Molecular Determinants of Na+ Channel Function in the Extracellular Domain of the beta 1 Subunit*

Kimberly A. McCormick, Lori L. IsomDagger , David Ragsdale§, David Smith, Todd Scheuer, and William A. Catterall

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The rat brain voltage-gated Na+ channel is composed of three glycoprotein subunits: the pore-forming alpha  subunit and two auxiliary subunits, beta 1 and beta 2, which contain immunoglobulin (Ig)-like folds in their extracellular domains. When expressed in Xenopus oocytes, beta 1 modulates the gating properties of the channel-forming type IIA alpha  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 beta 1 subunit in the modulation of alpha 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 beta 1 function. Mutations of hydrophobic residues in the core beta  sheets of the Ig fold disrupted beta 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 beta 1 subunit in modulating the rate of inactivation but did not significantly affect the association of the mutant beta 1 subunit with the alpha IIA subunit or its effect on recovery from inactivation. Our data suggest that the Ig fold of the beta 1 extracellular domain serves as a scaffold that presents the charged residues of the A/A' strands for interaction with the pore-forming alpha  subunit.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The rat brain voltage-gated sodium (Na+) channel is composed of three glycoprotein subunits: the pore-forming alpha  subunit with a relative molecular mass of 260 kDa, and two auxiliary subunits, beta 1 (36 kDa) and beta 2 (33 kDa) (for review, see Refs. 1 and 2). The alpha  subunit has four internally homologous domains, each containing six potential transmembrane-spanning regions and a pore-forming loop (3). The beta 1 and beta 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 beta 1 and beta 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 beta 2 subunit is linked covalently to the alpha  subunit by disulfide bonds, whereas the beta 1 subunit associates with the alpha 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 beta 1 with the alpha ·beta 2 complex (7).

Although the auxiliary subunits are not required for the formation of functional Na+ channels (8, 9), coexpression of the beta 1 subunit with the rat brain type IIA alpha  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 beta 1 with the type IIA alpha  subunit results in hyperpolarizing shifts in the voltage dependence of Na+ channel activation and inactivation and increased channel expression (11). The beta 1 subunit modulates the gating and expression of a variety of Na+ channel alpha  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 beta 1 subunit which are required for efficient modulation of rat brain type IIA alpha  subunits. We demonstrate that the intracellular domain of beta 1 is unnecessary and that the extracellular domain is essential for expression and function of the alpha ·beta 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 beta 1 expression and function. Through analysis of these mutants we have implicated the A/A' strand of the Ig fold in the beta 1 extracellular domain in interactions between beta 1 and alpha  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 beta 1 subunits were reported (15, 16). These results are compared with our data under "Discussion."

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Construction-- The deletion of the beta 1 intracellular domain (Fig. 1A, Delta 1) and the large internal deletion of the beta 1 extracellular domain (Fig. 1A, Delta 2) were constructed in the Bluescript pSK- phagemid (Stratagene, La Jolla, CA). Subsequent analyses of wild-type and mutant beta 1 subunits were performed in the pbeta 1.SK+ vector in which the entire coding sequence of the rat brain beta 1 subunit and 3'-untranslated region, including polyadenylation sequences, were cloned into Bluescript pSK+ (Stratagene). The pbeta 1.SK- and pbeta 1.SK+ vectors were constructed in a manner that eliminated a portion of the beta 1 5'-untranslated region which had been found previously to interfere with expression (10).


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Fig. 1.   Deletion of the intracellular domain of beta 1 has no detectable effect on beta 1 function, whereas partial deletions of the extracellular domain abolish function. Panel A, the mature rat brain Na+ channel beta 1 subunit, without the signal sequence, is depicted by a line drawing. The proposed transmembrane-spanning segment is labeled (tm), and putative N-linked glycosylation sites are indicated (psi ). Lines under the beta 1 diagram represent the fragments deleted from either the intracellular (Delta 1) or extracellular (Delta 2-Delta 8) domains. The letters in parentheses indicate the traces in panel B corresponding to each deletion mutant. Panel B, RNA encoding deletion mutant beta 1 subunits was coinjected with alpha IIA RNA into Xenopus oocytes, and Na+ currents were analyzed using a two-microelectrode voltage clamp. Na+ currents were elicited by a 30-ms test pulse to 0 mV from a holding potential of -90 mV in this and all subsequent figures. Representative normalized current traces are given for Delta 1 (c), Delta 3 (d), and Delta 6 (e). Traces a and b are normalized averages of current traces from oocytes injected with alpha IIA alone (n = 6) and oocytes coinjected with alpha IIA + wild-type beta 1 transcripts (n = 5), respectively. The dotted lines represent 1 S.D. for these control traces.

Mutagenesis-- The deletion of the beta 1 intracellular domain (Fig. 1A, Delta 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, Delta 2) of the beta 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 beta 1 subunit were produced by oligonucleotide-directed "loop out" deletion mutagenesis (Fig. 1A, Delta 3-Delta 8). The NH2-terminal signal sequence was maintained in all deletion mutants to facilitate proper membrane insertion and post-translational processing. Single stranded pbeta 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 beta 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 pbeta 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 alpha IIA subunit was transcribed using T7 RNA polymerase from plasmid pVA2580 (18), which had been linearized with ClaI. The beta 1Delta 1 construct was linearized with HindIII, and RNA was transcribed with T7 RNA polymerase. The beta 1Delta 2 construct was linearized with EcoRI, and RNA was transcribed with T7 RNA polymerase. Wild-type and mutant beta 1 constructs in pbeta 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 alpha IIA transcript and a 5-20-fold excess of mutant beta 1 subunit transcript, unless otherwise stated in the figure legends. Competition experiments were employed to determine if the ability to associate with the alpha IIA subunit was retained in mutant beta 1 subunits that showed decreased modulatory beta 1 function. In these experiments, alpha IIA and wild-type beta 1 subunits were injected in combination with the mutant beta 1 subunit. If the mutant beta 1 subunit is capable of binding to the alpha IIA subunit, less than maximum beta 1 function should be observed when analyzed by electrophysiological methods. Alternatively, wild-type beta 1 function in the expressing mutant beta 1 oocytes would indicate an inability of the mutant beta 1 to bind to the alpha IIA subunit. In all experiments, oocytes injected with only alpha IIA transcript or alpha IIA coinjected with wild-type beta 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 alpha IIA and mutant beta 1 RNA transcripts were plotted with normalized averaged current traces from a contemporaneous pool of control oocytes containing only alpha IIA transcript or alpha IIA in combination with wild-type beta 1 transcripts. The standard deviation of the control oocyte traces was calculated and is plotted as dotted lines in each figure.

Additional statistical analyses were performed on current traces obtained from oocytes coinjected with alpha IIA and beta 1(E4Q/D6N/E8Q). To determine the mean fractions of Na+ channels in the slow and fast gating mode (Fs and Ff) and the corresponding time constants (tau s and tau f), the decay phase of Na+ current traces was fit to a single exponential or a sum of two exponentials. Current traces obtained from oocytes injected with only the alpha IIA transcript were well fit by a single exponential. Two exponentials were used to fit the traces obtained from oocytes coinjected with transcripts encoding alpha IIA and wild-type beta 1 or with alpha IIA and beta 1(E4Q/D6N/E8Q) transcripts. Through these analyses it was determined that the time constants obtained from current traces from oocytes expressing the alpha IIA and beta 1(E4QD6NE8Q) transcripts (tau s = 4.58 ± 1.42 ms,tau f = 0.917 ± 0.474 ms, n = 6) did not vary significantly from tau s values for alpha IIA control cells (tau s = 4.74 ± 0.62 ms, n = 6) or tau f values obtained from control oocytes that coexpressed the alpha IIA and wild-type beta 1 subunits (tau f = 0.892 ± 0.156 ms, n = 7). Student's t test p values for comparisons of the time constants for alpha IIA + beta 1(E4QD6NE8Q) with control time constants were 0.90 and 0.46 for tau s and tau f, respectively, confirming that the differences between these values were not significant. To obtain accurate values for the fraction of oocytes gating in the fast mode (Ff) and slow mode (Fs), current traces from oocytes expressing alpha IIA alone, alpha IIA and wild-type beta 1, or alpha IIA and beta 1(E4QD6NE8Q), and from oocytes expressing combinations of these subunits in competition experiments were refit using mean values of the control cell tau s and tau f values defined above. The values for Ff and Fs obtained from these fits were compared, and the significance of the observed differences was determined by t test analyses.

Data obtained from experiments measuring recovery from inactivation were normalized and fit with either one or two exponentials, as described in the figure legends. The Ff, Fs, and corresponding tau  values of channel recovery from inactivation were determined from the fits.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The Intracellular Domain of the Rat Brain beta 1 Subunit Is Not Required for Modulation of alpha  Subunit Function-- An important step in understanding the mechanism by which the beta 1 subunit modulates alpha  subunit function is to determine which domains of beta 1 participate in alpha -beta 1 interactions and subsequent modulation of channel gating. To test the role of the intracellular domain, a truncated rat brain beta 1 subunit, lacking the intracellular domain, was constructed by the insertion of a stop codon at the position normally encoding Ile166 (Fig. 1A, Delta 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 alpha  subunit alone or in combination with wild-type or mutant beta 1 subunits. Na+ currents were recorded from the injected oocytes at room temperature using a two-electrode voltage clamp. Na+ channels containing only alpha IIA subunits inactivated slowly (Fig. 1B, trace a), whereas those containing alpha IIA and wild-type beta 1 inactivated more rapidly (Fig. 1B, trace b). Coinjection of RNA encoding the truncated beta 1 subunit and alpha IIA subunit in Xenopus oocytes resulted in rapidly inactivating Na+ currents that were similar to those seen when the alpha IIA was coexpressed with the wild-type beta 1 subunit (Fig. 1B, trace c). Because both the wild-type and truncated beta 1 subunits induced predominantly fast inactivating channels, the intracellular domain of the beta 1 subunit is not necessary for either the association of Na+ channel alpha  and beta 1 subunits or for functional modulation of the alpha  subunit by the beta 1 subunit.

Deletion Analysis of the Extracellular Domain of beta 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, Delta 2). When the alpha  subunit was coexpressed with the Ile51-Lys122 deletion mutant in Xenopus oocytes, the resulting Na+ currents were similar to those of channels composed of alpha  subunits alone (data not shown), indicating that the large internal deletion of the extracellular domain prevented expression and/or function of the beta 1 subunit. To investigate further the importance of the extracellular domain, nested deletions in the cDNA encoding the extracellular domain of the beta 1 subunit were produced by oligonucleotide-directed loop out mutagenesis (Fig. 1A, Delta 3-Delta 8). The time courses of Na+ currents resulting from coinjection of alpha  with the smallest of these beta 1 deletion mutants (Delta 3, Gly1-Phe40, Fig. 1B, trace d; Delta 6 Val119-Ser140, Fig. 1B, trace e) indicated that they decayed at a rate similar to channels consisting of only the alpha  subunit. Similar results were obtained for each electrophysiological parameter tested for these and the remaining deletion mutants. In addition, beta 1 subunits harboring either Delta 3 (Gly1-Phe40) or Delta 6 (Val119-Ser140) were unable to compete with the wild-type beta 1 subunit for binding to alpha 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 beta 1 subunit.

Analysis of N-Linked Glycosylation Sites-- The extracellular domain of the beta 1 subunit contains four potential sites for N-linked glycosylation at Asn74, Asn91, Asn95, and Asn116 (4), and biochemical experiments show that beta 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 beta 1 subunit, the Asn residues of all four sites were mutated individually to Gln. These mutant subunits showed no significant loss of beta 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 beta 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 beta 1 subunits.


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Fig. 2.   Disruption of putative N-linked glycosylation sites in the extracellular domain of beta 1 did not affect its function. Representative normalized current traces from Xenopus oocytes coinjected with alpha IIA RNA and RNA carrying the mutations beta 1N74Q (trace c), beta 1N91Q (trace d), beta 1N95Q (trace e), beta 1N116Q (trace f), and beta 1N91Q/N95Q (trace g) are shown. Traces a and b are averages of current traces from oocytes injected with alpha IIA alone and oocytes coinjected with alpha IIA + wild-type beta 1 transcripts, respectively, as described in the legend to Fig. 1B.

The Extracellular Domain of beta 1 Is Predicted to Form an Ig Fold-- Data-base searches found sequence similarities between the beta 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 beta 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.

Alignment of the amino acid sequences of the beta 1 subunit and P0ex (Fig. 3A) reveals that both conform closely to a consensus sequence for V-like Ig folds, like many cell adhesion molecules (23). The beta  strands A-G that form the Ig fold are highlighted in Fig. 3A and are illustrated in their three-dimensional organization in Fig. 3B. In this alignment Cys21 of the beta 1 subunit is shown in register with the Cys residues that form the disulfide bond in the Ig fold of P0 rather than Cys24 as in our previous alignment (6), because this fits the new x-ray structure (25) and the new V-like consensus (23) more closely. The remaining Cys residues of the beta 1 extracellular domain, Cys2 and Cys24, are in close proximity in our Ig fold model and may form an additional disulfide bridge between the beginning of the A strand and the end of the B strand. In addition to the invariant Cys residues in beta  strands B and F, other important Ig- fold residues, such as the Gly preceding strand B, the Trp in strand C, and the Arg in strand D, are aligned precisely in the folding model of beta 1 (Fig. 3B), and many other requirements for a V-like Ig fold are fulfilled (Fig. 3A). Myelin protein P0 therefore provides a molecular template for analysis of the structure of the beta 1 subunit based on its three-dimensional structure illustrated in Fig. 3B.


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Fig. 3.   The extracellular domains of the Na+ channel beta 1 subunit and myelin P0 share sequence similarities that are characteristic of Ig V-like motifs. Panel A, the primary sequence of the extracellular domains of the rat brain Na+ channel beta 1 subunit (bottom line) and myelin P0 from rat and shark (top and middle lines, respectively) were aligned using the Multiple Alignment Program (34). beta  strand assignments, shown above the alignment, were determined from the crystal structure of the rat myelin P0 extracellular domain (25). A consensus sequence (23) for Ig V-like motifs is given below the alignment with the following legend: *, hydrophobic amino acids; +, basic amino acids; #, Gly, Ala, or Asp; ×, any amino acid. Disulfide-linked Cys residues of myelin P0 are in bold, as are the corresponding beta 1 Cys residues. Based upon secondary structure predictions, the F strand of beta 1 may be longer than that of P0. Identities are highest in beta  strands B, C, E, and F, which contribute to the hydrophobic core of the Ig fold. Panel B, ribbon diagram of the Ig V-like motif of the rat myelin P0 extracellular domain, adapted from Ref. 25. The relative positions of beta 1 amino acids that are discussed in detail in the text are indicated.

Analysis of the Core beta  Strands by Alanine-scanning Site-directed Mutagenesis-- The Ig fold motif is a "sandwich" of two beta  sheets. Side chains from both beta  sheets form a hydrophobic core at the interface between the beta  sheets by interaction of the alternating hydrophobic amino acids in the primary sequence of core beta  strands. The central hydrophobic core, composed primarily of beta  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.

Alanine-scanning mutagenesis (26) was employed to test whether the central core of the Ig fold is necessary for expression and function of the beta 1 subunits. Ala was chosen as the replacement amino acid because it has a small side chain and does not introduce electrostatic effects or alter the main chain conformation. Although Ala is a strong alpha -helix former, it shows a significant propensity for beta  sheet formation and is found in both buried and exposed positions (27-30). Replacement of hydrophobic amino acid residues in the core of proteins with Ala has been shown not to cause global structural change (31). However, in the case of an Ig fold, substitution of Ala for multiple hydrophobic amino acids in a beta  strand that contributes to the hydrophobic core of the Ig fold should disrupt the tertiary structure of the domain.

The alanine-scanning mutagenesis survey of proposed beta  strands in the beta 1 extracellular domain is summarized in Fig. 4A. The substitution of Ala for multiple large, hydrophobic amino acids in the mutants AS-1, AS-2, AS-3, AS-5, and AS-6 resulted in slowly inactivating Na+ currents resembling those exhibited by the alpha  subunit in the absence of beta 1 (Fig. 4B). In AS-2, the substitution of Ala for three amino acids in the C strand, Phe35, Trp38, and Phe40, resulted in a loss of beta 1 function (Fig. 4B, traced). A Trp residue at the position of Trp38 is highly conserved among V-like domains (22, 23). In myelin P0, the side chain of this Trp residue packs against the disulfide bond formed between beta  strands B and F in the hydrophobic interface between the two beta  sheets (25). The substitution of Ala for Trp38, combined with the F35A and F40A mutations most likely resulted in a perturbation of the Ig fold hydrophobic core. A similar disruption of hydrophobic interactions in the central core of the Ig fold is likely to be responsible for the effects of mutations AS-1, AS-3, AS-5, and AS-6. None of beta 1 subunits harboring the deleterious alanine cluster mutations was capable of competing with the wild-type beta 1 subunit for binding to alpha IIA (data not shown), which suggests that the alanine substitutions either dramatically inhibited alpha -beta 1 interactions or prevented beta 1 folding and cell surface expression.


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Fig. 4.   Analysis of the core and non-core beta  strands of the beta 1 Ig fold motif by alanine-scanning mutagenesis. Panel A, Ala cluster mutations (AS-1 through AS-9) were constructed in putative central core and non-core beta  strands of the beta 1 extracellular Ig fold domain. Represented by A are mutations that had effects on beta 1 function, and a represents mutations that showed no detectable effects on function. The labels (c-i) correspond to the traces in panels B and C. Putative N-linked glycosylation sites are indicated by bold type (N). The juxtamembrane mutation AS-9 is analyzed in Fig. 6. Panel B, representative normalized current traces are shown for mutations in core beta  strands B, C, C', E, and F, which caused a loss of all detectable beta 1 function. Traces c, d, e, g, and h were recorded from oocytes injected with the mutant beta 1 subunits defined in panel A. Panel C, current traces from Ala cluster mutations in non-core beta  strands D and G, which did not affect beta 1 function, are shown in traces f and i, as defined in panel A. Traces a and b of panels B and C are averages of current traces from oocytes injected with alpha IIA alone and oocytes coinjected with alpha IIA + wild-type beta 1 transcripts, respectively, as described in the legend to Fig. 1B.

In contrast to the effects of mutations in these central core beta  strands, the mutation of three hydrophobic amino acid residues in the D strand in the AS-4 mutant (Fig. 4C, trace f) had no effect on expression and function of beta 1 subunits. A similar result was observed for the AS-8 mutant, which targeted the peripherally located G strand (Fig. 4C, trace i). Our findings show that perturbation of the central hydrophobic core structure of the Ig fold prevents beta 1 function whereas similar mutations in peripheral beta  strands have little effect. Thus, it is likely that hydrophobic amino acids in the central segments contribute to the stability of the core of the Ig fold and that disruption of the central core of the Ig fold prevents beta 1 expression and function. These results are consistent with the hypothesis that the Ig fold forms a scaffold to present interaction site(s) to the alpha  subunit for specific association.

Mutations of Loops Connecting the beta  Strands of the Ig Fold of the beta 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 beta  strands (22, 23). Many of the loop regions of the beta 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 beta 1 function, chimeric proteins were constructed in which the loops connecting the strands of the rat brain Na+ channel beta 1 subunit were replaced with the corresponding amino acids of myelin P0 protein. Although predicted to be topologically similar to beta 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 alpha  subunits.

Substitutions of clusters of amino acid residues from the myelin P0 protein into the B-C, C-C', C'-C", C"-D, and D-E loops were made as diagrammed in Fig. 5A. Surprisingly, all five beta 1/P0 chimeras functioned like wild-type beta 1 (Fig. 5, B and C). In addition, because the proposed D-E loop of beta 1 is longer than the corresponding loop of myelin P0, we also studied deletion and single amino acid mutations in that loop. Three amino acids, Thr79, Lys80, and Asp81 were deleted from the D-E loop, with no discernible effect on beta 1 function (data not shown). In addition, the simultaneous substitution of Leu82 and Leu85 with Ala resulted in no detectable loss of beta 1 function (data not shown). Taken together, these results suggest that the amino acid residues replaced or deleted in the loop regions we examined do not contribute significantly to alpha -beta 1 interactions and that the additional length of the beta 1 D-E loop is not an important factor for these interactions.


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Fig. 5.   Effects of chimeric beta 1/P0 proteins targeting the loops connecting the beta  strands of the beta 1 Ig fold motif. Panel A, substitutions of clusters of amino acid residues from the shark myelin P0 protein into the proposed Ig fold loops B-C, C-C', C'-C", C"-D, and D-E loops of the rat brain Na+ channel beta 1 subunit were made as diagrammed. Letters in parentheses indicate the current traces in panels B and C corresponding to each mutant. Putative N-linked glycosylation sites are indicated by bold type (N). Panels B and C, representative normalized current traces from Xenopus oocytes coinjected with alpha IIA RNA and RNA encoding chimeras P0-1 through P0-5, are shown (traces c-g). Traces a and b of panels B and C are averages of current traces from oocytes injected with alpha IIA alone and oocytes coinjected with alpha IIA + wild-type beta 1 transcripts, respectively, as described in the legend to Fig. 1B.

Hydrophobic Amino Acid Residues in the Juxtamembrane Region of the beta 1 Subunit Are Not Required for beta 1 Function-- The Ig fold of the beta 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 alpha IIA subunit because it is located near the membrane surface, where it could potentially interact with even the smallest extracellular loops of alpha 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 beta 1 function that was comparable to wild-type beta 1 (Fig. 6, traces c, d, e, and f). These results indicate that the hydrophobic amino acids of the beta 1 juxtamembrane region do not play an important role in either the association of beta 1 with the alpha  subunit or modulation of channel gating by beta 1.


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Fig. 6.   Hydrophobic amino acid residues in the proposed juxtamembrane region of the beta 1 extracellular domain are not required for beta 1 function. Channels containing alpha IIA together with beta 1(M135A/I138A/V139A) (trace c), beta 1(M135A) (trace d), beta 1(I138A) (trace e), or beta 1(V139A) (trace f) displayed inactivation kinetics that fell into the range observed for alpha IIA with wild-type beta 1 (trace b). Traces a and b are averages of current traces from oocytes injected with alpha IIA alone and oocytes coinjected with alpha IIA + wild-type beta 1 transcripts, respectively, as described in the legend to Fig. 1B.

Neutralization of Negative Charges in the A/A' Strands Results in a Partial Loss of beta 1 Function-- As in the variable domain of antibodies, the A strand of the myelin P0 Ig fold motif is broken into two short beta  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 beta 1 subunit contains a beta  strand-breaking Asp residue at position 6, indicating that this segment of beta 1 may also be broken into the smaller A and A' beta  strands. In Ig folds of cell adhesion molecules and antibody variable domains, the A and A' strands form one edge of the beta  sheet sandwich. In myelin P0, this edge is predicted to lie near the membrane surface, thus the corresponding region of the Na+ channel beta 1 subunit is a good candidate for interaction with extracellular loops of the pore-forming alpha  subunit. The primary sequence of the A and A' strands of beta 1 is unusual because alternating amino acid residues between positions 4 and 10 are negatively charged. In a beta  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 alpha  subunit and may contribute to the ionic interactions that were found to play a large role in the stability of the purified alpha ·beta 1 complex (7).

We examined the importance of the negative charges in the A and A' strands by site-directed mutagenesis. A beta 1 mutant was created in which three of the four negatively charged residues in the A and A' strands were neutralized (E4Q/D6N/E8Q). Care was taken to replace the native residues with amino acids of equal or better beta  sheet-forming propensity (30). The analysis of oocytes coinjected with alpha IIA and beta 1(E4QD6NE8Q) transcripts (1:1 w/w) showed that the beta 1 A/A' strand mutant was capable of only partial modulation of alpha  subunit function, as assessed from the macroscopic kinetics of Na+ channel inactivation (Fig. 7A, compare traces a-c). The rate of inactivation of channels containing the A/A' strand mutant beta 1 (trace c) was faster than for alpha  subunits alone (trace a), but significantly slower than those of channels comprised of alpha IIA and wild-type beta 1 (trace b). To test whether this difference was caused by poor expression of beta 1(E4QD6NE8Q), oocytes were injected with alpha IIA transcript plus a 10-fold excess of the beta 1(E4QD6NE8Q) transcript. As with the lower concentration of beta 1(E4QD6NE8Q) transcript, the rate of inactivation of the beta 1 A/A' strand mutant was substantially slower than that observed for cells injected with a 1:10 weight ratio of alpha IIA to wild-type beta 1 (Fig. 7B, compare traces b and c). Similar results were obtained with a 50-fold excess of beta 1(E4QD6NE8Q) injected mRNA (data not shown). In addition, the rate of inactivation for channels containing the beta 1 A/A' strand mutant channels was consistently slower than for channels containing wild-type beta 1 at test pulses from 0 mV to +20 mV (data not shown).


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Fig. 7.   Neutralization of negatively charged residues in the A/A' strand reduces the extent of modulation of Na+ channel inactivation but does not prevent association of beta 1 with the alpha  subunit. Panel A, normalized averaged current traces were obtained from oocytes expressing alpha IIA alone (n = 6, trace a), alpha IIA and wild-type beta 1 (1:1 w/w, n = 4, trace b), and alpha IIA and beta 1(E4Q/D6N/E8Q) (1:1 w/w, n = 5, trace c). The ability of beta 1(E4Q/D6N/E8Q) to compete with wild-type beta 1 for binding and modulation of alpha IIA was determined by injection of transcripts encoding alpha IIA, wild-type beta 1, and beta 1(E4Q/D6N/E8Q) at weight ratios of 1:1:1 (n = 3, trace d) and 1:1:10 (n = 3, trace e). Currents were elicited as described in the legend to Fig. 1B. 1 S.D. of the control current traces is represented by dotted lines. Panel B, normalized average current traces were obtained from oocytes expressing alpha IIA alone (n = 6, trace a), alpha IIA and wild-type beta 1 (1:10 w/w, n = 7, trace b), alpha IIA and beta 1(E4Q/D6N/E8Q) (1:10 w/w, n = 6, trace c). 1 S.D. of the control current traces is represented by dotted lines. Panel C, the decay time courses of the individual traces from panels A and B were fit to a sum of two exponentials, and the fraction of current represented by the fast exponential (Ff) was determined as described under "Experimental Procedures." The Ff range for each subunit combination is depicted by a box plot, in which the median is given by a solid line, and the 25th and 75th percentiles are represented by the lower and upper boundaries of the box, respectively. The error bars indicate the 10th and 90th percentiles in the data sets. Open circles represent outlier data points. Ff was calculated using fixed fast and slow time constants (see "Experimental Procedures"). Mean values for Ff were: alpha IIA + wild-type beta 1 (1:1 w/w), 0.9089 ± 0.0470; alpha IIA + beta 1(E4QD6NE8Q) (1:1 w/w), 0.686 ± 0.125; alpha IIA + wild-type beta 1 + beta 1(E4QD6NE8Q) (1:1:1 w/w/w), 0.829 ± 0.031; alpha IIA + wild-type beta 1 + beta 1(E4QD6NE8Q) (1:1:10 w/w/w), 0.742 ± 0.053; alpha IIA + wild-type beta 1 (1:10 w/w), 0.927 ± 0.035; alpha IIA + beta 1(E4QD6NE8Q) (1:10 w/w), 0.763 ± 0.085. Na+ current traces obtained from oocytes expressing only the alpha IIA subunit were found to have only a slow time constant (Ff = 0.0). Data sets indicated by an asterisk (*) were significantly different (p < 0.05) from the control data sets, alpha IIA alone and alpha IIA + wild-type beta 1 (at the appropriate weight ratio) but were not significantly different from each other. A p value of 0.053 resulted from the t test of Ff values of alpha IIA + wild-type beta 1 (1:1 w/w) versus alpha IIA + wild-type beta 1 + beta 1(E4QD6NE8Q) (1:1:1 w/w/w), indicating that these data may be significantly different. Panel D, normalized rates of recovery from inactivation are illustrated for oocytes expressing alpha IIA alone (bullet ), alpha IIA injected with a 10-fold excess of wild-type beta 1 RNA (black-square), or alpha IIA injected with a 10-fold excess of beta 1(E4Q/D6N/E8Q) (black-triangle). Recovery protocols were performed as described under "Experimental Procedures." Recovery data were fit with two exponentials for oocytes expressing alpha IIA alone. The mean fraction of current represented by the faster exponential (Ff), the slower exponential (Fs), the time constant of the fast fraction (tau f), and the time constant of the slow fraction (tau s) were: alpha IIA, Ff = 0.556 ± 0.045, tau f = 2.3 ± 0.3 ms, Fs = 0.414 ± 0.042, tau s = 245.0 ± 8.6 ms (n = 4). Recovery data for oocytes expressing alpha IIA + wild-type beta 1 (n = 5) and alpha IIA + beta 1 (E4Q/D6N/E8Q) (n = 4) were fit with a single exponential, with tau  values of 3.1 ± 0.9 ms and 3.1 ± 1.1 ms, respectively.

The differences in the effects of wild-type and mutant beta 1 subunits were confirmed by statistical analysis of the inactivation kinetics of channels containing the beta 1(E4QD6NE8Q) subunit compared with control Na+ channels during a 30-ms test pulse to 0 mV from a holding potential of -90 mV. Oocytes expressing alpha IIA in combination with either the wild-type or mutant beta 1 subunit exhibited two exponential decay components with distinct fast and slow time constants. In contrast, channels comprised of only the alpha IIA subunit displayed only the slower time constant. Our analysis found no statistically significant differences between the time constants of the fast and slow components of Na+ current decay for channels containing the beta 1(E4QD6NE8Q) mutation and those of control channels (see "Experimental Procedures"). This is consistent with our hypothesis that the beta 1 subunit causes a shift in the fraction of Na+ channels in fast and slow gating modes without changes of the kinetics within each gating mode (4, 10). However, channels containing the A/A' strand mutant beta 1 subunit exhibited a 22% reduction in the fraction of channels inactivating with the fast time constant relative to channels composed of alpha IIA and wild-type beta 1 subunits at a 1:1 ratio of alpha  and beta 1 transcript and a 16% reduction at a 1:10 ratio of alpha  and beta 1 transcripts. The mean values for Ff and their standard errors are illustrated as a box plot in Fig. 7C. This statistical analysis shows that these differences in the Ff are statistically significant.

Competition experiments were carried out to assess the ability of the beta 1 A/A' strand mutant to bind to alpha IIA and block the effect of wild-type beta 1. The beta 1 A/A' mutant competed with the wild-type beta 1 subunit for binding to alpha IIA when coinjected in equal amounts or in a 10-fold excess (Fig. 7A, traces d and e), producing channels that inactivated with the intermediate rate characteristic of the mutant beta 1 subunit rather than the faster rate characteristic of wild-type. As in the previous experiments, the slower inactivation rate was caused by a decrease in the fraction of channels inactivating with the fast time constant (Fig. 7C).

Further evidence that the A/A' strand beta 1 mutant interacts effectively with the alpha IIA subunit was obtained from analysis of the recovery from inactivation of Na+ channels (Fig. 7D). Recovery from inactivation of Na+ channels containing wild-type beta 1 subunits was fast, with essentially complete recovery in a single exponential time course with tau  = 3.1 ± 0.9 ms (n = 5). In contrast, Na+ channels containing only alpha IIA subunits have a biphasic recovery from inactivation with a prominent fast phase having tau  = 2.3 ± 0.3 ms and a slow phase having tau  = 245 ± 9 ms (n = 4). Na+ channels containing the beta 1 A/A' strand mutant recovered predominantly fast, with tau  = 3.1 ± 1.1 ms (n = 4) and were indistinguishable from channels containing the wild-type beta 1 subunit on the basis of recovery from inactivation (Fig. 7B). The results of experiments on recovery from inactivation show that the beta 1 A/A' strand mutant binds to the alpha IIA subunit and that the charge neutralization mutations in the mutant beta 1 subunit did not significantly affect the ability of beta 1 to increase the proportion of channels that recover rapidly from inactivation.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The Protein Component, but Not the Carbohydrate Component, of the Extracellular Domain Is Required for beta 1 Function-- Deletion of the intracellular domain of the beta 1 subunit does not affect modulation of the brain type IIA alpha  subunit, but deletion of segments of the extracellular domain does prevent Na+ channel modulation by beta 1 subunits. Similarly, deletion analysis showed that the intracellular domain of human beta 1 is not required for modulation of the skeletal muscle alpha  subunit by beta 1 subunits, whereas the extracellular domain is sensitive to deletion mutagenesis (15). In addition, the beta 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 beta 2 subunits (16). Taken together, these studies demonstrate that the extracellular domain is required for modulation of both brain and skeletal muscle alpha  subunits.

The extracellular domain of the beta 1 subunit contains four potential sites for N-linked glycosylation, in good agreement with biochemical results (21). In the structurally related myelin P0 protein, the carbohydrate chain linked to the E-F loop is required for homophilic association and is thought to be located in a pocket of the Ig fold motif where it may serve to orient the molecule in relation to the membrane surface (24, 33). As in myelin P0, the putative glycosylation sites of beta 1 are localized to loops between the beta  strands of the Ig fold, including two sites in the E-F loop. In contrast to myelin P0, our results show that the deletion of any of the four potential sites, as well as the simultaneous deletion of tandem E-F loop sites, does not significantly compromise the expression or function of beta 1. Therefore, we infer that carbohydrate-protein interactions are not significantly involved in either the association of the beta 1 subunit with the alpha  subunit or the subsequent modulation of Na+ channel gating by beta 1, as determined by the expression of Na+ channels in Xenopus oocytes. Evidently, although the extracellular domain is very sensitive to alteration of protein structure by deletion mutagenesis, N-linked glycosylation of these specific sites is not required for function of the beta 1 subunit.

Core beta  Sheets of the Ig Fold, but Not the Connecting Loops, Are Required for beta 1 Function-- The results of the alanine-scanning mutagenesis of hydrophobic residues within the core beta  strands support the hypothesis that the extracellular domain of beta 1 contains an essential Ig fold motif. All mutations that altered clusters of hydrophobic residues in the core beta  strands prevented beta 1 function, as expected if they interrupt the beta  sheet interactions in the core of the Ig fold. If the hydrophobic core of the Ig fold was disrupted by the cluster mutations, crucial alpha -beta 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 beta 1. In contrast, most mutations of the peripheral beta  sheets or connecting loops within the Ig fold did not alter beta 1 function. Normal beta 1 function was also observed for beta 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 alpha  subunit.

Our studies of chimeric proteins in which putative loop segments connecting the beta  strands of beta 1 were replaced with the corresponding myelin P0 sequence lend additional support to the Ig-fold hypothesis. In contrast to our deletion and alanine-scanning analyses, it is noteworthy that substitutions of up to seven amino acids are tolerated in the connecting loops in the Ig fold of the beta 1 extracellular domain. These results are consistent with the Ig fold structure because these loops are not required for the structural integrity of the Ig fold. Mutations in loop regions not directly involved in interactions with the alpha  subunit would be expected to have little or no effect on beta 1 function. From this analysis, we can exclude many of the amino acid residues in the connecting loops of the Ig fold from participating directly in interactions with the alpha subunit.

Acidic Residues in the A/A' Strand Are Required for Full beta 1 Function-- A region on the surface of the Ig fold was identified which is essential for optimum modulation of gating of the alpha  subunit. Neutralization of negative charges (E4QD6NE8Q) in the proposed A/A' strand of the beta 1 Ig fold domain yielded Na+ channels with very interesting electrophysiological characteristics. Two types of data argue that this mutant beta 1 subunit assembles properly and binds effectively to the alpha IIA subunit. First, it competes for expression with the wild-type beta 1 subunit. Second, in experiments studying recovery from inactivation, no slow phase of recovery was observed, indicating that there were no free alpha subunits present. Despite being bound to each alpha  subunit, this mutant beta 1 subunit consistently conferred rates of Na+ channel inactivation which were intermediate between those seen for the alpha IIA subunit coexpressed with the wild-type beta 1 subunit and those of channels consisting of only the alpha 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 beta 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 beta 1 subunit without preventing the association of beta 1 with the alpha  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 alpha IIA subunit rather than altered interactions with the G strand and the core of the Ig fold. The edge of the beta 1 Ig fold domain that carries these negatively charged residues may interact with the alpha  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 beta 1 and beta 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 beta 2 has a region with significant homology to the cell adhesion molecule contactin (5). It is interesting that the A/A' strand of the beta 1 Ig fold, which we found to be important for interactions with the alpha  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 alpha -beta 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.

    ACKNOWLEDGEMENT

Plasmid pSN63, containing cDNA encoding rat myelin P0, was a generous gift from Dr. Greg Lemke, Salk Institute.

    FOOTNOTES

* 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.

Dagger 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; tau f and tau s, time constants in fast and slow mode, respectively; P0, protein zero.

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Abstract
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