

might further stabilize the protein structure [43]. Our

results showed that supplementation of l-fucose



increased cell surface expression of Kv1.3 channels.

We speculate that increasing l-fucose concentration

increases the number of fucosylated glycans, which in

turn increases protein stability and surface expression

of Kv1.3. It is possible that fucosylation increased

expression of other glycoproteins that affected Kv1.3

expression.

Sialic acids are commonly linked a2,3 or a2,6 to the

termini of complex glycans or are polymerized in the

form of an a2,8 or an a2,9 linkage [44,45]. The influence

of sialic acids on the expression and stability of glyco-

proteins varies. Sialylation modification is crucial for

stabilization of stem cell marker CD133 proteins, and

removal of sialic acids accelerated CD133 degradation

via a lysosome-dependent pathway [46]. Sialic acids also

contributed to the maintenance of SRIF receptors in a

high affinity biologically active conformation [47] and

improved and prolonged cell surface expression of thy-

rotropin receptor [48]. In contrast, removal of terminal

sialic acid stabilized TRPV5 channels and enhanced

their level in plasma membrane by restricting endocyto-

sis [49]. Our data suggest that supplementation of

Neu5Ac, the most common form of sialic acids, pro-

moted surface expression of Kv1.3. We speculate that

increasing Neu5Ac concentration enhanced sialylation

of Kv1.3, which in turn increased its protein stability

and cell surface expression. The differential effect of

sialylation on glycoproteins is presumably due to the

difference in sialic acid structures and protein structures.

In summary, we have presented evidence that an

N-glycan is predominantly attached to N229 on the

S1–S2 linker of rat Kv1.3 and found that N-glycosyla-

tion promoted cell surface expression of Kv1.3 chan-

nels. Furthermore, supplementation of GlcNAc

increased the surface protein level and decreased the

protein internalization of Kv1.3, presumably by a com-

bined effect of a decreased branch size and increased

degree of branching in N-glycans. The GlcNAc effect

appears to apply to mainly membrane proteins with

complex type N-glycans.

Experimental procedures

Cell lines and culture conditions

CHO pro5 cells were obtained from the American Type

Culture Collection. CAD neuronal-like cell line was a gift

from J. Wang at the Department of Neuroscience, Tufts

University School of Medicine. CHO cells were maintained

in MEM a, supplemented with 10% fetal bovine serum,

penicillin (100 unitsÆmL)1), streptomycin (100 lgÆmL)1) and

100 mm glutamine. CAD cells were maintained in Ham’s

F-12 medium, supplemented with 10% fetal bovine serum,

penicillin (100 unitsÆmL)1), streptomycin (100 lgÆmL)1),

100 mm glutamine and proline. All cells were cultured at

37 �C under 5% CO2.

PCR and quick change mutagenesis

Rat Kv1.3 cDNAs were engineered using PCR to contain a

5¢ Kozak enhanced ribosomal binding sequence (CCACC)

before the start methionine and to remove endogenous

5¢- and 3¢-untranslated regions. Mutants N228Q or N229Q,

which had one N-linked site mutated from Kv1.3, and the

double mutant N228Q ⁄N229Q, which had two N-linked

sites mutated from Kv1.3, were generated by quick-change

mutagenesis following the manufacturer’s protocol (Strata-

gene, La Jolla, CA, USA). cDNAs generated were subcl-

oned into pcDNA3, a mammalian expression vector

(Invitrogen, Carlsbad, CA, USA). cDNAs were sequenced

(Cornell University, Ithaca, NY, USA) to confirm that the

mutation was introduced without other unwanted changes.

Transient transfection

CHO or CAD cells were transiently transfected with 0.5 lgcDNA of Kv1.3 constructs in 35 mm culture dishes using

Lipofectamine Plus following the manufacturer’s protocol

(Invitrogen, Carlsbad, CA, USA). Cells were cultured for

20 h post-transfection before they were processed.

Monosaccharide supplementation

Monosaccharides used include d-mannose, d-glucose,

GlcNAc, l-fucose, d-galactose and Neu5Ac. After trans-

fection, monosaccharides were diluted in pre-warmed cul-

ture medium to different concentrations and added for

20 h to cells. Upon confluency, cells were subjected to

membrane isolation, surface protein biotinylation or other

processes.

Membrane isolation

Once cells were confluent, an ice-cold hypotonic solution

containing protease inhibitors was added. Cells were har-

vested by pipetting up and down and then homogenized.

The homogenate was centrifuged at 5000 g at 4 �C for

5 min to pellet the nuclei. The supernatant was centrifuged

again at 16 000 g at 4 �C for 1 h to pellet the membranes,

which include rough endoplasmic reticulum, Golgi appara-

tus and plasma membranes. The membranes were collected

and analyzed by SDS ⁄PAGE and immunoblotting.

SDS/PAGE and immunoblotting

Protein samples obtained from different processes were

each mixed with Laemmli sample buffer and heated.



Similar amounts of proteins were used and fractionated

on a 9% SDS ⁄PAGE at 90 V for � 1.5 h. Proteins were

then transferred to polyvinylidene difluoride (PVDF)

membranes by using the HEB 2020 semi-dry blotter at

12 V for 1 h. The PVDF membranes were blocked with

5% non-fat milk in NaCl ⁄Pi plus 0.1% Tween 20 for

30 min and shaken overnight in primary antibodies

[Kv1.3 monoclonal antibodies (UC Davis ⁄NINDS ⁄NIMH

NeuroMab Facility, Davis, CA, USA), GLUT1 poly-

clonal antibodies (Abcam, Cambridge, MA, USA) or

actin monoclonal antibodies (Sigma-Aldrich, St. Louis,

MO, USA)]. Then horseradish-peroxidase-linked anti-rab-

bit or anti-mouse secondary IgG was added. The specific

proteins were detected on blots using an enhanced chemi-

luminescence detection kit (Amersham, Piscataway, NJ,

USA) and X-ray film AR5 (Eastman Kodak, Rochester,

NY, USA).

Cell surface biotinylation

Cell surface expression levels of Kv1.3 wild type and

mutants in transfected cells were analyzed by biotinylation

using hydrazide-LC-biotin, which is specific for carbohy-

drates, or EZ-link Sulfo-NHS–SS-Biotin, which is specific

to primary amines, following the manufacturer’s protocol

(Pierce, Rockford, IL, USA). Cells were collected in 1 mL

of ice-cold lysis buffer and shaken for 30 min at 4 �C.Aliquots were routinely taken from the solubilized cells to

estimate actin amounts by immunoblotting to control for

any difference in cell density between wells (data not

shown). The remaining lysates were then centrifuged to

remove the nuclei and other insoluble materials. The

supernatant was incubated with 70 lL of streptavidin-aga-

rose beads (Pierce, Rockford, IL, USA) overnight at 4 �C.The beads were washed and eluted with Laemmli sample

buffer and samples were run on SDS ⁄PAGE for immuno-

blotting.

Surface protein half-life estimation

Intact transfected cells were biotinylated as described above

and washed to remove the biotinylation reagent, and a

sample for time zero was taken and processed for streptavi-

din precipitation and immunoblotting analysis as described

above. Other cells in dishes were returned to complete med-

ium and incubated at 37 �C for increasing ‘chase’ times, in

the absence of biotinylation reagent, before streptavidin

precipitation and immunoblotting. Signals were normalized

to the value at time zero (start of chase) and plotted as a

function of time. Protein half-life values were estimated

from a curve fitted to a decaying two-phase exponential

time course, mean ± SE. A decrease in the cell surface

Kv1.3 signal was indicative that biotinylated Kv1.3 proteins

were internalized and degraded via the endosomal ⁄ lysosmal

pathway. However, if a fraction of the internalized biotiny-

lated Kv1.3 proteins were recycled intact back to the

surface, then the surface protein half-life of Kv1.3 would be

overestimated.

Glycosidase treatment

Endo H, PNGase F and sialidase (Sigma-Aldrich, St. Louis,

MO, USA) were used for detecting the presence and types of

N-glycans on Kv1.3 channels. Rat brain membranes (3 lL)or cell lysates (10 lL) were incubated with 0.3 units of Endo

H for 30 min at 37 �C or with 0.05 units of sialidase or 3

units of PNGase F overnight at 37 �C. These incubation

times were sufficient to give the maximum effect. Reactions

were terminated by adding Laemmli sample buffer, and

detected by SDS ⁄PAGE and immunoblotting.

Patch clamping

Cells were co-transfected with 0.1 lg Kv1.3 cDNA con-

structs and 0.1 lg green fluorescence protein cDNA con-

structs to allow visualization of transfected cells by

fluorescence microscopy. Whole cell currents were recorded

at room temperature (23–25 �C) with an Axopatch 200B

amplifier (Axon Instruments, Union City, CA, USA). The

bath solution contained 150 mm NaCl, 5 mm KCl, 1 mm

MgCl2, 2 mm CaCl2, 5 mm glucose and 10 mm HEPES

(pH 7.3). The intracellular solution contained 70 mm KCl,

65 mm KF, 5 mm NaCl, 1 mm MgCl2, 10 mm EGTA,

5 mm glucose and 10 mm HEPES (pH 7.3). Patch pipettes

were fashioned from 8161 Corning glass (Warner Instru-

ments, Hamden, CT, USA) to have tip resistances of 1.2–

2.0 mX when filled with the intracellular solution.

Activation protocols involved evoking currents by depolar-

izing voltage pulses (80 ms) from a holding potential of

)80 mV to levels ranging from )70 to 50 mV in 10 mV

increments. Leak and capacitance currents were subtracted

by a standard P ⁄ n procedure, and series resistance

was compensated to 80%. Maximum peak conductance

values (G) were measured from the mean value of the

leak-subtracted peak current (I) using Ohm’s law

[G = I ⁄ (Vp ) EK)]. Vp stands for the test voltage value,

and the predicted Nernst K+ equilibrium potential (EK) is

)83 mV.

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N-glycosylation promotes the cell surface expression of Kv1.3 potassium channels

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