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INTRODUCTION |
The anion-conductive inhibitory glycine receptor
(GlyR)1 is a member of the
ligand-gated ion channel superfamily of
neurotransmitter receptors that includes the closely related
inhibitory
-aminobutyric acid, type A receptors, as well as
the cation-permeable nicotinic acetylcholine receptors and
5-hydroxytryptamine type 3 receptors. Four GlyR genes encoding ligand
binding
subunits (
1-
4) and a single gene for the structural
subunit are known in vertebrates (for review see Refs. 1 and 2).
These subunits form homopentameric and heteropentameric chloride
channels (3, 4), which mediate postsynaptic inhibition in the spinal
cord and other regions of the mammalian central nervous system, thus
controlling motor and sensory pathways. All
subunit isoforms
assemble into functional homopentameric GlyRs upon heterologous
expression in Xenopus oocytes or mammalian cells (5-8).
Like other proteins of the ligand-gated ion channel superfamily, GlyR
subunits are multispanning (polytopic) type I membrane proteins with an
N-terminal cleavable signal sequence, which targets the nascent
polypeptide to the ER and drives its insertion into the lipid bilayer.
Mature GlyR subunits as released by signal peptidase cleavage are
modular polypeptides, composed of 1) a large glycosylated N-terminal
ectodomain that forms the agonist binding site; 2) four transmembrane
segments (M1-M4), which between M1 and M3 are connected by short
hydrophilic loops and between M3 and M4 by a large cytoplasmic loop of
~85 amino acids (designated M3-M4 loop in this paper); and 3) a short
extracellular C-terminal tail.
The classic model of how multispanning membrane proteins insert
cotranslationally into the ER membrane assumes that the overall topology of the mature protein is determined by the orientation of the
signal sequence, which is inserted first and initiates the
translocation of the following peptide segments (9, 10). Accordingly,
downstream hydrophobic sequences simply serve as alternate stop
transfer and signal anchor sequences, which cause the nascent
polypeptide to passively follow the lead of the preceding transmembrane
segment and thereby direct the sequential insertion of polytopic
proteins. In prokaryotes, the transmembrane orientation of the most
N-terminal hydrophobic sequence, i.e. the cleavable signal
sequence in case of type I proteins, has been found to depend on the
flanking charged residues; the more positively charged end is retained
on the cytoplasmic (Cis) side of the membrane, as described by
the "inside- or Cis-positive rule" (11). Electrostatic interaction
of arginine and lysine residues with negatively charged head groups of
phospholipids (12), and, at least in prokaryotes, the negative-inside
transmembrane potential (13) determines the transmembrane orientation.
Because of their low average degree of ionization at physiological pH,
histidines have almost no effect on peptide topology (14). Negatively
charged residues affect the topology of prokaryotic proteins only when
present in high numbers (15). In contrast, the orientation of the first
transmembrane segment of eukaryotic proteins correlates best with the
charge difference hypothesis, which considers both positively and
negatively charged amino acids by proposing that a net negative
cytoplasmic charge dictates a luminal disposition (16). Besides
charges, the length and hydrophobicity of the signal sequence (17, 18), as well as glycosylation at sites near the signal sequence (19), have
all been documented to affect transmembrane topology. Experimental support for the classical insertion model has been provided for a
variety of membrane proteins (20-23). There is, however, increasing evidence that the initial translocation events may not necessarily dictate the topology of the entire mature protein (24-26). Rather, additional topogenic sequence information in subsequent transmembrane segments or internal loops appears to be required for the correct positioning of some transmembrane segments of multispanning proteins (for a recent review see Ref. 27).
In this paper, we show that a cluster of basic residues located in the
cytoplasmic loop of the GlyR
1 subunit approximately eight residues
C-terminal to the transmembrane segment M3 constitutes an important
determinant of proper membrane insertion. Based on the usage of three
naturally occurring, though normally inaccessible N-glycosylation sites of the M3-M4 loop, we demonstrate that
the unbiased orientation of M3 and the ensuing hydrophilic loop depends on the presence of these positively charged residues. The other positively charged amino acids of the M3-M4 loop located outside of
this basic cluster exert little or no effect, indicating that a high
density of positive charges rather than a net charge difference determines topology.
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EXPERIMENTAL PROCEDURES |
cDNA Constructs--
The cDNA construct encoding
1-His with a C-terminal hexahistidyl tag (His) has been described
previously (8). Mutations were inserted using the QuikChangeTM
site-directed mutagenesis kit (Stratagene) and confirmed by sequencing.
All amino acids were numbered according to their position in the mature
protein sequence.
Oocyte Expression--
Defolliculated Xenopus
oocytes were injected with ~50-nl aliquots of capped cRNAs (0.5 µg/µl) and kept at 19 °C in sterile frog Ringer's solution
(90 mM NaCl, 1 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, and 10 mM Hepes, pH 7.4) supplemented with 50 mg/liter gentamycin
as described (28). One to three days after cRNA injection, glycine
responses were measured by two-electrode voltage-clamp recording at a
holding potential of
70 mV as described previously (4).
Protein Purification, SDS-PAGE and BN-PAGE--
cRNA-injected
and non-injected control oocytes were metabolically labeled by
overnight incubation with L-[35S]methionine
(>40 TBq/mmol; Amersham Biosciences) at about 100 MBq/ml (0.1 MBq per oocyte) in frog Ringer's solution at 19 °C and chased with
1 mM unlabeled methionine as indicated. His-tagged proteins
were then purified by Ni2+-NTA-agarose (Qiagen)
chromatography from digitonin (1%) (w/v) extracts of oocytes as
detailed previously (29) with the following modification. Iodoacetamide
was routinely included at 10 and 1 mM in the lysis and
washing buffers, respectively, to prevent artificial cross-linking of
polypeptides by disulfide bonds. Bound proteins were released from the
Ni2+-NTA-agarose with non-denaturing elution buffer
consisting of 0.5% (w/v) digitonin and 250 mM
imidazole/HCl, pH 7.6, and kept at 0 °C until analyzed by PAGE.
For SDS-PAGE or Tricine-PAGE (30), proteins were supplemented with the
appropriate SDS sample buffer containing 20 mM
dithiothreitol and electrophoresed in parallel with
[14C]labeled molecular mass markers (Rainbow; Amersham
Biosciences) on linear SDS-polyacrylamide gels. Where indicated,
samples were treated prior to SDS-PAGE with either endoglycosidase H
(Endo H) or peptide-N-glycosidase F (PNGase F; both enzymes
were purchased from New England Biolabs) in the presence of 1% (w/v)
Nonidet P-40 to counteract SDS inactivation of PNGase F.
BN-PAGE (31) was performed as described previously (29)
immediately after protein purification. Purified proteins were supplemented with blue native sample buffer to final concentrations of
10% (v/v) glycerol, 0.2% (w/v) Serva blue G, and 20 mM
sodium 6-amino-n-caproate and applied onto polyacrylamide
gradient slab gels. Where indicated, samples were treated prior to
BN-PAGE with 8 M urea to induce partial dissociation of
receptor complexes into lower order intermediates. Both SDS- and
BN-polyacrylamide gels were fixed, dried, and exposed to BioMax
MS film (Eastman Kodak Co.) at
80 °C. For quantification,
the dried gels were exposed to a PhosphorImager screen and scanned
using a Storm 820 PhosphorImager (Amersham Biosciences). Individual
bands were quantified with the ImageQuant software.
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RESULTS |
Alanine Substitution of Basic Residues of the
1-His Chain
Downstream to M3 Results in a Mixed Orientation of the M3-M4
Loop--
Between the M3 and M4 segments, the GlyR
1 polypeptide
contains a cytoplasmic loop of ~85 amino acids that includes a total of 23 basic (ten Lys, ten Arg, three His) and nine acidic (three Asp, six Glu) residues (Fig.
1A). Particularly remarkable
is a cluster of basic amino acids, 316RFRRKRRHHK, a few
residues downstream to M3. To examine a possible functional role of
this sequence motif, we replaced four or seven of its basic residues by
alanines, yielding 316AAAAARRHHK
(316-320A-
1-His mutant) or 316AAAAAAAHHA
(316-322,325A-
1-His mutant), respectively (Fig.
1B). Two-electrode voltage-clamp analysis of
Xenopus oocytes injected with the respective mutant cRNAs
revealed no effect of these alanine replacements on the shape of the
glycine-induced currents or on glycine potency (results not shown).
However, a statistically significant reduction of the amplitude of the
maximal current elicited by glycine from 4.7 ± 1.2 to 2.3 ± 0.8 µA was observed for the 316-320A-
1-His receptor
as compared with the parent
1-His receptor, respectively, in three
independent experiments, each with five to ten oocytes.

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Fig. 1.
Sequence and topology of the human GlyR
1 subunit. A, the membrane topology
and predicted boundaries of the transmembrane segments are based on the
original model (see Ref. 2). NX(T/S) sequons are indicated
with gray symbols combined with white lettering.
Notably, only one of the sequons (38NVS) is located on the
predicted ectodomain, whereas the three others (335NFS,
358NNS, 361NTT) are located on the cytoplasmic M3-M4 loop. Basic residues (Lys, Arg) are highlighted
with filled black symbols and white lettering,
whereas acidic residues (Glu, Asp) are highlighted in gray.
B and C, survey about the GlyR 1 subunit
mutants used in this study. Amino acid residues are designated by the
single letter code with basic residues highlighted in
boldface. Numbers indicated by indices correspond
to positions of the mature 1 sequence, i.e. after
cleavage of the 28-amino acid-long signal peptide. B, GlyR
1 subunit constructs carrying mutations solely in the M3-M4 loop.
The entire sequence of the M3-M4 loop is shown without the flanking
transmembrane segments M3 and M4. Charged residues were replaced by
alanine, whereas asparagine was replaced by glutamine. C,
the amino acid sequence of the hydrophobic stretches thought to
represent the transmembrane segments M2 and M3 (boxed) are
shown together with the linking sequence (M2-M3 ectodomain) and the
N-terminal part of the M3-M4 loop encompassing the cluster of basic
residues.
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Next we compared the synthesis and post-translational processing
of the parent
1-His GlyR and the charge neutralization mutants. The
parent
1-His polypeptide isolated as a control migrated as a single
band at 48 kDa when analyzed by reducing Tricine-PAGE subsequent to
isolation by Ni2+-NTA-agarose chromatography from
[35S]methionine-labeled Xenopus oocytes (Fig.
2, A and B,
lane 1). Treatment with Endo H reduced the mass of the
1-His subunit isolated directly after the pulse by 3 kDa to the
45-kDa protein core (Fig. 2B, lane 2). This mass
shift is consistent with the presence of a single N-glycan,
resulting from usage of the sole N-glycosylation motif,
38NVS, in the N-terminal extracellular domain of the
1
polypeptide (Fig. 1A) (8). After replacement of the acceptor
Asn38 by glutamine, the
1-His polypeptide migrated at 45 kDa, and no mass shift upon Endo H treatment was observed (results not shown).

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Fig. 2.
N-Glycan status of GlyR
1-His subunits with neutralized basic charges
downstream to M3. Oocytes were injected with indicated cRNAs,
labeled overnight with [35S]methionine, and extracted
with digitonin. Proteins were natively purified by
Ni2+-NTA-agarose chromatography, denatured with Tricine-SDS
sample buffer, and resolved by reducing Tricine-SDS-PAGE (4%/10%/13%
acrylamide). Autoradiographs of the gels are shown. A, the
wild-type GlyR 1-His subunit migrates as a 48-kDa polypeptide.
Neutralization of positively charged amino acids leads to the
appearance of an additional 54-kDa polypeptide. B, the same
samples as in A were denatured with reducing Tricine-SDS
sample buffer and then incubated for 2 h with Endo H or PNGase F
as indicated. The 54-kDa form of the 316-320A- 1-His
mutant was reduced to the 45-kDa protein core by deglycosylation with
Endo H or PNGase F. C, the indicated polypeptides were
incubated with increasing amounts of Endo H (in percent of maximum
amount of enzyme used). D, elimination of
N-glycosylation sequons located in the M3-M4 loop results in
mass shifts, which corroborate that the misfolded 54-kDa polypeptides
carries N-glycans at Asn335 and
Asn358 (or Asn361).
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In contrast to the parent
1-His polypeptide, both the
316-320A-
1-His and the
316-322,325A-
1-His mutants migrated as double bands of
apparent masses of 48 and 54 kDa when analyzed under the same
conditions (Fig. 2A, lanes 2 and 3).
Strikingly, Endo H treatment of the 316-320A-
1-His
(Fig. 2B, lane 5) and the
316-322,325A-
1-His (not shown) mutants shifted both the
48- and the 54-kDa polypeptides to the 45-kDa
1-His protein core.
The same result was obtained with PNGase F (Fig. 2B,
lane 6). This indicates that the 48- and the 54-kDa
polypeptides possess the same protein mass but differ in their number
of N-glycans.
To determine the number of N-glycans that give rise to the
54-kDa polypeptide, the 316-320A-
1-His mutant was
incubated with increasing concentrations of Endo H (Fig. 2C,
lanes 7-12). Because of partial deglycosylation at
intermediate Endo H concentrations, a ladder-like pattern of four bands
was generated (lanes 8 and 9). Neighboring bands
differ in mass by 3 kDa, the mass of an N-linked
oligosaccharide side chain. The 45-kDa band represents the core
protein, whereas the 48-, 51-, and 54-kDa bands correspond to
polypeptides with one, two, and three N-glycans,
respectively. Because 38NVS is the only potential acceptor
site for N-linked glycosylation on the extracellular part of
the
1-His polypeptide, the two additional N-glycans must
originate from usage of other endogenous sequons that are topologically
inaccessible in the non-mutated GlyR
1 polypeptide. Indeed, the GlyR
1 subunit sequence carries a total of four consensus
N-glycosylation sites, three of which reside on the
cytoplasmic M3-M4 loop, 335NFS, 358NNS, and
361NTT (cf. Fig. 1A). These sites
should be glycosylated only when the M3-M4 loop translocates into the
ER lumen, thus allowing for formation of the hyperglycosylated 54-kDa form.
The glycosylation of only two of the three sequons of the M3-M4 loop
could signify either that one of the closely adjacent sequons
358NNS and 361NTT remains unused for steric or
other reasons or that acceptor Asn335 is exposed to the
cytoplasm. To discriminate between these possibilities, we substituted
Asn335 of the 318,321A-
1-His mutant with
glutamine. The 318,321A-
1-His mutant behaved virtually
identical to the 316-320A-
1-His mutant (Fig.
2D, lane 2) (see also below). Consistent with the
occupancy of Asn335 of the 54-kDa form with an
N-glycan, elimination of Asn335 resulted in a
3-kDa shift to 51 kDa (Fig. 2D, lane 3). After further 3-kDa shift was observed when 358NNS and
361NTT were simultaneously eliminated in addition to
Asn335 (Fig. 2D, lane 4). This
indicates that the two N-glycans of the M3-M4 loop of the
54-kDa
1-His mutant polypeptides are located at Asn335
and Asn358 (or Asn361), each contributing a
3-kDa oligosaccharide side chain to the total mass of 54 kDa. Because
of the highly polar nature of the amino acid residues between the end
of M3 (position 307) and Asn335, the simultaneous usage of
both Asn335 and Asn358 (or Asn361)
further implies that virtually the entire M3-M4 loop was translocated into the ER lumen upon charge neutralization in mutants. Accordingly, the preceding lipophilic segment, M3, fails to integrate properly into
the membrane upon neutralization of positive charges of the M3-M4 loop.
GlyRs with Mixed Topology of the M3-M4 Loop Have an Impaired
Assembly Capacity and Are Unable to Leave the Endoplasmic
Reticulum--
To analyze the effect of a mixed topology of the M3-M4
loop on subunit assembly, we resolved natively purified
1-His
mutants by BN-PAGE, a method which displays the oligomeric nature of
receptor proteins (8, 32). Regardless of the number of basic charges neutralized downstream to M3, all mutants analyzed migrated as perfectly assembled homopentamers when the
[35S]methionine pulse was followed by an overnight chase
interval (results not shown). If, however, the GlyRs were purified
directly after a 4-h [35S]methionine pulse, a propensity
of the mutants to aggregate became apparent, as indicated by the
appearance of high molecular weight
1-His protein that
migrated at a broad range of masses above that of the pentameric
receptor (Fig. 3A).
Quantitative scanning of the protein bands resolved by BN-PAGE showed
also that the wild-type GlyR
1-His subunits existed
partially in an aggregated form shortly after synthesis
(Fig. 3B). However, the amount of aggregates was markedly
higher when one charge of the basic cluster downstream of M3 was
neutralized (R319A, K320A; see lanes 3 and 5) and
increased further upon neutralization of four basic charges (RFRRK316-320AFAAA; see lane 7). Most likely,
these aggregates are formed primarily of
1 subunits with luminally
exposed M3-M4 loop. The aggregates of both the wild-type and the mutant
GlyR
1 subunits disappeared during a subsequent chase interval
despite the continued presence of the hyperglycosylated 54-kDa form
(not shown) (cf. Fig. 3C). Evidently, the
luminally exposed M3-M4 loop delays but does not prevent proper GlyR
assembly. The lack of a marked effect of the wrongly folded M3-M4 loop
on assembly can be reconciled with the known location of the assembly
domains of ligand-gated ion channel subunits in the N-terminal
ectodomain (1).

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Fig. 3.
Assembly and ER exit of GlyR
1-His subunits with neutralized basic charges
downstream to M3. A, 1-His GlyRs natively purified
from cRNA-injected oocytes immediately after a 4-h
[35S]methionine pulse were resolved by BN-PAGE (4-12%
acrylamide). Where indicated, samples were partially denatured by a 1-h
incubation at 8 M urea and 56 °C. B,
quantitative profiles of the protein bands of the lanes
shown in A obtained by PhosphorImager analysis reveal an
increased propensity of the 1-His mutants to aggregate
(hatched areas). C, 1-His GlyRs natively
purified from cRNA-injected oocytes after a 4-h
[35S]methionine pulse and an additional 36-h chase
interval were denatured with reducing Tricine-SDS sample buffer and
then incubated for 2 h with Endo H or PNGase F as indicated. The
monoglycosylated 48-kDa polypeptide was entirely Endo H-resistant,
indicating that it had reached the Golgi apparatus. In contrast, the
54-kDa polypeptide persisted in the Endo H-sensitive form, consistent
with retention in the ER.
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To examine whether
1-His GlyRs with aberrantly folded M3-M4
loop are able to leave the ER, we exploited that N-glycans
in general become complex-glycosylated and hence resistant to Endo H
during passage of the Golgi apparatus en route to the cell
surface. Accordingly, the glycosylation status of the 48- and the
54-kDa polypeptides was determined after a chase interval (Fig.
3C). Consistent with previous observations, the wild-type
48-kDa
1-His chain was entirely Endo H-resistant when isolated after
a 24-h chase interval (Fig. 3C, lane 2). In
contrast, the hyperglycosylated 54-kDa polypeptide generated from the
316-320A-
1-His construct persisted entirely in the Endo
H-sensitive form, indicating that
1-His chains with aberrantly
folded M3-M4 loop are incapable of leaving the ER. This view is
supported by a relative decrease in the amounts of the additional 35- and 13-kDa polypeptides, which represent proteolytic cleavage products
generated in a lysosomal compartment from GlyRs that are
endocytotically retrieved from the cell surface, and hence are
indicative of the plasma membrane insertion of GlyRs (33).
Quantification by PhosphorImager analysis revealed that 57% of the
wild-type
1-His subunit, but only 16% of the
316-320A-
1-His mutant, was proteolytically cleaved into
the 35- and 13-kDa products. We conclude from these results that GlyRs
with aberrantly folded M3-M4 loop are not exported to the cell surface.
Neutralization of a Single Basic Residue Downstream to M3 Is
Sufficient to Disturb Topology of the M3-M4 Loop--
To determine how
many basic residues can be removed without disturbing membrane
topology, GlyR
1 mutants with only one or two alanine substitutions
in the 316RFRRKRRHHK motif were generated. Surprisingly,
substitution of only a single basic residue was already sufficient to
create a mixed topology of the M3-M4 loop, as evidenced by the
synthesis of the hyperglycosylated 54-kDa polypeptide (Fig.
4A). The faint 54-kDa band
isolated with the parent GlyR
1-His subunit indicates that even a
minor portion of the wild-type
1 subunit adopts the incorrectly
folded conformation (Fig. 4A, lane 1; see also
Fig. 2D, lane 1). Quantification by
PhosphorImager analysis revealed that 5-10% wild-type
1 subunits
possessed a luminally oriented M3-M4 loop shortly after
synthesis.

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Fig. 4.
Positional effects of charged residues on
M3-M4 loop topology of the GlyR 1 subunit
revealed by alanine scanning mutagenesis. cRNA injected oocytes
were incubated with [35S]methionine for 4 h
(A) or 14 h (B) prior to extraction with
digitonin. Proteins were resolved by reducing Tricine-SDS-PAGE.
Autoradiographs of the gels are shown. C and D,
protein bands shown in A or B were quantified by
PhosphorImager analysis to determine the fraction of misfolded 54-kDa
polypeptide of single mutants (C) and double mutants
(D) as a function of the position of the neutralized basic
residues. For the double mutants, the arithmetic mean of the amino acid
position of the two mutations is plotted. The dotted line
indicates the relative level of misfolded 54-kDa wild-type 1 subunit
in these experiments.
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Positional Effect of Basic Residues on M3-M4 Loop
Topology--
Quantification by PhosphorImager analysis of the 48- and
54-kDa bands revealed a striking positional effect of single charge neutralizations of the M3-M4 loop. Neutralization of
Lys320, located at a distance of ~12 residues from the
end of the M3 domain (cf. Fig. 1), produced the largest
effect among all single mutants investigated, with a fraction of 48%
aberrantly folded 54-kDa polypeptide (Fig. 4C). To examine
the effect of charge neutralization over the entire M3-M4 loop, a set
of double mutants was generated in which two consecutive positively
charged amino acids were systematically replaced by alanine. The
aberrantly folded 54-kDa polypeptide was most abundant relative to the
normal 48-kDa polypeptide when Arg318 and
Arg321 or Arg319 and Lys320 were
neutralized by alanine substitution (Fig. 4, B (lane
8) and D). Thus, the positive charges in the center of
the basic cluster are of particular importance for the topology of the
M3-M4 loop. Neutralization of >two positive charges within the cluster (up to seven positive charges as in the
316-322,325A-
1-His mutant; see Fig. 4B,
lane 3) did not further increase the relative amount of the
54-kDa polypeptide. Notably, charge neutralizations close to the M3
segment (Fig. 4B, lane 6) or in the C-terminal
half of the M3-M4 loop (lanes 11-14) had only little or no
effect. Even when a total of five basic residues was replaced by
alanines in the C-terminal half of the M3-M4 loop, no 54-kDa polypeptide was formed (lane 15).
In a further attempt to assess the role of charges for the disposition
of the M3-M4 loop, we used the 318,321A-
1-His-mutant to
also neutralize three negative charges, EDE, at positions
326-329 immediately downstream of the basic cluster. The resulting
318,321,326-328A-
1-His mutant displayed a mixed
topology of the M3-M4 loop (Fig. 4B, lane 5).
Quantification revealed a relative decrease of the misfolded 54-kDa
polypeptide from 60% (parent 318,321A-
1-His mutant) to
48% (318,321,326-328A-
1-His mutant), indicating that
increasing the net charge difference (positive charges minus negative
charges) can only partially compensate for neutralization of residues
within the basic cluster.
Neutralization of Basic Charges in the M2-M3 Ectodomain Rescues
GlyR
1 Mutant Topology--
The presence of several positive
charges on both sides of a transmembrane segment can prevent its
membrane insertion (34). We, therefore, considered that positively
charged residues in the 14-amino acid-long hydrophilic loop connecting
M2 and M3 may impede membrane insertion of M3 in our charge
neutralization mutants. Indeed, alanine substitution of one of these
charges, Lys276 or Lys281 (Fig. 1C),
markedly reduced the fraction of newly synthesized 318,321A-
1-His mutant with luminal M3-M4 loop
orientation (Fig. 5, A (lanes 3 and 4) and B). Simultaneous
neutralization of both Lys276 and Lys281 almost
fully abolished formation of the 54-kDa polypeptide (lane 5), suggesting that the basic cytoplasmic cluster C-terminal to M3
is required to counteract the three basic charges in the M2-M3 ectodomain. Two-electrode voltage-clamp measurements in oocytes demonstrated that the K276A and K281A mutations result in 29-fold decrease in glycine potency (Fig. 5C). A related mutation,
K276A, which is associated with startle disease, a rare neurological disorder, is known to produce a similar substantial decrease in glycine
sensitivity (35) by impairing the opening of the channel rather than
the binding of glycine (36).

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Fig. 5.
Effect of simultaneous neutralization of
positive charges of the M2-M3 ectodomain and the basic cluster on the
disposition of the M3-M4 loop. A, cRNA-injected oocytes
were pulse-labeled with [35S]methionine for 4 h.
Proteins were purified from digitonin extracts of these cells and
resolved by reducing Tricine-SDS-PAGE. B, the relative
amount of the misfolded 54-kDa polypeptide in A was
quantified by PhosphorImager analysis. C, glycine
dose-response curves. The lines drawn through data
points represent non-linear fits of the Hill equation to the data,
which yielded EC50 values of 0.22 mM (Hill
coefficient of 2.7) and 6.4 mM (Hill coefficient of 1.2)
for the parent 318,321A- 1-His mutant ( ) and the
276,281,318,321A- 1-His mutant ( ), respectively.
Error bars, S.D. values of 11 (parent construct) and four
(mutant) recordings in oocytes of different females.
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DISCUSSION |
The present study shows that the correct topology of the M3-M4
loop of the GlyR
1 subunit depends on a cluster of positively charged residues, 316RXRRKRR, immediately
downstream of the hydrophobic region M3. If one or more positive
charges are neutralized within this motif, the GlyR
1 subunit adopts
two topologies, the correct one with the hydrophobic region M3 spanning
the membrane and the connecting loop being localized cytoplasmically,
and an aberrant one with M3 being non-spanning and the M3-M4 loop being
translocated into the ER lumen. This indicates that the cluster of
basic residues contains topogenic information. Both the correctly and
the incorrectly folded
1-His subunits assembled to homopentamers,
but solely homopentamers consisting of correctly folded
1 subunits
were exported to the plasma membrane, suggesting that the aberrant M3-M4 loop is recognized by the cellular quality control system. Also,
when in our experiments the metabolic labeling period was followed by a
20-h chase, the intensity of the aberrant 54-kDa polypeptide decreased
relative to that of the correctly folded
1 subunit. Together these
data indicate that incorrectly folded receptor proteins are not only
unable to reach the plasma membrane but are also subject to increased
proteolytic degradation. The plasma membrane-bound mutant GlyRs, on the
other hand, exhibited an electrophysiological phenotype identical to
that of the parent
1 GlyR, indicating that the basic charges
C-terminal to M3 are not directly involved in receptor functioning.
Functional Importance of the Basic Cluster for Correct GlyR Subunit
Topogenesis--
According to the classic model of membrane
integration of polytopic proteins, the hydrophobic M3 region should
follow the lead of the preceding transmembrane segment, M2. Because M2
has a Ncyt-Cexo orientation, M3 should adopt
passively the opposite Nexo-Ccyt orientation,
thus acting as a stop-transfer sequence that halts further
translocation of the polypeptide chain across the membrane. Here,
failure of the M3 segment of 30-80% of the newly synthesized
1
chains to span the membrane was demonstrated by the aberrant
N-glycosylation of the M3-M4 loop after neutralization of
one or more basic residues 8-14 amino acids downstream to the C-terminal end of the M3 region. This indicates that proper membrane integration of the apolar M3 region depends critically on the presence
of the highly charged basic motif. A similar role of positively charged
residues in halting translocation of a hydrophobic segment has been
described previously (37).
There is increasing evidence for polytopic proteins that topogenic
information is not restricted to the most N-terminal hydrophobic domain
but spread throughout the protein (for a recent review see Ref. 27).
One of the determinants is apparently the transmembrane distribution of
positive charges, which prevent not only the translocation of N- and
C-terminal segments (38) but also of connecting loops of polytopic
membrane proteins (39). In addition, internal loops of polytopic
membrane proteins have on average a higher content of positively
charged residues as compared with external ones (23). The failure of
the hydrophobic M3 region to integrate into the membrane after
neutralization of only a single positively charged residue was
nevertheless unexpected, because seven positively charged residues are
left within the 15 residues flanking the C-terminal end of the
hydrophobic M3 region. When considering all charges within the flanking
15-residue windows on each side of the M3 segment, there remains a
marked net positive charge difference
(C-N) of +4 (with net charges
of +2 and +6, respectively) for the single charge neutralization
mutants. According to the charge difference rule, this should be more
than sufficient to dictate an Nexo-Ccyt
orientation of a signal anchor sequence. However, despite this excess
of positive charges only a minor fraction of the M3 segment adopted the
correct Nexo-Ccyt orientation, and most of the
M3-M4 loop was translocated incorrectly. This is also surprising in
view of an excess of 11 positive charges within the entire M3-M4 loop,
which harbors a total of 20 positively and nine negatively charged
residues. In conclusion, neither the charge difference rule nor the
positive inside rule provide any indication for a biased topology of
the apolar M3 domain subsequent to neutralization of one or several
positive charges of the cluster. This suggests that the M3 segment by
itself is unable to reliably halt translocation.
Positive Charges on the Short M2-M3 Ectodomain Seem to Impair the
Stop-transfer Function of the Apolar M3 Segment--
Hydropathy
analysis based on the Kyte-Doolittle algorithm with a window of five
amino acids provided no indication that the M3 domain may not be
sufficiently hydrophobic to partition by itself into the lipid bilayer.
From our observation that unbiased folding can be restored by
neutralization of positive charges on the M2-M3 ectodomain, we infer
that the positive charges on the M2-M3 ectodomain prevent the M3 domain
from positioning itself correctly. The three positive amino acids of
the 14-amino acid-long M2-M3 loop may impose constraints to the M3
segment to adopt a Ncyt-Cexo orientation, which
is obviously incompatible with the Ncyt-Cexo
orientation of the preceding M2 domain. Therefore, the hydrophobic M3
domain may remain in an unstable state unless the downstream topogenic
cluster of basic residues imposes the correct Nexo-Ccyt orientation. The extreme sensitivity
of M3 transmembrane orientation to neutralization of single basic
residues within the 316RXRRKRR sequence suggests
that the particular high density of positive charges is essential for
keeping the M3-M4 loop on the Cis-side of the membrane. Hence,
the basic cluster seems to serve as an accessory cytoplasmic
stop-transfer signal which, by blocking translocation of the M3-M4
loop, forces the apolar M3 segment to insert properly into the
membrane. Once inserted, hydrophobic interactions with the lipid
bilayer may suffice to stabilize the M3 domain in the correct
Nexo-Ccyt orientation.
Positive charges appear to be more easily translocated through the ER
than through bacterial membranes, most likely because of the absence of
a membrane potential in the ER (23). Our findings imply that the more
frequent translocation of positive charges in eukaryotic systems
requires additional topogenic signals, such as the basic cluster
described here. Interestingly, clusters of basic charges have been
shown recently (40) by computer analysis to occur more frequently in
cytoplasmic loops of proteins near the cytoplasmic membrane surface
than predicted from the abundance of Arg and Lys residues. It is likely
that topogenic basic clusters act through electrostatic interactions
with negative charges of lipids or proteins (or both). Proteins of the
translocation machinery appear not to harbor relevant charged residues
(27), but anionic phospholipids, at least in prokaryotes, are
determinants of membrane protein topology because of the electrostatic
interaction of their head groups with arginine and lysine residues (12,
41, 42). Interestingly, successful solubilization of the GlyR but not
nicotinic acetylcholine receptors has been found to depend stringently
on the presence of exogenous phospholipids (43). This is consistent with a role of lipids in the conformational stabilization of GlyR subunits. Whether the topogenic basic cluster identified here contributes to phospholipid stabilization of GlyR structure by ensuring
its proper topology remains to be determined.