From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
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ABSTRACT |
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The rat brain voltage-gated Na+
channel is composed of three glycoprotein subunits: the pore-forming
subunit and two auxiliary subunits,
1 and
2, which contain
immunoglobulin (Ig)-like folds in their extracellular domains. When
expressed in Xenopus oocytes,
1 modulates the gating
properties of the channel-forming type IIA
subunit, resulting in an
acceleration of inactivation. We have used a combination of deletion,
alanine-scanning, site-directed, and chimeric mutagenesis strategies to
examine the importance of different structural features of the
1
subunit in the modulation of
IIA function, with an
emphasis on the extracellular domain. Deletion analysis revealed that
the extracellular domain is required for function, but the
intracellular domain is not. The mutation of four putative sites of
N-linked glycosylation showed that they are not required
for
1 function. Mutations of hydrophobic residues in the core
sheets of the Ig fold disrupted
1 function, whereas substitution of
amino acid residues in connecting segments had no effect. Mutations of
acidic residues in the A/A' strand of the Ig fold reduced the
effectiveness of the
1 subunit in modulating the rate of
inactivation but did not significantly affect the association of the
mutant
1 subunit with the
IIA subunit or its effect
on recovery from inactivation. Our data suggest that the Ig fold of the
1 extracellular domain serves as a scaffold that presents the
charged residues of the A/A' strands for interaction with the
pore-forming
subunit.
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INTRODUCTION |
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The rat brain voltage-gated sodium (Na+) channel is
composed of three glycoprotein subunits: the pore-forming subunit
with a relative molecular mass of 260 kDa, and two auxiliary subunits,
1 (36 kDa) and
2 (33 kDa) (for review, see Refs. 1 and 2). The
subunit has four internally homologous domains, each containing six
potential transmembrane-spanning regions and a pore-forming loop (3).
The
1 and
2 subunits from rat brain are not closely related in
terms of amino acid sequence, but each contains a single membrane-spanning segment that separates a large
NH2-terminal extracellular domain from a smaller
COOH-terminal intracellular domain (4, 5). In addition, the
extracellular domains of the
1 and
2 subunits have sequences
similar to those of proteins of the immunoglobulin
(Ig)1 superfamily and are
proposed to contain an Ig-like motif (5, 6). The
2 subunit is linked
covalently to the
subunit by disulfide bonds, whereas the
1
subunit associates with the
subunit in a noncovalent manner (7).
Biochemical analyses of detergent-solubilized rat brain Na+
channels suggest that both ionic and hydrophobic interactions are
important in association of
1 with the
·
2 complex (7).
Although the auxiliary subunits are not required for the formation of
functional Na+ channels (8, 9), coexpression of the 1
subunit with the rat brain type IIA
subunit in Xenopus
oocytes increases the proportion of Na+ channels that
function in a fast gating mode (4, 10). Na+ channel
inactivation is accelerated 5-fold, the voltage dependence of
inactivation is shifted in the negative direction, and a larger fraction of channels recovers rapidly from inactivation. In addition, the amplitude of peak Na+ current is increased, consistent
with an increase in channel expression. In Chinese hamster lung cells,
coexpression of
1 with the type IIA
subunit results in
hyperpolarizing shifts in the voltage dependence of Na+
channel activation and inactivation and increased channel expression (11). The
1 subunit modulates the gating and expression of a variety
of Na+ channel
subunits (10, 12-14), suggesting that
the interaction domains of these two proteins are well conserved.
In the present study we examine the structural features of the 1
subunit which are required for efficient modulation of rat brain type
IIA
subunits. We demonstrate that the intracellular domain of
1
is unnecessary and that the extracellular domain is essential for
expression and function of the
·
1 complex in Xenopus
oocytes. A combinatorial approach using deletion mutagenesis, alanine-scanning mutagenesis, and chimeric protein analyses supports the hypothesis that the extracellular domain forms an Ig fold that is
essential for
1 expression and function. Through analysis of these
mutants we have implicated the A/A' strand of the Ig fold in the
1
extracellular domain in interactions between
1 and
subunits.
This region is localized on a presumed surface-exposed edge of the Ig
fold motif. While our experiments were in progress, deletion and
chimeric mutagenesis studies of the modulation of skeletal muscle
Na+ channels by
1 subunits were reported (15, 16). These
results are compared with our data under "Discussion."
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction--
The deletion of the 1 intracellular
domain (Fig. 1A,
1)
and the large internal deletion of the
1 extracellular domain (Fig. 1A,
2) were constructed in the Bluescript
pSK
phagemid (Stratagene, La Jolla, CA). Subsequent
analyses of wild-type and mutant
1 subunits were performed in the
p
1.SK+ vector in which the entire coding sequence of the
rat brain
1 subunit and 3'-untranslated region, including
polyadenylation sequences, were cloned into Bluescript pSK+
(Stratagene). The p
1.SK
and p
1.SK+
vectors were constructed in a manner that eliminated a portion of the
1 5'-untranslated region which had been found previously to
interfere with expression (10).
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Mutagenesis--
The deletion of the 1 intracellular domain
(Fig. 1A,
1) was accomplished via polymerase
chain reaction mutagenesis by the substitution of a termination signal
(TAA) for the Ile166 codon. The large internal deletion
(Fig. 1A,
2) of the
1 extracellular domain
was constructed by an in-frame removal of a BstYI fragment. Two sets of three nested deletions in the cDNA encoding the
extracellular domain of the
1 subunit were produced by
oligonucleotide-directed "loop out" deletion mutagenesis (Fig.
1A,
3-
8). The NH2-terminal signal sequence was maintained in all deletion mutants to facilitate proper membrane insertion and post-translational processing. Single stranded p
1.SK+ DNA was prepared by helper phage
infection and served as the mutagenesis template. Deletion mutagenesis
reactions were performed using the Oligonucleotide-directed In
Vitro Mutagenesis System Version 2 or SculptorTM
protocols (Amersham Corp.). Ala substitutions, A/A' strand charge neutralizations, and
1/P0 chimeras were constructed in
B1.M13Mp18 by oligonucleotide-directed mutagenesis. Mutagenesis
reactions were according to the protocol of Kunkel et al.
(17). Mutants were identified by DNA sequencing or by incorporation of
a silent restriction endonuclease site and then subcloned into the
p
1.SK+ phagemid. Sequences of the mutant constructs were
determined by automated sequence analysis performed by the DNA Core of
the Molecular Pharmacology Facility, Department of Pharmacology,
University of Washington.
In Vitro Transcription of RNA and Expression of Na+
Channels in Xenopus Oocytes--
RNA transcripts were obtained using
the mMessage mMachine in vitro transcription protocol
(Ambion, Inc., Austin, TX). Transcripts were purified by sedimentation
through columns of Sephadex G-50. The rat IIA subunit
was transcribed using T7 RNA polymerase from plasmid pVA2580 (18),
which had been linearized with ClaI. The
1
1 construct
was linearized with HindIII, and RNA was transcribed with T7
RNA polymerase. The
1
2 construct was linearized with EcoRI, and RNA was transcribed with T7 RNA polymerase.
Wild-type and mutant
1 constructs in p
1.SK+ were
linearized with HindIII, and RNA was transcribed using T3 RNA polymerase. Xenopus laevis oocytes were harvested,
defolliculated with collagenase, and maintained as described (19).
Oocytes were injected with a 50-nl volume of an RNA mixture containing 0.5-1.25 ng of
IIA transcript and a 5-20-fold excess
of mutant
1 subunit transcript, unless otherwise stated in the
figure legends. Competition experiments were employed to determine if
the ability to associate with the
IIA subunit was
retained in mutant
1 subunits that showed decreased modulatory
1
function. In these experiments,
IIA and wild-type
1
subunits were injected in combination with the mutant
1 subunit. If
the mutant
1 subunit is capable of binding to the
IIA
subunit, less than maximum
1 function should be observed when
analyzed by electrophysiological methods. Alternatively, wild-type
1
function in the expressing mutant
1 oocytes would indicate an
inability of the mutant
1 to bind to the
IIA subunit. In all experiments, oocytes injected with only
IIA
transcript or
IIA coinjected with wild-type
1 RNA
served as controls.
Electrophysiological Recording--
After 48 h at 20 °C,
expressed Na+ channels were examined at room temperature by
two-electrode voltage clamp (Dagan CA1, Minneapolis, MN; see Ref. 19).
Voltage pulses were applied, and data were recorded using the
Basic-Fastlab data acquisition system (Indec Systems, Capitola, CA).
Linear capacity currents were canceled using the internal voltage clamp
circuitry. Residual linear currents were subtracted using the P/4
procedure (20). Signals were low pass filtered at 2 kHz. The amplitude
of expressed Na+ currents was typically 1-10 µA. The
bath was perfused continuously with Frog Ringer containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl2, 10 mM HEPES, pH 7.2. Na+
currents were elicited by step depolarizations to 0 mV from a holding
potential of 90 mV. Prepulses of 100-ms duration to various voltages,
followed by a test pulse to 0 mV, were used to examine the voltage
dependence of steady-state fast inactivation of Na+
channels. Na+ channel current-voltage relationships were
determined from peak currents elicited by 30-ms steps to various test
voltages from a holding potential of -90 mV. Recovery from inactivation
was examined by applying a 15-ms conditioning pulse to 0 mV from a holding potential of -90 mV followed by a recovery interval of variable
duration and a test pulse to 0 mV.
Statistical Analysis of Electrophysiological
Data--
Representative normalized current traces obtained from
oocytes coinjected with IIA and mutant
1 RNA
transcripts were plotted with normalized averaged current traces from a
contemporaneous pool of control oocytes containing only
IIA transcript or
IIA in combination with
wild-type
1 transcripts. The standard deviation of the control
oocyte traces was calculated and is plotted as dotted lines
in each figure.
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RESULTS |
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The Intracellular Domain of the Rat Brain 1 Subunit Is Not
Required for Modulation of
Subunit Function--
An important step
in understanding the mechanism by which the
1 subunit modulates
subunit function is to determine which domains of
1 participate in
-
1 interactions and subsequent modulation of channel gating. To
test the role of the intracellular domain, a truncated rat brain
1
subunit, lacking the intracellular domain, was constructed by the
insertion of a stop codon at the position normally encoding
Ile166 (Fig. 1A,
1). Lys residues
at positions 164 and 165, immediately following the proposed
transmembrane segment, were retained to maintain proper membrane
orientation. Xenopus oocytes were prepared and coinjected
with in vitro transcribed RNA encoding the adult rat type
IIA
subunit alone or in combination with wild-type or mutant
1
subunits. Na+ currents were recorded from the injected
oocytes at room temperature using a two-electrode voltage clamp.
Na+ channels containing only
IIA subunits
inactivated slowly (Fig. 1B, trace a), whereas
those containing
IIA and wild-type
1 inactivated more
rapidly (Fig. 1B, trace b). Coinjection of RNA
encoding the truncated
1 subunit and
IIA subunit in
Xenopus oocytes resulted in rapidly inactivating
Na+ currents that were similar to those seen when the
IIA was coexpressed with the wild-type
1 subunit
(Fig. 1B, trace c). Because both the wild-type
and truncated
1 subunits induced predominantly fast inactivating
channels, the intracellular domain of the
1 subunit is not necessary
for either the association of Na+ channel
and
1
subunits or for functional modulation of the
subunit by the
1
subunit.
Deletion Analysis of the Extracellular Domain of 1--
The
importance of the extracellular domain was investigated further by a
series of deletion mutations (Fig. 1A). An internal deletion
of the extracellular domain was constructed which removed amino acids
Ile51 through Lys122 (Fig. 1A,
2). When the
subunit was coexpressed with the
Ile51-Lys122 deletion mutant in
Xenopus oocytes, the resulting Na+ currents were
similar to those of channels composed of
subunits alone (data not
shown), indicating that the large internal deletion of the
extracellular domain prevented expression and/or function of the
1
subunit. To investigate further the importance of the extracellular
domain, nested deletions in the cDNA encoding the extracellular
domain of the
1 subunit were produced by oligonucleotide-directed loop out mutagenesis (Fig. 1A,
3-
8). The
time courses of Na+ currents resulting from coinjection of
with the smallest of these
1 deletion mutants (
3,
Gly1-Phe40, Fig. 1B, trace
d;
6 Val119-Ser140, Fig. 1B,
trace e) indicated that they decayed at a rate similar to
channels consisting of only the
subunit. Similar results were
obtained for each electrophysiological parameter tested for these and
the remaining deletion mutants. In addition,
1 subunits harboring
either
3 (Gly1-Phe40) or
6
(Val119-Ser140) were unable to compete with the
wild-type
1 subunit for binding to
IIA (data not
shown; for a description of the competition experiments, see
"Experimental Procedures"). These results highlight the requirement
for the integrity of the extracellular domain for function of the
1
subunit.
Analysis of N-Linked Glycosylation Sites--
The extracellular
domain of the 1 subunit contains four potential sites for
N-linked glycosylation at Asn74,
Asn91, Asn95, and Asn116 (4), and
biochemical experiments show that
1 subunits have at least three
N-linked carbohydrate chains (21). To investigate the
importance of these sites in the modulation of Na+ channel
gating by the
1 subunit, the Asn residues of all four sites were
mutated individually to Gln. These mutant subunits showed no
significant loss of
1 function, as assessed in the Xenopus oocyte expression system (Fig.
2). The adjacent glycosylation sites at
Asn91 and Asn95 were also altered
simultaneously. The resulting
1 double mutant behaved normally, like
the single site mutants (Fig. 2). These results show that none of the
N-linked glycosylation sites is required for expression and
function of
1 subunits.
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The Extracellular Domain of 1 Is Predicted to Form an Ig
Fold--
Data-base searches found sequence similarities between the
1 extracellular domain and members of the Ig fold superfamily (5,
6). The resemblance is closest with the V-like family of Ig fold
proteins, which contain domains resembling the variable regions of
antibodies and include many cell adhesion molecules and cell
recognition proteins (22, 23). The
1 extracellular domain has the
highest degree of similarity to that of myelin protein zero
(P0), with 36% identity and 49% similarity over a 70-amino acid segment (5). P0 is the major protein in
peripheral nerve myelin and mediates the wrapping of myelin sheaths via
homophilic interactions (23). Its structure has been studied by
molecular modeling (24), and the crystal structure of the rat
P0 extracellular domain (P0ex) was determined
recently at 1.9 Å resolution (25). Both analyses show that
P0ex is folded as a V-like Ig domain.
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Analysis of the Core Strands by Alanine-scanning Site-directed
Mutagenesis--
The Ig fold motif is a "sandwich" of two
sheets. Side chains from both
sheets form a hydrophobic core at the
interface between the
sheets by interaction of the alternating
hydrophobic amino acids in the primary sequence of core
strands.
The central hydrophobic core, composed primarily of
strands B and E
in one sheet and strands C, C', and F in the other, is crucial for the maintenance of the Ig fold (22). The central core of the Ig fold serves
as a scaffold for the presentation of sites involved in molecular
recognition, cell adhesion, and ligand binding.
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Mutations of Loops Connecting the Strands of the Ig Fold of the
1 Subunit--
In Ig fold proteins such as antibody variable
domains and cell adhesion molecules, the sites of interaction with
ligands are generally confined to the hypervariable segments connecting
the core
strands (22, 23). Many of the loop regions of the
1 extracellular domain do not contain large hydrophobic amino acids and
therefore are not investigated easily by alanine-scanning mutagenesis.
To investigate the importance of these loops for
1 function,
chimeric proteins were constructed in which the loops connecting the
strands of the rat brain Na+ channel
1 subunit were
replaced with the corresponding amino acids of myelin P0
protein. Although predicted to be topologically similar to
1, the
rat isoform of myelin P0 does not modulate Na+
channel gating in the Xenopus oocyte expression system (data not shown), indicating that differences in specific amino acid residues
within these two closely similar structures must be responsible for
interaction with Na+ channel
subunits.
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Hydrophobic Amino Acid Residues in the Juxtamembrane Region of the
1 Subunit Are Not Required for
1 Function--
The Ig fold of
the
1 subunit is connected to its transmembrane domain by a
juxtamembrane domain of 11 residues (Fig. 3A). We felt that
this region was a good candidate for interaction with the
IIA subunit because it is located near the membrane surface, where it could potentially interact with even the smallest extracellular loops of
IIA. Positions
Met135, Ile138, and Val139 in the
juxtamembrane region were targeted because of the propensity of
hydrophobic residues for protein interaction sites (32). Individual Ala
substitutions of these amino acid positions were constructed, as well
as an Ala cluster mutation (AS-9) in which all three hydrophobic amino
acid residues in this segment were replaced by Ala (Fig.
4A). The cluster mutation and each of the individual Ala
substitutions retained
1 function that was comparable to wild-type
1 (Fig. 6, traces c,
d, e, and f). These results indicate
that the hydrophobic amino acids of the
1 juxtamembrane region do
not play an important role in either the association of
1 with the
subunit or modulation of channel gating by
1.
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Neutralization of Negative Charges in the A/A' Strands Results in a
Partial Loss of 1 Function--
As in the variable domain of
antibodies, the A strand of the myelin P0 Ig fold motif is
broken into two short
strands, A and A' (Fig. 3B and
Ref. 25). In such an Ig fold, the A strand is hydrogen-bonded to the B
strand of the BED sheet, whereas the A' strand forms hydrogen bonds
with the G strand of the opposite GFC sheet (23, 25). Through these
hydrogen bonds, the A and A' strands contribute to the stability of the
core of the Ig fold. As in rat myelin P0, the A strand of
the Na+ channel
1 subunit contains a
strand-breaking
Asp residue at position 6, indicating that this segment of
1 may
also be broken into the smaller A and A'
strands. In Ig folds of
cell adhesion molecules and antibody variable domains, the A and A'
strands form one edge of the
sheet sandwich. In myelin
P0, this edge is predicted to lie near the membrane
surface, thus the corresponding region of the Na+ channel
1 subunit is a good candidate for interaction with extracellular loops of the pore-forming
subunit. The primary sequence of the A
and A' strands of
1 is unusual because alternating amino acid residues between positions 4 and 10 are negatively charged. In a
sheet structure where the intervening amino acids are involved in
intersheet hydrogen bonding, these negative charges would be arrayed on
the external surface of the Ig fold. Therefore, these charged residues
may be accessible for interaction with extracellular regions of the
Na+ channel
subunit and may contribute to the ionic
interactions that were found to play a large role in the stability of
the purified
·
1 complex (7).
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DISCUSSION |
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The Protein Component, but Not the Carbohydrate Component, of the
Extracellular Domain Is Required for 1 Function--
Deletion of
the intracellular domain of the
1 subunit does not affect modulation
of the brain type IIA
subunit, but deletion of segments of the
extracellular domain does prevent Na+ channel modulation by
1 subunits. Similarly, deletion analysis showed that the
intracellular domain of human
1 is not required for modulation of
the skeletal muscle
subunit by
1 subunits, whereas the
extracellular domain is sensitive to deletion mutagenesis (15). In
addition, the
1 extracellular domain, together with proximal
residues of the transmembrane domain, was found to be sufficient for
modulation of the skeletal muscle Na+ channel in chimeric
subunits formed with Na+ channel
2 subunits (16). Taken
together, these studies demonstrate that the extracellular domain is
required for modulation of both brain and skeletal muscle
subunits.
Core Sheets of the Ig Fold, but Not the Connecting Loops, Are
Required for
1 Function--
The results of the alanine-scanning
mutagenesis of hydrophobic residues within the core
strands support
the hypothesis that the extracellular domain of
1 contains an
essential Ig fold motif. All mutations that altered clusters of
hydrophobic residues in the core
strands prevented
1 function,
as expected if they interrupt the
sheet interactions in the core of
the Ig fold. If the hydrophobic core of the Ig fold was disrupted by
the cluster mutations, crucial
-
1 interaction sites may be lost
because of the misfolding of the Ig fold scaffold, or the mutations may have conferred a decrease in the expression or stability of
1. In
contrast, most mutations of the peripheral
sheets or connecting loops within the Ig fold did not alter
1 function. Normal
1 function was also observed for
1 subunits carrying mutations in the
juxtamembrane region, which is proposed to lie outside of the Ig fold.
Therefore, our working hypothesis is that the core of the Ig fold is
required to position specific molecular determinants appropriately for
interaction and modulation of the Na+ channel
subunit.
Acidic Residues in the A/A' Strand Are Required for Full 1
Function--
A region on the surface of the Ig fold was identified
which is essential for optimum modulation of gating of the
subunit. Neutralization of negative charges (E4QD6NE8Q) in the proposed A/A'
strand of the
1 Ig fold domain yielded Na+ channels with
very interesting electrophysiological characteristics. Two types of
data argue that this mutant
1 subunit assembles properly and binds
effectively to the
IIA subunit. First, it competes for
expression with the wild-type
1 subunit. Second, in experiments
studying recovery from inactivation, no slow phase of recovery was
observed, indicating that there were no free
subunits present.
Despite being bound to each
subunit, this mutant
1 subunit
consistently conferred rates of Na+ channel inactivation
which were intermediate between those seen for the
IIA
subunit coexpressed with the wild-type
1 subunit and those of
channels consisting of only the
IIA subunit (Fig. 7,
panels A and B). Further analysis revealed that
the fraction of channels that gate in the fast mode was significantly
reduced in channels containing the
1(E4QD6NE8Q) mutation (Fig.
7C). Taken together, the data suggest that the
neutralization of negative charges in the A/A' strand reduces the
change in gating mode effected by the
1 subunit without preventing
the association of
1 with the
subunit. Interestingly, because
the A' and G strands interact with each other in Ig V-like motifs (23,
25), the lack of effect of mutations in the G strand of mutant AS-8
(Fig. 4C, trace i) supports the conclusion that
the effects of mutations of charged amino acid residues in the A/A'
strand result from altered interactions with the
IIA
subunit rather than altered interactions with the G strand and the core
of the Ig fold. The edge of the
1 Ig fold domain that carries these
negatively charged residues may interact with the
subunit in a
manner that affects its rate of inactivation and gating mode.
Comparison with Cell Adhesion Molecules--
Members of the Ig
superfamily are typically involved in cell recognition and cell
adhesion. The inclusion of the Na+ channel auxiliary
subunits 1 and
2 in this superfamily suggests that these subunits
may have additional functions beyond modulation of channel expression
and gating properties. This hypothesis is supported by the observation
that the primary sequence of
2 has a region with significant
homology to the cell adhesion molecule contactin (5). It is interesting
that the A/A' strand of the
1 Ig fold, which we found to be
important for interactions with the
subunit, is not among the Ig
fold regions that are most often implicated in ligand binding or cell
adhesion. For example, in antibodies and T-cell receptors, two variable
domains dimerize to form an antigen binding site composed of residues
within the B-C, C'-C", and F-G loops, known as complementarity
determining regions. Head-to-head homophilic adhesion of Ig V-like
domains, such as those from CD2, typically involves the broad A'GFCC'C" face, and binding to integrins occurs primarily at the GFC face of cell
adhesion molecules (23). In myelin P0, hydrogen bonds between residues in the C' strand of partner molecules are believed to
be the most important determinants of homophilic adhesion (25). The
localization of
-
1 modulation determinants to a discrete edge of
the Ig fold leaves the faces of the Ig fold and the loops analogous to
the complementarity determining regions free for interactions with
proteins from the extracellular matrix or opposing cell membranes.
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ACKNOWLEDGEMENT |
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Plasmid pSN63, containing cDNA encoding rat myelin P0, was a generous gift from Dr. Greg Lemke, Salk Institute.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Research Grant NS25704 (to W. A. C.) and by National Research Service Award postdoctoral research fellowships from the National Institutes of Health (to K. A. M. and D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Pharmacology, University of Michigan,
Ann Arbor, MI 48109.
§ Present address: Montreal Neurological Inst., Montreal, Quebec H3A 2B4, Canada.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280.
1
The abbreviations used are: Ig, immunoglobulin;
Ff and
Fs, fraction of channel in fast and
slow gating mode, respectively; f and
s, time constants in fast and slow mode, respectively; P0, protein zero.
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REFERENCES |
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