However, whole cell currents of N220Q⁄ N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1.. However, whole cell cur
Trang 1in the Kv3.1 channel
Natasha L Brooks, Melissa J Corey and Ruth A Schwalbe
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, NC, USA
Voltage-gated K+ channel (Kv3.1) plays a
fundamen-tal role in neuronal excitability and lymphocyte
differ-entiation [1–6], and belongs to the Kv3 subfamily of
the voltage-gated K+ channel (Kv) supergene family
[7] Upon stimulation, the voltage-dependent gate
opens and potassium ions flow out of the cell,
indu-cing negative intracellular voltage, and termination of
excitation [8] Based on hydropathy plots, Kv3.1 has
six transmembrane segments (S1–S6) and cytoplasmic
N- and C-termini (Fig 1A) The segments between S1–S2 and S3–S4 are extracytoplasmic loops, and those between S2–S3 and S4–S5 are cytoplasmic loops
Each of the Kv3.0 family members and their splice variants contain two conserved, native N-glycosylation sites in the S1–S2 linker Rat and human Kv3.1 pro-teins have two native N-glycosylation consensus sequences running from amino acid residues 220 to
Keywords
brain; glycosylation; K + channel; topology,
trafficking
Correspondence
R A Schwalbe, Department of
Biochemistry and Molecular Biology, Brody
School of Medicine at East Carolina
University, 600 Moye Boulevard, Greenville,
NC 27834, USA
Fax: +1 252 744 3383
Tel: +1 252 744 2034
E-mail: schwalber@mail.ecu.edu
(Received 25 August 2005, revised 18 May
2006, accepted 23 May 2006)
doi:10.1111/j.1742-4658.2006.05339.x
N-Glycosylation is a cotranslational and post-translational process of pro-teins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1) Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin Also, par-tially purified proteins were digested with either PNGase F or endoglycosi-dase H Attachment of simple-type oligosaccharides at positions 220 and
229 was directly shown by single (N229Q and N220Q) and double (N220Q⁄ N229Q) Kv3.1 mutants Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane Unitary conductance of N220Q⁄ N229Q was similar to that of the wild-type Kv3.1 However, whole cell currents of N220Q⁄ N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1 The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q⁄ N229Q These results demonstrate that the S1–S2 linker is topologically extracellular, and that N-glycosylation influen-ces the opening of the voltage-dependent gate of Kv3.1 We suggest that occupancy of the sites is critical for folding and maturation of the func-tional Kv3.1 at the cell surface
Abbreviations
CDGS, carbohydrate-deficient glycoprotein syndromes; Endo H, endoglycosidase H; ER, endoplasmic reticulum; G–V plot, conductance– voltage plot; Kv, voltage-gated K + channel; KvAP, voltage-gated K + channel of Aeropyrum pernix; PM, plasma membrane; PNGase F, peptide N glycosidase F; Sf9, Spodoptera frugiperda; TM, tunicamycin.
Trang 2222 (NKT) and from 229 to 231 (NGT) in the S1–S2
linker, and they share 100% sequence identity in this
region (Fig 1B) A recent X-ray structure of a Kv
from Aeropyrum pernix (KvAP) suggested that the
S1–S2 linker resides in the membrane for all Kvs [9]
Comparison between the S1–S2 linker of KvAP and
mammalian Kvs may be difficult because of differences
in the length of their S1–S2 linkers and the
N-glycosy-lation consensus sequences within this segment for
Kv3.0s and Kv1.0s (Fig 1C)
N-Glycosylation is a cotranslational and
post-trans-lational modification found on extracellular segments
of membrane proteins and is important for protein
maturation, trafficking, and function [10–13] The
N-glycosylation consensus sequence is AsnXxxSer⁄ Thr,
where the central residue cannot be a Pro residue
Membrane protein segments are only glycosylated
when they are translocated to the luminal side of the
endoplasmic reticulum (ER) membrane, and therefore
occupancy of an N-glycosylation site designates a
region of extracellular topology [11,12] Defects in the
attachment of oligosaccharides to protein give rise to
mental and psychomotor retardation, dimorphisms,
and blood coagulation defects [14,15]
Carbohydrate-deficient glycoprotein syndromes (CDGS) I–IV are a
group of disorders characterized by the presence of
abnormal oligosaccharides on many glycoproteins
[16,17] The occurrence of CDGS emphasizes that
proper glycosylation of both membrane and secretory
glycoproteins are essential for normal development
and health [18,19] More recently, it has been sugges-ted that ER stress is linked to several human
neuron-al diseases [20], and therefore it may be that abnormal glycosylation processing of proteins contri-butes to these diseased states as well
Here we have examined whether the native N-gly-cosylation sites are utilized in rat brain and infected Sf9 cells, and the role that occupancy of these sites has in the expression of functional Kv3.1s at the cell surface of Sf9 cells Immunoband patterns of wild-type Kv3.1, N220Q⁄ N229Q, N229Q, and N220Q, in the absence and presence of tunicamycin (TM), endo-glycosidase H (Endo H), or peptide N endo-glycosidase F (PNGase F), revealed that both sites in Kv3.1 were occupied by N-linked oligosaccharides Patch clamp measurements and cell fractionation showed that the unglycosylated Kv3.1, N220Q⁄ N229Q, is targeted to the plasma membrane, like wild-type Kv3.1 However, whole cell currents of N220Q⁄ N229Q revealed slower activation kinetics and a small positive shift in voltage dependence compared to wild-type Kv3.1 The voltage dependence of activation for the partially glycosylated Kv3.1s, N229Q and N220Q, appeared similar to that of N220Q⁄ N229Q Our findings demonstrate that the S1–S2 linker of Kv3.1 is in an extracellular aqueous environment Additionally, they demonstrate that N-glycosylation influences the open-ing of the voltage-dependent gate of Kv3.1, suggest-ing that vacant sites alter the foldsuggest-ing and maturation
of Kv3.1 at the cell surface
220 229
S1 S2 S3 S4 S5 S6
A
Rat 210 ETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTY
Human 210 ETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTY
B
Kv3.1 P25122 210 ETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTY
Kv1.1 P10499 187 ETLPELKDDKDFTGTIHRIDNTTVIYTSNIFTDP
Kv1.2 P63142 183 ETLPIFRDENEDMHGGGVTFHTYSNSTIGYQQSTSFTDP
Kv1.4 P15385 329 ETLPEFRDDRDLIMALSAGGHSRLLNDTSAPHLENSGHTIFNDP
Kv1.5 P19024 261 ETLPEFRDERELLRHPPVPPQPPAPAPGINGSVSGALSSGPTVAPLLPRTLADPF
KvAP Q9YDF8 64 SGEY
C
Fig 1 Topological model of Kv3.1 and amino acid sequences of the S1–S2 linker
of Kvs (A) Topology of a Kv3.1 monomeric unit Black circles represent the Asn of native N-glycosylation consensus sites N220 and N229 Branched structures represent the attachment of oligosaccharide at native sites (B) Sequence identity between Kv3.1 S1–S2 linkers from rat (P25122) and human (P48547) (C) Comparison of the S1–S2 amino acid sequence of eukaryotic Kvs and prokaryotic KvAPs The Kv name corres-ponding to the adjacent S1–S2 amino acid sequence is indicated in bold and is fol-lowed by the accession number Conserved, native N-glycosylation sites are shown as underlined font The italicized number indi-cates the first residue of the S1–S2 linker.
Trang 3Occupancy of the two native N-glycosylation
sites
Rat brain membranes were digested with PNGase F,
and then analyzed by western blotting PNGase F is
an enzyme that removes a wide range of N-linked
oligosaccharides from proteins [21] Native Kv3.1
migrates as a diffuse immunoband (about 109 kDa)
which is much larger than its calculated molecular
mass of 66 kDa (Fig 2) This migration pattern of
native Kv3.1 suggests that the protein undergoes a
cotranslational or post-translational modification To
show that the modification was indeed a result of
attachment of N-linked oligosaccharides, rat brain
membranes were incubated with PNGase F The
Kv3.1 immunoband (about 81 kDa) migrated much
faster, indicating that Kv3.1 undergoes N-glycosylation
in rat brain membranes To further verify specificity of
the Kv3.1b antibody, membranes isolated from Sf9
cells infected with recombinant baculovirus that
enco-ded the Kv3.1b cDNA were immunoblotted (Fig 2)
The electrophoretic migration pattern of wild-type
Kv3.1 revealed a predominant immunoband at about
87 kDa and two lower faint bands The lowest band
migrated to about 77 kDa, and the middle band was
at about 81 kDa Only the two lowest bands were
detected when Sf9 cell membranes were treated with
PNGase F, suggesting that the top two bands are
gly-cosylated protein Taken together, these results
demon-strate that Kv3.1 is N-glycosylated in rat and insect
cells, and that the type of N-linked oligosaccharide differs
To directly demonstrate that both of the absolutely conserved N-glycosylation consensus sequences were utilized, they were removed independently (N229Q and N220Q) and simultaneously (N220Q⁄ N229Q) by conserved substitutions of the Asn residues with Gln residues In addition, an M2 FLAG epitope was attached to the C-terminus and was utilized for purifi-cation and identifipurifi-cation of the various Kv3.1 proteins Wild-type Kv3.1, N229Q, N220Q and N220Q⁄ N229Q were M2 immunoaffinity purified from whole cell lysates of Sf9 cells infected in the absence and presence of TM, and then immunoblotted using
_
Anti-Kv3.1b rat brain
membranes
Sf9 cell membranes
PNGase F:
Fig 2 N-Glycosylation of Kv3.1 in rat brain tissue and Sf9 cell
membranes Rat brain membranes and partially purified Sf9
pro-teins were untreated (–) or treated (+) with PNGase F, resolved by
SDS ⁄ PAGE and immunoblotted, as indicated The arrows of each
panel indicate migration of glycosylated (upper arrow) or
unglycosyl-ated (lower arrow) Kv3.1 protein Ovals represent KaleidoscopeTM
protein standards (top to bottom in kDa): 250, 150, 100, and 75.
Endo H:
TM:
N229Q
Kv3.1:
Anti-FLAG
Wt N229Q N220Q N220Q/N229Q
Kv3.1:
TM:
A
B
Anti-Kv3.1b
Fig 3 Detection of high-mannose oligosaccharides in Sf9 cell membranes Sf9 cells were infected with recombinant baculovirus containing wild-type Kv3.1, N229Q, N220Q or N220Q ⁄ N229Q in either the absence (–) or the presence (+) of 25 lgÆmL)1 tunicamy-cin (TM) Proteins were transferred and probed with anti-Kv3.1b (A)
or anti-FLAG (B, upper panel) Partially purified Kv3.1 protein was treated (+) with endoglycosidase H (Endo H) (B, lower panel) The arrows in each panel indicate migration of fully glycosylated (upper), partially glycosylated (middle) or unglycosylated (lower) Kv3.1 protein In all cases, proteins were partially purified from whole cell lysates using M2-agarose Ovals represent molecular mass stand-ards (top to bottom in kDa): 250, 150, 100, and 75.
Trang 4Kv3.1b (Fig 3A) and M2 anti-FLAG (Fig 3B) TM
inhibits the oligosaccharyltransferase that carries out
the initial step of the N-glycosylation pathway in the
ER lumen [22] As mentioned above, wild-type Kv3.1
migrates as three immunobands, with the upper band
as the predominant band When Sf9 cells expressing
wild-type Kv3.1 were treated with TM, only the lowest
immunoband was observed The single Kv3.1 mutants,
N229Q and N220Q, migrated as doublets which
appear to correspond to the lower two immunobands
of wild-type Kv3.1 In both cases, the upper band was
darker than the lower band Additionally, the upper
band was not visible in the presence of TM A single
immunoband was detected for N220Q⁄ N229Q, which
migrated to a similar position as the lowest faint band
observed for wild-type Kv3.1 and the lower faint band
of the single mutants, and furthermore, the
immuno-band did not shift in the presence of TM To verify
that wild-type Kv3.1 was modified by a high-mannose
oligosaccharide typical of Sf9 cells, not a complex
oligosaccharide [23], N-linked oligosaccharide was
removed by Endo H treatment of partially purified
Kv3.1 protein (Fig 3B) When partially purified
wild-type Kv3.1, N229Q and N220Q proteins were digested
with Endo H, the lowest band becomes the
predomin-ant form in all three instances The band observed for
N220Q⁄ N229Q does not shift in the presence of Endo
H These results indicate that the upper band of
wild-type Kv3.1 represents the situation when both
glycosylation sites are occupied by high-mannose-type
oligosaccharides, the middle band represents one
occu-pied site, and the lowest band is the unglycosylated
monomer
Glycosylated and unglycosylated forms of Kv3.1
are targeted to the plasma membrane
Infected Sf9 cells expressing wild-type Kv3.1 and
N220Q⁄ N229Q were fractionated into three distinct
fractions [24,25] Subsequently, Kv3.1 protein was M2
immunoaffinity purified from each fraction (Fig 4A)
A predominant immunoband was detected for
wild-type Kv3.1 in all three distinct fractions Two faint
lower bands were clearly observed in the ER fraction,
while in the other two fractions only the lower faint
band was observed The totally unglycosylated form of
Kv3.1, generated by mutating Asn residues at positions
220 and 229 to Gln residues (N220Q⁄ N229Q, Fig 4A)
or by treating Sf9 cells expressing wild-type Kv3.1 with
TM (Fig 4B), was also observed in the plasma
mem-brane These results indicate that N-glycosylation is
not required to transport Kv3.1 to the plasma
mem-brane
Functional unglycosylated Kv3.1 is at the cell surface
Whole cell currents of infected Sf9 cells expressing either wild-type Kv3.1 or N220Q⁄ N229Q were observed when the membrane potential was depolar-ized beyond ) 10 mV and current amplitudes reached saturation at membrane potentials beyond +40 mV (Fig 5A,B, top panel) The patterns of these inactivat-ing, voltage-dependent K+ currents were typical of a delayed rectifier, and were similar to those expressed
by wild-type Kv3.1 in Xenopus oocytes [1,3,4,26–28] and other heterologous expression systems [29–31] To show that channel densities at the cell surface for wild-type Kv3.1 (Imax⁄ cap is 140 ± 29 pA ⁄ pF, n ¼ 13) and N220Q⁄ N229Q (Imax⁄ cap is 156 ± 36 pA ⁄ pF, n ¼ 11) were comparable, the maximum current amplitude was determined and divided by the cell capacitance Differ-ences between the two forms could be identified when the voltage dependence for channel activation was analyzed The membrane conductance vs applied test potential indicated that more depolarization was required for 50% of the N220Q⁄ N229Q channels (V0.5 [test potential at which g/gmax ¼ 0.5] is 20.5 ± 0.6 mV, n¼ 11) to reach activation than for wild-type
Kv3.1:
A
B
Wt Kv3.1 +TM
Kv3.1:
Fig 4 Glycosylated and unglycosylated Kv3.1 proteins are deliv-ered to the plasma membrane (A) Plasma membrane (PM), Golgi apparatus (Golgi) and endoplasmic reticulum (ER) fractions from Sf9 cells infected with the indicated Kv3.1 baculovirus were isola-ted by sucrose density gradients Protein was M2-affinity agarose purified from each fraction and immunoblotted (B) M2-agarose affinity purified Kv3.1 protein from membrane fractions of Sf9 cells expressing wild-type Kv3.1 in the presence of 25 lgÆmL)1 tunica-mycin (TM) The top two arrows denote where glycoforms would
be and the bottom arrow represents the aglycoform.
Trang 5Kv3.1s (V0.5 is 16.6 ± 0.7 mV, n¼ 13) Additionally,
slightly fewer channels were activated as the applied
voltage was increased for unglycosylated Kv3.1 (slope
of normalized current voltage relationship, dV, is
9.3 ± 0.4 mV for N220Q⁄ N229Q, n ¼ 11) than for
glycosylated Kv3.1 (dV is 8.1 ± 0.4 mV for wild-type
Kv3.1, n¼ 13) A range of values for Vm0.5 from
10 mV to 18 mV, and for dV from 8 mV to 11 mV, of
wild-type Kv3.1 have previously been reported in
various heterologous expression systems [30]
The activation kinetics of wild-type Kv3.1
expressed in vitro and in vivo is quite rapid [30,31]
When the whole cell current tracings were normalized
at each potential from + 20 mV to + 50 mV for
wild-type Kv3.1 and N220Q⁄ N229Q, and then placed
on top of each other, it was observed that the
activation kinetics were somewhat slower for
N220Q⁄ N229Q than for wild-type Kv3.1 (Fig 6A)
25 ms
25 ms
Voltage (mV)
C
0.0
0.2
0.4
0.6
0.8
1.0
Fig 5 Functional expression of wild-type Kv3.1 and the
N220Q ⁄ N229Q mutant Whole cell currents were produced by
depolarizing voltage pulses from a holding potential of ) 50 mV to
levels ranging from ) 40 to +100 mV in 10 mV increments
Repre-sentative tracings are shown from Sf9 cells infected with (A)
wild-type Kv3.1 and (B) N220Q ⁄ N229Q (C) Corresponding Boltzmann
plots Wild-type Kv3.1 (V 0.5 ¼ 16.6 ± 0.7, dV ¼ 8.1 ± 0.4, n ¼ 13)
data are represented by (d) and N220Q ⁄ N229Q (V0.5¼
20.56 ± 0.6, dV ¼ 9.3 ± 0.4, n ¼ 11) data are represented by n.
The Boltzmann isotherm G ¼ G max ⁄ [1 + exp(V 0.5 ) V) ⁄ q] was used
to fit the data, which represent ± SEM.
A
25 ms
B
0 10 20 30 40 50 60
Voltage (mV)
0 10 20 30 40
Voltage (mV)
C
Fig 6 Comparison of activation rates in wild-type Kv3.1 and N220Q ⁄ N229Q (A) Whole cell currents from Sf9 cells expressing wild-type Kv3.1 (solid line) and N220Q ⁄ N229Q (dashed line) were normalized at 20 mV (red), 30 mV (blue), 40 mV (purple) and 50 mV (black), and the resulting normalized currents were placed on top of each other (B) Rise times and (C) activation time constants are shown for wild-type Kv3.1 (d) and N220Q ⁄ N229Q (n) Rise times represent the time required for the current to rise from 10% to 90% of its peak current at the indicated applied voltage Activation time constants were determined by fitting the current traces at each potential to a single exponential Data represent SEM.
Trang 6In both cases, the activation time decreased as the
applied potential increased, which indicates the
volt-age dependence of channel activation The time for
the current to rise from 10% to 90% of its maximum
value was less for N220Q⁄ N229Q than for wild-type
Kv3.1 at the various applied potentials (Fig 6B)
Time constants for activation at similar potentials
were also determined by fitting each current with a
single exponential (Fig 6C) Again, it was
demonstra-ted that the activation rate for N220Q⁄ N229Q is
slower than that for wild-type Kv3.1 The
deactiva-tion kinetics of wild-type Kv3.1 (time constant
deacti-vation, soff is 2.4 ± 0.9 at ) 40 mV, n ¼ 3) and
N220Q⁄ N229Q (soff is 3.6 ± 0.3 at ) 40 mV, n ¼ 3)
were rapid, and similar to those previously reported
in heterologous expression systems and neurons [31]
These results indicate that differences in the voltage
dependence of channel activation can be measured
between the glycosylated Kv3.1 (wild-type Kv3.1) and
its unglycosylated counterpart (N220Q⁄ N229Q)
Previously, it has been reported that whole cell
current recordings of wild-type Kv3.1 in mammalian
expression systems display little saturation in current
amplitude in response to large depolarization steps
[29,32,33] This noninactivating type of behavior was
also observed for both glycosylated and
unglycosylat-ed Kv3.1s expressunglycosylat-ed in Sf9 cells (Fig 7A,B) but
occurred less often than the inactivating currents In
the case of the noninactivating behavior, the channel
densities for wild-type Kv3.1 (Imax⁄ cap is 368 ± 29
pA⁄ pF, n ¼ 11) and N220Q ⁄ N229Q (Imax⁄ cap is
314 ± 50 pA⁄ pF, n ¼ 9) were quite similar
How-ever, both forms of Kv3.1 had higher channel
densi-ties than those that had inactivating behavior Like
the cells that expressed the inactivating type of
behavior for wild-type Kv3.1 and N220Q⁄ N229Q,
the rise times at the various potentials were slower
for the unglycosylated Kv3.1 than for glycosylated
Kv3.1 (Fig 7C) Moreover, the rise times were faster
in those cells that expressed the noninactivating type
of behavior than in those that expressed the
inacti-vating type of behavior for either wild-type Kv3.1 or
N220Q⁄ N229Q
Single-channel recordings of wild-type Kv3.1 and
N220Q⁄ N229Q have long openings, and long and brief
closures (Fig 8A,B) Current amplitudes and unitary
conductances of wild-type Kv3.1 and N220Q⁄ N229Q
were virtually identical (Fig 8C), and quite similar to
those in previous reports of wild-type Kv3.1 in
Xenopus oocytes [26,28] and mammalian cells [29,32]
These results indicate that the current amplitudes and
single-channel conductances are similar for
glycosylat-ed and unglycosylatglycosylat-ed Kv3.1s
Partially glycosylated Kv3.1 mutants at the cell surface are functional
The single N-glycosylation Kv3.1 mutants (N229Q and N220Q) expressed whole cell currents at applied poten-tials of ) 10 mV, and current amplitudes increased as the applied potential increased until currents reached saturation at membrane potentials beyond + 40 mV (Fig 9A,B) The Boltzmann equation indicates that
a little more depolarization is required to activate 50% of the partially glycosylated Kv3.1s (V0.5 is 18.7 ± 1.2 mV and 20.9 ± 1.3 mV for N229Q and N220Q, respectively, n¼ 5) than for the fully glycosyl-ated Kv3.1 (wild-type Kv3.1; Fig 9C) The conduct-ance–voltage (G–V) slopes for N229Q (dV is 8.7 ± 0.6 mV, n¼ 5) and N220Q (dV is 9.0 ± 0.7 mV,
n¼ 5) appear to be more similar to those for N220Q⁄ N229Q than wild-type Kv3.1, but they were not statistically different As for the N220Q⁄ N229Q channel (rise times at + 20 mV, 50.5 ± 3.3 ms, and + 40 mV, 29.6 ± 2.6 ms, n¼ 10; and activation time constants
at + 20 mV, 27.7 ± 3.1 ms, and + 40 mV, 16.0 ±
0 10 20 30 40
Voltage (mV)
C
Fig 7 Whole cell analysis of noninactivating currents from wild-type Kv3.1 and N220Q ⁄ N229Q Representative tracings are shown from Sf9 cells infected with (A) wild-type Kv3.1 and (B) N220Q ⁄ N229Q (C) Rise times are shown for wild-type Kv3.1 (n ¼
11, d) and N220Q ⁄ N229Q (n ¼ 8, n) Data represent ± SEM.
Trang 71.2 ms, n¼ 11), the activation kinetics of N229Q (rise
times at +20 mV, 54.9 ± 6.4 ms, and +40 mV,
26.3 ± 2.3 ms; and activation time constants at
+20 mV, 29.6 ± 5.6 ms, and +40 mV, 12.8 ± 1.0 ms,
n¼ 5) and N220Q (rise times at +20 mV,
54.5 ± 5.2 ms; and +40 mV, 34.4 ± 2.8 ms; and
acti-vation time constants at +20 mV, 30.8 ± 3.8 ms, and
+40 mV, 16.7 ± 0.9 ms, n¼ 5) were slower than those
of wild-type Kv3.1 (rise times at +20 mV,
41.0 ± 2.7 ms and +40 mV, 19.4 ± 1.1 ms; and
acti-vation time constants at +20 mV, 20.4 ± 1.6 ms, and
+40 mV, 10.5 ± 0.8 ms, n¼ 13) These results
indi-cate that the absence of one N-linked
high-mannose-type oligosaccharide can produce small changes in the
voltage dependence of channel activation
Discussion
The findings reported here demonstrate that both of
the conserved N-glycosylation consensus sequences, in
the S1–S2 linker, of Kv3.1 can be occupied by various types of oligosaccharide Three distinct immunobands were identified for wild-type Kv3.1 expressed in Sf9 cells The upper band was the predominant band, and the two lower bands were of minor intensity (Fig 3)
In the presence of PNGase F, Endo H, or TM, the upper band was no longer observed When digestion was complete or cells expressing wild-type Kv3.1 were treated with TM, only the lowest faint immunoband was observed (Fig 3) In addition, when the conserved N-glycosylation sites were removed either independ-ently or together, the immunobands corresponded to the two faint bands of wild-type Kv3.1 or the lowest faint band, respectively These results indicate that the most prominent band of wild-type Kv3.1 expressed in insect cells represents occupancy of both glycosylation sites by simple oligosaccharides, the upper faint band represents occupancy of one site by a simple oligosac-charide, and the lowest faint band represents vacancy
of both sites
A
+60 mV
+80 mV
+100 mV
100 ms
1 pA
O C
1
2
3
4
C
Voltage (mV)
Fig 8 Single-channel analysis of wild-type Kv3.1 and N220Q ⁄ N229Q Representative single-channel recordings from infected Sf9 cells expressing (A) wild-type Kv3.1 (d) and (B) N220Q ⁄ N229Q (n) at various test potentials as indicated The open state of the channel is indica-ted by O and the closed state is represenindica-ted by C (C) Current–voltage relationship of wild-type Kv3.1 and N220Q ⁄ N229Q Single-channel conductance was 27 pS (n ¼ 8) for wild-type Kv3.1 and 24 pS (n ¼ 6) for N220Q ⁄ N229Q Open circles denote wild-type Kv3.1 and closed circles represent N220Q ⁄ N229Q Linear regression fit of the data was performed with a dashed line for wild-type Kv3.1 and a solid line for N220Q ⁄ N229Q Data represent ± SEM.
Trang 8On the basis of conservation of initial steps in the
N-glycosylation pathway and divergence of this
path-way following synthesis of the common N-glycan
inter-mediate, GlcNAc2Man3GlcNAc2-N-Asn, in insects and
mammals [34], we would expect Kv3.1 to be
glycosyl-ated in native tissue A diffuse immunoband was
observed in rat brain membranes that migrated much
slower than would be expected from its calculated
molecular mass, or that detected in Sf9 cells (Fig 2)
Previous studies are in agreement that Kv3.1 in rat
brain migrates much slower than would be expected
from its predicted molecular mass [35], and
further-more, its migration appears to differ in various regions
of the brain [36,37] This slow migration pattern of
Kv3.1 was not shown to be due to N-glycosylation
Our results show that digestion of rat brain
mem-branes with PNGase F produces a much
faster-migra-ting band that moves to a similar position as
unglycosylated Kv3.1 expressed in Sf9 cells Taken
together, we conclude that Kv3.1 isolated from rat
brain is N-glycosylated (Fig 2), and the
oligosaccha-rides are of either hybrid or complex type in composi-tion Additionally, it may be that the composition of N-linked oligosaccharides is different in various regions of the brain
Many glycosylation studies have indicated that in order for the oligosaccharyltransferase to have access
to an N-glycosylation consensus sequence of a mem-brane protein, the segment containing the tripeptide sequence must enter the lumen of the ER, which becomes the extracellular segment of the protein once
it is transferred to the plasma membrane [11,38,39] Additionally, if the site is within an extracytoplasmic loop, the segment must be larger than 30 residues, and this site must be at least 11 residues away from the membrane Utilization of a site is also greater at an earlier time point during protein synthesis In conjunc-tion, glycosylation of sites at positions 220 and 229 of Kv3.1 confirms the extracellular placement of the S1– S2 linker identified by hydropathy plots (Fig 1A) This finding is also in agreement with utilization of the native glycosylation site in the S1–S2 linker of other Kvs (Fig 1C) For example, the native glycosylation site in the S1–S2 linker of Shaker H4 [40], Kv1.1 [41], Kv1.2 [42], Kv1.4 [43] and Kv1.5 [44] were shown to
be occupied by N-linked oligosaccharides Addition-ally, utilization of introduced glycosylation sites throughout the S1–S2 segment of Kv1.2 suggested that the majority of this segment resides in the extracellular aqueous environment and that its conformation is flex-ible [42] The Kv3.1 results demonstrated that both glycosylation sites in the S1–S2 linker were utilized, indicating that both of these regions can accommodate
a conformation accessible to the oligosaccharyltransf-erase Moreover, the large hydrophilic oligosaccharides attached to Asn220 and Asn229 would place the entire region of the S1–S2 linker outside the lipid bilayer The structural model of bacterial KvAP places this segment in a lipid environment, and suggests that this was the case for all Kvs [9] It is possible that the S1– S2 linker may reside in the membrane of a bacterial
Kv, which would not undergo N-glycosylation, but it
is unlikely that this orientation applies to all
eukaryot-ic Kvs The S1–S2 linker of KvAP is very short (four residues) compared to the longer linkers of Kv3.1 (38 residues), Kv1.1 (34 residues), Kv1.2 (39 residues), Kv1.4 (44 residues) and Kv1.5 (55 residues) (Fig 1C) Therefore, this region in the bacterial channel is not very comparable to that of eukaryotes The aforemen-tioned glycosylation reports, along with our report, provide strong evidence that the conserved S1–S2 linker of Kv1.1, Kv1.2, Kv1.4 and Kv1.5, along with that of Kv3.1, is topologically extracellular The reports also suggest that the orientations of the S1 and
C
0.0
0.2
0.4
0.6
0.8
1.0
Voltage (mV)
25 ms
A
25 ms
N229Q
Fig 9 Functional expression of N-glycosylation single mutants.
Whole cell currents are shown for Sf9 cells infected with (A)
N229Q and (B) N220Q (C) Corresponding Boltzmann plots for
N229Q ( solid line; V0.5¼ 18.7 ± 1.2, dV ¼ 8.7 ± 0.6, n ¼ 5) and
N220Q (m dashed line; V 0.5 ¼ 20.9 ± 1.3, dV ¼ 9.0 ± 0.7, n ¼ 5)
are shown here.
Trang 9S2 segments are similar in all eukaryotic Kv1.0 and
Kv3.0 subfamilies, but not necessarily the same
between prokaryotic and eukaryotic domains
N-Glycosylation, a cotranslational and
post-transla-tional process, of proteins may influence protein
fold-ing, maturation, stability, traffickfold-ing, and consequently
cell surface expression of functional channels [10–12]
Cell fractionation results of unglycosylated Kv3.1
produced by elimination of the two native sites,
N220Q⁄ N229Q mutant, or by treating cells expressing
wild-type Kv3.1 with TM, demonstrated that targeting
to the plasma membrane was not abolished In
addi-tion, whole cell current measurements of
unglycosylat-ed Kv3.1 (N220Q⁄ N229Q) and partially glycosylated
Kv3.1 (N229Q and N220Q) show that they form
func-tional channels at the cell surface These results
indi-cate that N-glycosylation is not required for transport
of Kv3.1 to the cell surface, and thus suggest that the
protein can fold and assemble into a stable functional
homomultimer However, the small changes in the
acti-vation kinetics suggest that when the sites are vacant,
Kv3.1 is folded slightly different in its mature structure
compared to when the sites are occupied Previous
studies of Kv1.4 have also demonstrated that
glycosy-lation of the native site in the S1–S2 linker was
required for proper trafficking and stability [43] On
the other hand, glycosylation of the native site in the
S1–S2 linker of Kv1.0s, including Shaker [40] and its
mammalian homologue, Kv1.1 [41], did not affect cell
surface expression
In this article, we demonstrate that the voltage
dependence of Kv3.1 activation is altered by the
absence of N-linked oligosaccharides More
depolar-ization (about 6 mV) was required to activate 80% of
the unglycosylated Kv3.1s (N220Q⁄ N229Q; V>80% is
38 mV) than to activate 80% of the glycosylated
Kv3.1s (wild-type Kv3.1; V>80% is 32 mV)
Addition-ally, the fraction of channels that were activated as the
applied voltage increased was significantly lower for
unglycosylated Kv3.1s than for their glycosylated
counterparts The time required for the unglycosylated
Kv3.1s to reach their peak current was also less at the
various applied potentials relative to the glycosylated
Kv3.1s Activation kinetic values of the partially
gly-cosylated Kv3.1s (N229Q and N220Q) appeared to be
more similar to those of unglycosylated Kv3.1 than to
that of the glycosylated channel Thus, these results
indicate that the occupancy of the N-glycosylation sites
is a determining factor for the voltage-dependent
acti-vation kinetics of Kv3.1
A recent report on Kv1.1 indicated that
N-glycosyla-tion did alter its gating funcN-glycosyla-tion, and this effect was
shown to result from sialic acid residues attached to
the oligosaccharide [45] It was suggested that when the composition of the N-linked oligosaccharide was
of a simple type, there was a positive shift in voltage dependence of activation and slower activation kinetics than when the oligosaccharide was of a complex type [45,46] Therefore, it is possible that the maturation processing of the high-mannose glycan of Kv3.1 to a complex carbohydrate may cause a negative shift in voltage dependence, and an increase in the rate of acti-vation When the channel activation of wild-type Kv3.1s expressed in Sf9 cells is compared to that expressed both in vivo and in mammalian heterologous expression systems (V>80% is 30 mV; ton at +40 mV
is 3.4 ms; ton at +20 mV is 3–4 ms), the voltage dependence of activation appears to be different [31,47] The results of the aforementioned studies of Kv3.1, along with our report, would suggest that the composition of the N-linked oligosaccharides may influence the voltage dependence of Kv3.1 activation
in a similar manner as occupancy of the glycosylation sites
In conclusion, our study indicates that decreases in the occupancy of N-glycosylation sites that may occur
in patients suffering from CDGS [15,19] or ER stress-related neurodegenerative diseases [20] could alter the expression of K+ currents at the cell surface of neu-rons that express Kv3.1 at high densities Future stud-ies will be needed to determine whether different compositions of the N-linked oligosaccharide alter the channel activation of Kv3.1
Experimental procedures
Materials
PharMingen, San Diego, CA, USA Hink’s TNM-FH insect medium was bought from MediaTech, Inc., Herndan,
VA, USA, FBS was from Invitrogen, Carlsbad, CA, USA, and Pluronic F-68 solution, as well as gentamicin, came from Sigma Chemical Co., St Louis, MO, USA The TA cloning kit and restriction enzymes were acquired from Invitrogen The BaculoGold transfection kit was purchased from BD Biosciences, San Diego, CA Plasmid purification columns were obtained from Qiagen, Valencia, CA, USA Anti-FLAG M2-agarose gel and mouse anti-FLAG M2 were purchased from Sigma, and goat anti-mouse IgG, alkaline phosphatase-conjugated, was purchased from MP Biomedicals, Inc., Irvine, CA, USA Rabbit anti-Kv3.1b was purchased from Alomone Laboratories, Jerusalem, Israel Protease inhibitor cocktail set III and Triton X-100 were from CalBiochem, San Diego, CA, USA Precast
Trang 10CA, USA Ultracentrifuge tubes for SW41 and 70Ti rotors
were purchased from Beckman, Palo Alto, CA, USA All
other chemicals used in this study were ordered from Sigma
or Fisher Scientific, Co., Hampton, NH, USA
Mutant constructs
PCR was used to attach the FLAG sequence
(DY-KDDDDK) to the 3¢-end of Rattus rattus Kv3.1 cDNA
(accession number P25122), referred to as 3¢FLAG-Kv3.1
The 3¢FLAG-Kv3.1–pCRII recombinant vector was kindly
provided by B Wible and A M Brown (Rammelkamp
Center for Education and Research, Case Western
was constructed by PCR overlap extension [48] using
3¢FLAG-Kv3.1–pCRII as template Forward and reverse
primers were designed to contain nucleotide mismatches
that eliminated the two native N-glycosylation sites at
posi-tions 220–222 (NKT) and 229–231 (NGT) of Kv3.1 cDNA
PCR products were subcloned into pCR2.1 for
sub-cloned into EcoRI-digested baculovirus transfer vector,
pACSG2 To generate the N220Q and N229Q single
mutants, the QuikChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA, USA) was used (manufacturer’s
protocol) Mutagenic forward and reverse primers were
designed to contain nucleotide mismatches that eliminated
either the first native N-glycosylation site at position
220–222 (NKT) or the second native site at position
229–231 (NGT) of Kv3.1 cDNA, respectively The dsDNA
template was Kv3.1-pacSG2 DNA sequences were verified
Standard procedures were followed for subcloning, and
DNA amplification, isolation and sequencing [49]
Cell culture and recombinant baculoviruses
Sf9 cells were maintained in Hink’s TNM-FH medium
Mono-layer Sf9 cultures were used to maintain Sf9 cells and were
passaged about twice a week Suspension cultures of Sf9
cells were seeded from monolayer cultures and stirred at a
constant rate of 80–120 r.p.m Fresh suspension cultures
were prepared every 3–5 days Recombinant baculoviruses
were produced by cotransfection of recombinant
Baculovi-rus transfer vectors and BaculoGold viral DNA [modified
(AcNPV)] The manufacturer’s instructions were followed
for this procedure (BD Biosciences) Viral seed stocks of
intermediate viral titer were generated using monolayer
cul-tures High viral titers were produced in suspension cultures
intermediate viral titer supernatant Expression of
recom-binant proteins required the addition of high viral titer
necessary for studying the occupancy of N-glycosylation
postinoculation
Cell fractionation and M2-agarose affinity purification
Sf9 cell fractionations were carried out as previously des-cribed [24,25] Adjustments of the cell fractionation proto-col involved reducing the starting material and the volume
of the sucrose layers but maintaining the relative ratios of
) were harvested by centrifugation at 1204 g in a Beckman SX
NaCl) at pH 7.4, and recentrifuged under the same
lyse the cells Ice-cold homogenizing buffer (250 mm
cocktail set III from Calbiochem; added volume, about 1.8 mL) was used to resuspend the thawed cell pellet The cells were disrupted in a dounce homogenizer (continuous strokes for at least 10 min) and centrifuged at 500 g for
Eppendorf 45-30-11 rotor, unless otherwise specified An equal volume of sucrose adjustment buffer (2.55 m sucrose,
10 mm Tris, 1 mm EDTA, pH 7.4) was added to the cleared lysate to increase the sucrose concentration to 1.4 m The sucrose gradient [2.0 m sucrose, 920 lL; 1.6 m sucrose, 1840 lL; 1.4 m sucrose (sample), about 3680 lL; 1.2 m sucrose, 3680 lL; 0.8 m sucrose, 1840 lL] was pre-pared and centrifuged at 83 472 g in a Beckman SW41
plasma membrane (PM), Golgi apparatus (Golgi), and ER fractions were removed and added to ultracentrifuge tubes, where they were diluted by addition of about 10 mL of imi-dazole buffer (25 mm imiimi-dazole, 1 mm EDTA, pH 7.4), and centrifuged at 117 734 g in a Beckman 70Ti rotor for 1.5 h to concentrate the fractions The pellet was solubilized
KCl, pH 7.4, 0.5% Triton X-100) and transferred to micro-centrifuge tubes The tube was rinsed with 800 lL of pellet resuspension buffer to recover any remaining sample Next,
M2-ag-arose gel slurry) was added to each solubilized sample (total volume, about 1 mL) and incubated on a rotator for 1 h at room temperature The resin was washed three times with
pH 7.4) by centrifugation at 425 g for 3 min The resin was
(same as previous step), and the washed resin was resus-pended in 100–125 lL of 2· SDS ⁄ PAGE sample buffer (62.5 mm Tris, pH 6.8, 2% SDS, 25% glycerol, 0.01%