1Neurosciences Program and 2Department of Biological Sciences, Hopkins Marine Station, Stanford University, Pacific Grove, California 93950
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ABSTRACT |
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Liu, Taylor I.,
Zora N. Lebaric,
Joshua J. C. Rosenthal, and
William F. Gilly.
Natural Substitutions at Highly Conserved T1-Domain Residues
Perturb Processing and Functional Expression of Squid Kv1
Channels.
J. Neurophysiol. 85: 61-71, 2001.
Shaker-type K-channel -subunits (SqKv1A, B, D) expressed
in neurons of the squid stellate ganglion differ in the length of their
N-termini and in the species of amino acid present at several points in
the T1 domain, an intracellular region involved in the tetramerization
process during channel assembly. Heterologous expression of wild-type
SqKv1A, B, and D in Xenopus oocytes reveals large
differences in the level of both functional channels (assayed by
whole-oocyte voltage clamp) and total channel protein (assayed by
immunoblotting). Functional expression is poorest with SqKv1A and by
far the best with SqKv1D. Biophysical properties of the three SqKv1
channels are essentially identical (assayed by cell-attached patch
clamp). Site-directed mutagenesis was used to determine whether the
observed differences in expression level are impacted by two residues
in the T1 domain at which SqKv1A and B (but not D) differ from the
consensus sequences found in many other taxa. In SqKv1A, glycine is
substituted for arginine in an otherwise universally conserved sequence
(FFDR in the T1B subdomain). In SqKv1B, glycine
replaces serine in a sequence that is conserved within the Kv1
subfamily (SGLR in the T1A subdomain).
Restoration of the consensus amino acid at these positions largely
accounts for the observed differences in expression level. Analysis of the glycosylation state of aberrant versus restored
-subunits suggests that the anomalous amino acids in SqKv1A and B exert their
influence during early steps in channel processing and assembly which
take place in the endoplasmic reticulum (ER).
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INTRODUCTION |
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A defined segment of the
cytoplasmic amino- (N-) terminus of voltage-gated K (Kv) channels,
known as T1 or NAB, contains contact points at which -subunits
oligomerize into tetrameric channels (Li et al. 1992
;
Shen et al. 1993
). Two important subdomains, T1A and T1B, have been
identified within T1. Conserved "distinct" sequences in
T1B were originally noted by Wei et al.
(1990)
and distinguish the Kv1-4 subfamilies (starred brackets
in Fig. 1B). Members of
different subfamilies do not form heteromultimeric channels
(Covarrubias et al. 1991
; Xu et al.
1995
), and this incompatibility is contributed to by both
subdomains (Shen and Pfaffinger 1995
). Consequently, the
T1 domain confers intra-subfamily specificity to the tetramerization of
Kv
-subunits and thereby directly influences the complement of
functional Kv channels expressed in an individual cell.
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We have previously described three cDNAs that encode Kv1 -subunits
expressed in the squid giant axon system (SqKv1A; Rosenthal et
al. 1996
) and in other, nongiant neurons of the stellate
ganglion (SqKv1B and D; Rosenthal et al. 1997
). All
three isoforms share a common core region (S1-S6) but differ in the
length and composition of the N-terminal domains (Fig. 1A)
with five differences in the T1 region (Fig. 1B; numbering
for SqKv1A).
Two of these five differences occur in short stretches of amino acids
that are extremely conserved in other Kv members. The T1B sequence FFDR is present in every entry in
Fig. 1B (bold) with the exception of SqKv1A, in which the
consensus arginine is replaced by glycine (G87). A second highly
conserved sequence is SGLR in T1A (Fig.
1B), which differentiates Kv1 members from those of the
other subfamilies. SqKv1B lacks the consensus serine (S47 in SqKv1A),
which again is replaced by glycine. This residue occupies a
structurally important position in the "tetramerization interface"
of the T1 domain of an Aplysia Kv1 -subunit (AKv1.1a or
AK01A) and may play a critical role in preventing tetramerization of
Kv1 and Kv3
-subunits (Bixby et al. 1999
;
Kreusch et al. 1998
).
These aberrant amino acids in SqKv1A (G87) and SqKv1B (G36) may
be expected to influence tetramerization and possibly other steps in
channel assembly. In this paper, we explore the impact of these
residues on expression of SqKv1 channels in Xenopus oocytes and make use of SqKv1D, which maintains the consensus amino acid at
both positions, as a standard for comparison. In each case, the
anomalous amino acid greatly reduces the density of functional K
channels assayed by two-electrode voltage clamp as well as the level of
mature, fully glycoslyated -subunit protein determined by
immunoblotting. Biochemical analysis of normal and defective
-subunits suggests that factors leading to poor expression act early
in the biosynthetic pathway, probably in the endoplasmic reticulum
(ER). Although glycosylation is not necessary to form functional SqKv1 channels, our results indicate that it may increase the efficiency of surface expression at early times following cRNA injection.
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Methods |
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Construction of expression plasmids and site-directed mutagenesis
SQKV1A, B, AND D.
Full-length cDNAs were isolated from a squid stellate ganglion library
as previously described (Rosenthal et al. 1996, 1997
). Polymerase chain reaction (PCR) was used to amplify the entire coding region of each cDNA with primers containing Bgl II
sites flanking the start and stop codons. SqKv1A-D have the identical C-terminal, and a common antisense primer was employed (SKC 16 of
Rosenthal et al. 1997
and Table
1). The sense primers were designed to
generate a consensus translation initiation sequence (Kozak
1989
). For SqKv1A (SKC37, Table 1), only the nucleotide at the
3 position was changed (C to A), and no amino acid (aa) codons were
thereby altered. In the cases of SqKv1B and D, however, an additional
change at the +4 position (T to G) resulted in alteration of aa 2 from
Ser to Ala and from Tyr to Asp, respectively.
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SQKV1A V5.
Three nucleotides (GTT) encoding Val at position 5 of SqKv1A were
omitted from the 5' (sense) primer SCK37; this primer was thus SKC 15 used in a previous study (Rosenthal et al. 1996
). Amplification thus introduced the
V5 mutation into the full-length product which was then ligated into pBSTA as described above.
SQKV1A G87R, SQKV1B R76G, AND SQKV1D R53G. A BstE II-Kas I fragment (109 nt) was removed from SqKv1A-pBSTA and replaced with the corresponding portion of SqKv1B. This changed the codon for aa 87 of SqKv1A from GGG (Gly) to AGG (Arg); the fragments were otherwise identical. To replace Arg with Gly at the equivalent position in SqKv1B-pBSTA (aa 76) and SqKv1D-pBSTA (aa 53), the same fragment was replaced with that from SqKv1A.
SQKV1A 1-34 G87R.
This mutant was created by replacing most of the coding region of
SqKv1D-pBSTA [from aa 4 (RsrII site) to the stop codon
(Bgl II)] with the corresponding fragment of SqKv1A
G87R-pBSTA. The resulting construct (called
1-34 G87R
for simplicity) lacks residues 1-34 and begins with the initiating Met
of SqKv1D. Cysteine at position 36 (i.e., aa 2 of the mutant) was
changed by this approach to Asp in order to maintain a Kozak consensus
sequence as described above.
SQKV1B G36S AND SQKV1D S13G. A Rsr II-Kas I fragment (195 nt) was removed from SqKv1B-pBSTA and replaced with the corresponding fragment of SqKv1D. This changed the codon for aa 36 of SqKv1B from GGC (Gly) to AGC (Ser); the fragments were otherwise identical. SqKv1D S13G was created by removing the same fragment in SqKv1D-pBSTA and replacing it with that from SqKv1B.
SQKV1A G87R HA-TAG. An in-frame, HA-epitope tag (YPYDVPDYASL) was incorporated into the C-terminal of SqKv1A G87R as follows. Hind III and BamH I sites were introduced into SqKv1A G87R before the start and stop codons, respectively, by PCR (sense primer SKC38 and antisense primer SKC36, Table 1) using SqKv1A G87R-pBSTA as a template. This amplification product was ligated into the Hind III and BamH I sites of the vector pOX (gift of T. Jegla, Stanford University), which contains the same Xenopus-untranslated regions as pBSTA. A synthetic, double-stranded oligonucleotide encoding the HA epitope with a BamH I site at the 5' end and a Xho I site at the 3' end was ligated into the same sites in SqKv1A G87R-pOX.
SQKV1A-D GLYCOSYLATION MUTANTS. All glycosylation mutations (Asp to Gln) were made using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Mutations were confirmed by sequencing.
Xenopus oocyte preparation and cRNA injection
Stage IV oocytes were isolated from adult Xenopus laevis and treated at room temperature with 1.5 mg/ml collagenase Type IA (Sigma, St. Louis, MO) in Ca-free OR-2 [(in mM) 82 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES; pH 7] until single oocytes were released (~2 h). Oocytes were then washed (room temperature) three times in Ca-free OR-2 and three times with ND96 [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES; pH 7.2] before incubation at 18°C in supplemented ND96 containing 2.5 mM Na-pyruvate and 50 µg/ml gentamycin. cRNA injection was carried out 12-24 h later.
Full-length cRNAs were transcribed from SqKv1 expression plasmids linearized with Not I using either T7 (for pBSTA) or T3 (for pOX) RNA polymerase (Message Machine, Ambion, Austin, TX). Products were quantified by absorbance at 260 nm prior to injection, and comparative concentrations were also checked by ethidium bromide staining of samples in 1% TAE gels. Oocytes were injected with 8 ng of cRNA (50 nl vol) through a glass micropipette using a manual microsyringe (Drummond Scientific, Broomall, PA) and were then cultured in supplemented ND96 as described above until recordings were carried out. Medium changes were made daily.
Tissue preparation and immunoblotting
Oocytes were harvested for analysis 2 days after cRNA injection.
Five to six oocytes were homogenized using a Dounce homogenizer in
ice-cold lysis buffer [(in mM) 5 Tris-HCl, 1 EDTA, 1 EGTA; pH 8]
containing freshly added protease inhibitors {1 µg/ml each of
aprotinin, leupeptin, and pepstatin A plus 50 mg/ml AEBSF
[4-(2-aminoethyl)-benzenesulfonyl fluoride] (all from Sigma)}. Yolk
and cellular debris were cleared from the homogenate by centrifugation
at 3000 g for 10-15 min at 4°C. A crude membrane fraction
was then pelleted by another centrifugation at 100,000 g for
30 min at 4°C. This pellet was resuspended in 12 µl SDS sample
buffer (Sambrook et al. 1989) plus 0.15 M
-mercaptoethanol. Samples were heated to 65°C for 5 min, separated
using SDS-PAGE on 7.5% gels, and electrophoretically transferred to
nitrocellulose membranes which were then soaked for 2 h at room
temperature in 10% nonfat dry milk in PBS [(in mM) 80 Na2HPO4, 20 NaH2PO4, 100 NaCl; pH
7.4]. Affinity-purified polyclonal antibodies to SqKv1A
(Rosenthal et al. 1996
) were used at 1:2000 dilution in
PBS with overnight incubation at 4°C. Goat anti-rabbit secondary
antibodies conjugated to horseradish peroxidase (Sigma, St. Louis, MO)
were used at 1:5000 dilution with a 45-60 min incubation time at room
temperature. An enhanced chemiluminescence detection system (Super
Signal Substrate, Pierce, Rockford, IL) was used to visualize the signals.
Immunoprecipitation and endoglycosidase digestion
These experiments employed the SqKv1A G87R HA-tag construct.
After oocytes were injected with cRNA, they were maintained in supplemented ND96 containing 500 µCi/ml
[35S]-methionine (Amersham) for 2 days at
18°C. Ten oocytes were collected and processed as described by
Ivanina et al. (1994) with minor modifications. Primary
antibody (monoclonal anti-HA, Berkeley Antibody Co., Berkeley, CA) was
used at a dilution of 1:150. Secondary antibodies coupled to magnetic
beads (BioMag goat anti-mouse IgG, PerSpective Diagnostics, Cambridge,
MA) were used at a final dilution of 1:200. Immunoprecipitated proteins coupled to the magnetic beads were suspended in 15 µl SDS sample buffer containing 0.15 M
-mercaptoethanol, heated to 65°C for 5 min, and loaded onto 7.5% SDS-PAGE. Gels were fixed in 45%
methanol-10% glacial acetic acid (10-15 min), treated for 30 min
with autoradiography enhancer (Enhance, NEN, Boston, MA), washed in
distilled H2O (10-15 min), dried, and exposed to
autoradiography film (X-OMAT AR, Kodak) overnight at
70°C.
For endoglycosidase digestion, magnetic beads were resuspended in 10 µl wash buffer (Ivanina et al. 1994) after the final wash. Proteins attached to beads were denatured by heating to 100°C
for 10 min. Digestion with endoglycosidase H (New England Biolabs,
Beverly, MA) was then carried out according to manufacturer's instructions at 37°C for 1.5 h. SDS sample buffer with 0.15 M
-mercaptoethanol was added before continuing with gel
electrophoresis as described above.
Two-microelectrode whole-oocyte voltage clamp
Recordings were made in ND96 at room temperature (22-25°C)
using a conventional amplifier (OC-725B, Warner Instruments, Hamden, CT) and procedures. Microelectrodes were filled with 3 M KCl and had
resistances of about 1 M. Analog K current
(IK) signals were filtered at 5 kHz
with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA).
Data acquisition, pulse generation, and on-line signal processing
(P/
4 treatment to remove linear leakage and capacity currents) were
carried out using a system designed by Dr. D. R. Matteson,
University of Maryland School of Medicine.
Maximum K slope conductance (gK) was
calculated for individual oocytes from the peak
IK-voltage (V) relation
between 0 and +60 mV using a linear regression fit. The
gK-V relation for all channels studied here is saturated over this range (Rosenthal et
al. 1997 and unpublished data). In some cases when
IK was extremely large,
IK at +10 mV was taken as an index of
gK.
Cell-attached macropatch recordings
All recordings were conducted 2-4 days after cRNA injection as
previously described (Rosenthal et al. 1997). The
vitelline membrane was manually removed after soaking an oocyte for
several minutes in a hypertonic solution containing (in mM) 200 K-aspartate, 20 KCl, 10 EGTA, 1 MgCl2, 10 HEPES;
pH 7.4. The oocyte was then immediately transferred to the recording
chamber filled with a bath solution [(in mM) 140 KCl, 2 MgCl2, 11 EGTA, 1 CaCl2, 10 HEPES; pH 7.2], in which all recordings were carried out at room temperature. Pipettes (1-3 M
) were coated with Sylgard (Dow
Corning, Midland, MI) and filled with external solution [(in mM) 127 NaCl, 15 KCl, 6 MgCl2, 1 CaCl2, 5 HEPES; pH 7.1]. After attaining a suitable high-resistance seal, the stability of
IK was verified over a 10 min period
before acquiring data for analysis. Signals were filtered (15 kHz),
sampled, and processed (P/
4) as described above.
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RESULTS |
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SqKv1 isoforms are expressed at different levels in Xenopus oocytes
Oocytes were injected on day 0 with an identical amount
(8 ng) of cRNA encoding either SqKv1A, B, or D -subunits, and
gK was determined from five oocytes of
each type over the next 5 days. Although the time course of
gK increase is roughly similar in each
case, the maximum level of gK attained
over days 3-4 differs tremendously (Fig.
2). SqKv1D channels are expressed at a
level about four times greater than that of SqKv1B and roughly 80 times that of SqKv1A.
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Identification of N-terminal elements in SqKv1A that decrease functional expression
The primary structure of SqKv1A, the most poorly expressed isoform, shows two obvious differences from that of SqKv1D which could be relevant to the low level of functional expression observed. First, the N-terminal of SqKv1A is substantially longer and unusually hydrophobic (8 of the first 11 amino acids). Second, of the four amino acids that differ in the T1 domain (Fig. 1B), G87 stands out as the only major anomaly in SqKv1A. This isoform contains the overwhelming consensus residue displayed by other Kv1 members at two of the other sites (V43 and N45), and the third site (K132) is not highly conserved. In an effort to increase the expression level shown by SqKv1A to that of SqKv1D, we therefore individually altered these two features.
ALTERATION OF G87 TO THE ARG CONSENSUS. Changing Gly 87 in SqKv1A to Arg dramatically affects the level of functional expression in oocytes without altering voltage-dependent properties of gK or single-channel conductance. Two-microelectrode recordings from oocytes injected with equal amounts of wild-type SqKv1A or G87R cRNA are displayed in Fig. 3, A and B. Activation kinetics and the gK-V relation (data not illustrated) are very similar in both cases. However, IK at +10 mV, a good indicator of maximum gK, is greatly increased by the G87R mutation (Fig. 3D).
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DELETION OF THE N-TERMINAL.
Although the G87R mutation increases functional expression, the maximum
level of gK attained is still
substantially less than that seen with expression of SqKv1D (Fig.
3D). To test the idea that the longer, hydrophobic
N-terminus of SqKv1A could have a deleterious effect on functional
expression, the first 34 amino acids of SqKv1A G87R were deleted in a
way that made the N-terminus identical to that of SqKv1D
(1-34 G87R; see METHODS). This manipulation increased functional expression to the level observed for SqKv1D (Fig.
3D), again with no significant effects on voltage-dependent properties (Fig. 3C).
DELETION OF VAL 5.
Although we have not carried out an extensive analysis of the
N-terminal elements of SqKv1A that impair functional expression, we
have identified a single amino acid that is important. Deletion of Val
at position 5 in the wild-type SqKv1A (SqKv1A V5) leads to an
increase in functional expression of about fourfold (Fig. 4D) with no apparent change in
voltage-dependent properties (Fig. 4, A and B).
Data in Fig. 4D indicate that the
V5 mutation in the G87R
background (SqKv1A
V5 G87R) increases the level of functional expression to the same degree as that observed with the complete deletion of the N-terminal (Fig. 3D). Replacement of Val
with other amino acids or additional deletions were not explored.
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Voltage-dependent properties of SqKv1A V5 G87R and SqKv1D
The combined V5 and G87R mutations in SqKv1A restores
functional expression to the high level observed for SqKv1D, and this permits reliable patch-clamp analysis of macroscopic voltage-dependent properties of IK through these SqKv1
channels. This is not possible with constructs that express at much
lower levels. Although the identical core structure of these
-subunits suggests the corresponding channels should have very
similar properties, this is an important point to confirm.
Basic voltage-dependent properties of SqKv1A V5 G87R channels
derived from cell-attached patch recordings are compared with those of
SqKv1D in Fig. 5.
IK records for both types of channels illustrate the similar natures of activation (Fig. 5A),
deactivation (Fig. 5B), and inactivation (Fig.
5C). A population analysis indicates that
gK-V relations for the two
channels are not significantly different (Fig. 5D), and the
same is true for activation kinetics (Fig. 5E), deactivation
kinetics (Fig. 5F), and inactivation kinetics (Fig.
5G).
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Identification of a residue in the T1 domain of SqKv1B that impairs expression
SqKv1B lacks the expression-inhibiting elements of SqKv1A discussed above. It has the consensus Arg in FFDR (at the site equivalent to G87 of SqKv1A) and lacks Val 5 and the other first 11 amino acids comprising the extreme N-terminus of SqKv1A. As indicated in Fig. 2, however, SqKv1B still expresses at a much lower level than does SqKv1D. There are only two amino acid differences in the entire N-terminal and T1 regions of the latter two isoforms (Fig. 1), and one of these (corresponding to K132 in SqKv1A) is not highly conserved among Kv1 members. However, at the site of S47 in SqKv1A, a consensus Ser exists in all Kv1 members except SqKv1B which has Gly.
Changing this anomalous residue of SqKv1B to the consensus Ser (SqKv1B G36S) increases the level of functional expression ~10-fold to the level shown by SqKv1D (Fig. 6A) with no significant effect on voltage-dependent properties of IK as determined in two-microelectrode recordings (Fig. 6, B and C). Cell-attached macropatch experiments confirmed that properties of IK for G36S channels are indistinguishable from those analyzed in conjunction with Fig. 5 for the other two high-expression variants (data not illustrated). The converse manipulation was carried out using SqKv1D, i.e., the consensus Ser at the equivalent position was changed to Gly (SqKv1D S13G). This mutation leads to a large decrease in the level of functional expression (Fig. 6A) without significantly affecting voltage-dependent properties determined by two-electrode voltage clamp (Fig. 6, D and E). Unfortunately the low level of expression preludes high-quality macropatch analysis. Thus, the identity of this single amino acid appears to completely account for the difference in expression levels shown by SqKv1B and D.
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Anomalous T1 residues also affect the level of -subunit
protein
Conventional immunoblotting methods were used to investigate the
effects on the total level of -subunit protein produced by the
manipulations of SqKv1A and B that increased surface expression of
functional K channels. The crude membrane fraction used for these
experiments contains material both from surface and internal membrane
pools, and analysis of the glycosylation state of bands detected is
presented below.
SQKV1A PROTEIN: EFFECTS OF THE G87R AND
V5 MUTATIONS.
Increased functional expression due to both manipulations of SqKv1A
discussed above is accompanied by a large increase in total
-subunit
protein. Immunoblots were prepared from equal numbers of oocytes
injected with cRNA for either wild-type SqKv1A or SqKv1A G87R, and
typical results are illustrated in Fig.
7A. The single band detected
with an apparent molecular weight of about 52 kDa represents
core-glycosylated protein (as demonstrated below) and is much more
prevalent when the native Gly (G87 lane) is replaced by Arg (R87 lane).
Introduction of the same G87R mutation into the background of SqKv1A
V5 leads to a similar large increase in the intensity of the 52-kDa
band, and multiple bands of higher apparent molecular weight become
very prominent (Fig. 7B). As demonstrated below, these later
bands represent glycosylated
-subunits in which carbohydrate
has been additionally processed. These higher molecular weight bands
are more prevalent with those constructs that support a high level of
functional expression.
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SQKV1B PROTEIN: EFFECTS OF THE G36S MUTATION. Immunoblots of SqKv1B samples typically show a large amount of a single, core-glycosylated band with little or no detectable higher molecular weight material. Introduction of the G36S mutation, which greatly increases functional expression, is accompanied by a reduction in intensity of the band of lower apparent molecular weight and enhancement of higher molecular weight forms (Fig. 7D). The reverse mutation made in SqKv1D at the analogous position (SqKv1D S13G) results in a dramatic increase in intensity of the lower molecular weight band (Fig. 7E). This mutation also greatly decreases functional expression (Fig. 4).
Glycosylation of SqKv1 -subunits
The strong correlation between the level of functional K-channel
expression and the prominence of higher molecular weight -subunit
protein described above for the three SqKv1 isoforms suggests that the
identified deviations from the T1 consensus may affect both assays by a
common mechanism. Here we analyze the glycosylation state of SqKv1A
protein and make use of this information to judge where in the
biosynthetic pathway the anomalous T1 residues exert their deleterious
effects on expression.
IDENTIFICATION OF CORE-GLYCOSYLATED AND MORE HIGHLY PROCESSED FORMS
OF SQKV1A -SUBUNITS.
N-linked glycosylation of membrane proteins occurs cotranslationally in
the ER where carbohydrate with a high mannose content is covalently
linked to asparagine residues of the core polypeptide. These
core-glycosylated polypeptides are then transported to the Golgi
apparatus where the core-carbohydrate trees are trimmed and processed
before delivery of the mature protein to the surface membrane.
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IDENTIFICATION OF N-LINKED GLYCOSYLATION SITES.
Three extracellular consensus sites for N-linked glycosylation exist in
each of the natural SqKv1 isoforms, and these are located in the S1-S2
and S3-S4 linkers (Fig. 9A;
numbering for SqKv1A). To determine which of these sites were actually
glycosylated in Xenopus oocytes, we used site-directed
mutagenesis to alter them individually in the background of SqKv1A
V5 G87R (N202Q, N272Q, and N281Q). In the case of the S1-S2 mutant,
all signs of glycosylation are eliminated (N202Q lane in Fig.
9B). The more fully processed bands are absent, and the
52-kDa core-glycosylated band is shifted downward to the same extent as
that produced by tunicamycin treatment (Fig. 7, A and
B). Tunicamycin treatment of oocytes expressing this N202Q
mutant produced no additional decrease in apparent molecular weight
(data not illustrated). Mutations at the sites in the S3-S4 linker
(N272Q and N281Q) did not significantly alter the banding patterns in
Western blots (Fig. 9B).
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EFFECTS OF GLYCOSYLATION ON FUNCTIONAL EXPRESSION.
Oocytes expressing the glycosylation mutants discussed above all
produced functional K channels. An analysis of cell-attached patch data
like that described in conjunction with Fig. 5 was carried out for the
N202Q mutation of SqKv1A V5 G87R. Voltage-dependent properties of
macroscopic IK were no different from
those in control oocytes (data not illustrated), and nonglycosylated
channels SqKv1A channels therefore appear to function normally.
Although a voltage shift in the
gK-V relation has been
reported for nonglycosylated mammalian Kv1.1 channels (Thornhill
et al. 1996
), this effect is not detected with SqKv1A or
Shaker (Santacruz-Toloza et al. 1994
).
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DISCUSSION |
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Control of Kv channel density in neurons and other cells may
involve regulation at the level of protein synthesis, processing, trafficking, or degradation. The general sequence of events in the
biochemical maturation of mammalian (Deal et al. 1994)
and insect (Nagaya and Papazian 1997
; Schulteis
et al. 1998
) Kv1 channels has been determined in heterologous
expression systems. N-linked glycosylation occurs during translation,
and assembly of core-glycosylated
-subunits into tetramers occurs in
the ER. Complex processing of carbohydrate, additional folding of the
mature channel, and sorting for delivery to the surface membrane take
place later in the Golgi. These basic features presumably also apply to
squid Kv1 channels.
Impaired expression of SqKv1A and B may result from defects early in biosynthesis
Aberrant amino acids in two extremely conserved segments of the T1 domain in SqKv1A (FFDG87 vs. FDDR, Fig. 1B) and SqKv1B (G36GLR vs. SGLR, Fig. 1B) greatly inhibit functional expression of the wild-type channels in Xenopus oocytes (Figs. 3, 4, and 6). The low level of gK observed reflects a reduced density of functionally normal K channels and is not due to changes in unitary conductance or gating properties.
Immunoblot analyses reveal that fully mature, glycosylated protein is
greatly reduced by glycine in either of the above positions and that
only core-glycosylated -subunits are detectable. This suggests that
very little wild-type protein moves from the ER to the Golgi for final
processing and ultimately to the surface membrane where functional
channels can be detected. The aberrant glycine residues therefore
appear to exert their influence early in biosynthesis, most likely in
the ER, where core-glycosylated subunits probably accumulate until
being degraded. Mechanisms leading to the proposed failure of wild-type
SqKv1A and B to efficiently exit the ER merit further investigation.
Effects of glycine at these two sites on the level of core-glycosylated
protein are not the same, however. In SqKv1A, the FFDG87 anomaly reduces the level of
core-glycosylated protein (Fig. 7, A-C), whereas the level
is increased by G36GLR in SqKv1B (Fig. 7,
D and E). This discrepancy may reflect different
degradation rates for each type of defective -subunit.
Structural implications of the aberrant T1 residues in SqKv1A and B
Exactly how the T1 domain confers sub-family specificity to
tetramerization of Kv channels is not well understood, but the positions studied here in SqKv1A (FFDG87) and
SqKv1B (G13GLR) correspond to structurally
important points identified in the tetrameric T1 complex of an
Aplysia Kv1 member (Bixby et al. 1999; Kreusch et al. 1998
), the equivalent Aplysia
positions being FFDR113 and
S73GLR. Based on this structure, it is likely
that glycine substitution at these positions generates different
structural defects.
In the first case, the FFDR113 site
(Aplysia nomenclature throughout) occupies a hinge-like
position in a linker connecting well-defined layers in the T1 tertiary
structure, and interaction of R113 is predicted with Gln126 on a
neighboring -subunit (His in SqKv1A-D). Although the functional
importance of R113 has not been determined, substitution of Gly for Arg
at this position might disrupt overall structural integrity to a point
that could compromise oligomerization.
The second site in Aplysia, S73GLR,
has been more thoroughly studied, and structural analysis indicates
that it interacts with both Gln126 and Glu78 (conserved in SqKv1s).
Substitution of Ser with Gly at this site greatly impedes binding of
the mutant and wild-type T1 proteins (Bixby et al.
1999), and this residue thus appears to be important in
conferring sub-family specificity to tetramerization of Kv1
-subunits. Self-association of G73GLR constructs was not tested in the cited study, but inefficient tetramerization due to this defect would be consistent with
observations presented in the present paper.
Impaired functional expression due to distal N-terminal elements in SqKv1A
Deletion of first 34 amino acids of SqKv1A, or even of just Val at position 5, results in a large increase in functional channel density (Fig. 3D). Neither mutation alters other functional channel properties, and both increase the total amount of channel protein accumulated (Figs. 7, A and B, and 8, A and B).
Mechanisms underlying these effects are not clear. One possibility
stems from the similarity of the first 12 amino acids of SqKv1A to
conventional leader sequences that are cleaved in the ER during
biosynthesis of many membrane and secretory proteins (Dalbey and
von Heijne 1992; von Heijne 1990
). Although the
N-terminus of SqKv1A is recognized as a signal-peptide (Nielsen
et al. 1997
; www.cbs.dtu.dk/services/SignalP/), the region
upstream of the predicted cleavage site (15 amino acids) is unusually
short and may therefore not serve as a very good leader. Nonetheless,
the N-terminus of SqKv1A might interact with the oocyte ER membrane in
an abnormal way and perturb the conformation of nascent
-subunits. Such defective subunits would probably be rapidly degraded. Although functional channels can form from true wild-type SqKv1A subunits, this
appears to occur with extremely low efficiency.
A vaguely defined "signal sequence" located in the first 34 amino
acids of a rabbit Kv1.3 -subunit also inhibits expression in oocytes
through an undefined mechanism (Segal et al. 1999
; Yao et al. 1998
). Although the extreme N-terminus of
Kv1.3 is rather hydrophobic, it is clearly not identified as a signal
peptide by the algorithm cited above. Furthermore, deletion of amino
acids 3-27 of Kv1.3 did not increase expression, and an inhibitory
mechanism of the type postulated for SqKv1A therefore seems unlikely.
Functional roles for glycosylation of SqKv1 channels
Kv1 -subunits from squid (Fig. 9), insects
(Santacruz-Toloza et al. 1994
), and mammals (Deal
et al. 1994
; Shi et al. 1996
) are all
glycosylated within a stretch of nine amino acids in the middle of the
S1-S2 linker, a region that shows no primary sequence similarity, even
between mammalian Kv1s (Stühmer et al. 1989
). Although the common location of this glycosylation site suggests that
carbohydrate attachment here is important, the functional significance
of this modification remains obscure.
A novel finding in this paper concerns the increased delay and decreased rate in the appearance of functional, nonglycosylated SqKv1 channels in the oocyte membrane following cRNA injection (Fig. 10B). At times later than 12 h postinjection, however, this inhibitory effect is no longer detectable (Fig. 10A), and gK rises at a similar rate to a comparable final level for both normal and nonglycosylated channels.
These kinetic manifestations suggest a functional role for glycosylation. The significance of the abnormally long delay in the appearance of functional, nonglycosylated channels after cRNA injection can be viewed in the same manner as the delay in IK activation after a voltage-clamp step. Sigmoidal IK kinetics indicates multiple closed states in the activation pathway. Similarly, the delayed appearance of nonglycosylated channels indicates that they have either passed through more steps during processing or through the same steps more slowly than their glycosylated counterparts do.
This analogy can also be extended to later times. Rate-limiting steps in channel activation act to determine the limiting "final" time course at which IK develops following a voltage step. Rates of appearance for glycosylated and nonglycosylated channels are identical more than 12 h postinjection, suggesting that the rate-limiting steps in channel processing are not glycosylation sensitive.
The gating analogy would suggest that the glycosylation-sensitive steps
precede the rate-limiting step, and this would be consistent with an
involvement of glycoprotein-specific chaperones, like calnexin
(Nagaya et al. 1999). Transient interaction between calnexin and glycosylated Shaker protein occurs early in
biosynthesis in the ER. The functional importance of this interaction
is not known for Kv1 proteins, but calnexin-binding might increase the efficiency of early processing steps or act to bypass certain steps. In
either case, nonglycosylated channels, which do not interact with
calnexin, would move through the ER more slowly. If a subsequent step,
e.g., sorting in the Golgi or delivery to the surface membrane, was
rate-limiting, the time course for the appearance of functional
channels would be affected by the lack of glycosylation in the way
observed for SqKv1A.
Expression of SqKv1 channels in the squid nervous system
Experiments described in this paper were carried out on cloned
squid channels expressed in a heterologous expression system. How
applicable are these results to natural expression of the corresponding
channels in GFL neurons (SqKv1A) and other neurons of the stellate
ganglion (SqKv1B and D)? Functional expression of SqKv1B and D channels
in oocytes is inversely proportional to the level of mRNAs for these
species detected in the stellate ganglion with in situ hybridizations
(Rosenthal et al. 1997). The functional significance of
this correlation is unclear. It is not known whether these two isoforms
are expressed in individual neurons, but the probability of
heteromultimer formation would appear to be greatly reduced by the
anomalous Gly36 of SqKv1B based on data discussed above (Bixby
et al. 1999
).
In the case of SqKv1A, which is only expressed in GFL neurons, two
major questions arise. The first concerns the putative sequence peptide
in the N-terminus. How this unusual feature impacts functional
expression in vivo is unknown and merits study. The second question
involves the FFDG87 anomaly in T1. Preliminary evidence suggests that editing of SqKv1A mRNA occurs at this position in vivo (Rosenthal and Bezanilla 2000). How the
resulting glycine substitution at a universally conserved Kv residue
affects SqKv1A expression in GFL neurons, and how mRNA editing might be
involved in controlling channel density in GFL neurons remain
outstanding questions.
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ACKNOWLEDGMENTS |
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We posthumously thank J. Kono for many years of support. This work constituted part of T. Liu's Ph.D. thesis from Stanford University.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-17510 to W. F. Gilly.
Present address of J.J.C. Rosenthal: Dept. of Physiology, UCLA School of Medicine, Los Angeles, CA 90095.
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FOOTNOTES |
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Address for reprint requests: W. F. Gilly, Hopkins Marine Station, Pacific Grove, CA 93950 (E-mail: lignje{at}leland.stanford.edu).
Received 8 May 2000; accepted in final form 15 September 2000.
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REFERENCES |
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