Introduction

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The mollusk genus Conus consists of a large number of carnivorous marine snails that produce a cocktail of small-peptide toxins, conotoxins, that are used to paralyze their prey and that can also be fatal to mammals (Gray et al., 1988). The conotoxin peptide is generally characterized by a compact structure of hypervariable amino acid residues superimposed over a scaffold of two to three disulfide bridges (Olivera et al., 1990). Members of a conotoxin family have been used as pharmacological reagents that distinguish between individual members of the respective target ion channel family. For example, GIIIA/B µ-conotoxins target rat skeletal muscle voltage-gated sodium channels (rNa_v1.4) but not neuronal sodium channels (Shon et al., 1998; McIntosh et al., 1999; Safo et al., 2000). GIIIA and GIIIB µ-conotoxins, which are often considered indistinguishable, block rNa_v1.4 with an IC₅₀ of 50 nM (Cruz et al., 1985; Moczydlowski et al., 1986; Yanagawa et al., 1987; Trimmer et al., 1989; Chen et al., 1992). However, cardiac and neuronal Na⁺ channels show much reduced affinity to the GIIIA/B toxins (Cruz et al., 1985; Moczydlowski et al., 1986; White et al., 1991; Chen et al., 1992; Gellens et al., 1992; Shon et al., 1998), and Chahine et al. (1994a) reported that human skeletal muscle (hNa_v1.4) channels expressed in human embryonic kidney (HEK) 293 cells are less sensitive to GIIIA (IC₅₀ of ~1500 nM) than rNa_v1.4.

Although it is not clear what determines the relative insensitivity of specific sodium channels to µ-conotoxins, several studies have identified specific toxin-channel interactions that are critical determinants of the toxin block of rNa_v1.4. Alanine-scanning mutagenesis revealed that the basicity of GIIIA (+6 at neutral pH) is crucial for the activity of the toxin, with the arginine 13 (R13) residue having the biggest effect on blocking the Na⁺ current (Sato et al., 1991; Becker et al., 1992). The GIIIA and GIIIB µ-conotoxins have a similar asymmetrical three-dimensional structure (Lancelin et al., 1991; Hill et al., 1996) and recent studies have shown that these µ-conotoxins might have a specific docking orientation (Dudley et al., 2000; Li et al., 2001a). Mutations of pore-lining residues close to the selectivity filter reduced the binding affinity of the toxin, with the largest changes reported for substitutions of the negatively charged residues, glutamic acid (E) at positions 758 and 765, and aspartic acid (D) at position 762 (Dudley et al., 1995; Chang et al., 1998; Li et al., 2000). These results are consistent with the suggestion that electrostatic interaction of the R13 residue of GIIIB with the negatively charged groups of the pore is important for the toxin block of rNa_v1.4 (Becker et al., 1992; Dudley et al., 1995; Chang et al., 1998; Li et al., 2000). Although electrostatic interactions within the pore are clearly important to the high-affinity block of rNa_v1.4 by GIIIA/B, the pore residues reported to interact with GIIIA/B are conserved at the comparable positions in nearly all members of the voltage-gated sodium channel family; hence, residues at these positions cannot determine the selectivity of GIIIA/B to rat Na_v1.4.

We report in this study the identification of naturally occurring amino acid variants at comparable positions in the S5-S6 linker of domain 2 of multiple channels (S729 in rNa_v1.4 to L735 in hNa_v1.4, and N732 in rNa_v1.4 to K928 and K903 in neuronal rNa_v1.1a and rNa_v1.7 channels, respectively), which are individually sufficient to confer resistance to GIIIB on recombinant rNa_v1.4 channels. We also demonstrate different kinetic properties of binding of GIIIA and GIIIB with wild-type rNa_v1.4 and a differential effect of the two toxins on mutant channels, demonstrating distinct features of these toxins. These results support a model of complex channel-toxin interaction whereby specific residues N-terminal to the SS1 segment of the P loop are critical to determine the toxin selectivity to rat skeletal muscle sodium channel.

	Materials and Methods

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Construction of Human-Rat Na_v1.4 Channel Chimera. The insert encoding hNa_v1.4 in the vector pRc/CMV (Chahine et al., 1994b) was subcloned in two steps into the vector RBG4 to facilitate linker swapping between human and rat channels and to use the same promoter to control the expression of the two inserts. The plasmid µ1-RBG4 (Ukomadu et al., 1992) carrying the rNa_v1.4 insert was digested with EcoICRI in the vector and SacII in the insert to remove the sequence encoding domains 1 to 3, and the remainder of the construct (vector plus domain 4) was band isolated. The plasmid hNa_v1.4-pRC-CMV was digested by NotI and the ends polished using T4 DNA polymerase (Roche Applied Science, Indianapolis, IN), and the fragment (4467 base pairs) encoding the respective human cognate of domains 1 to 3 was subsequently released by digestion with SacII. This partial hNa_v1.4 insert was band isolated and subcloned into the partial rNa_v1.4 construct described above to produce a chimera channel of human domains 1 to 3 and rat domain 4. Chimera inserts were confirmed by sequencing the junctions. The chimera channel showed toxin sensitivity (data not shown) identical to that of hNa_v1.4, in general agreement with published results (Chahine et al., 1998a) that domain 2 determines GIIIA/B sensitivity. The sequence encoding hNa_v1.4 domain 4 was isolated as a SacII/EcoRI fragment and used to replace the remaining rNa_v1.4 domain 4 in the chimera.

Exchange of Domain 2 S5-S6 Linker between Human and Rat Na_v1.4 Channels. A 244-base pair fragment that encodes D2/S5-S6 linker was amplified from both human and rat Na_v1.4 constructs using forward primer F1 (5'-GGCCATCATCGTCTTCATCTTCG-3', corresponding to nucleotides 2552 to 2574 and 2198 to 2220 of rat and human Na_v1.4, respectively) and reverse primer R1 (5'-CAATGACCATGACCATGAGGAAG-3', corresponding to nucleotides 2796 to 2774 and 2442 to 2420 of rat and human Na_v1.4, respectively). F1 has two nucleotide changes compared with the respective human sequence (C to T at position 4, and C to G at position 13), whereas the reverse primer sequence is identical in both channels. These changes do not affect the amplification of the product under the conditions that were used in this study and are eliminated from the respective constructs because they are distal to the restriction enzymes SphI and FseI (see below) used in swapping fragments encoding the linkers.

Point Mutation of rNa_v1.4. PCR-based mutagenesis (Horton et al., 1993) was used to introduce amino acid substitutions to D2/S5-S6 linker of Na_v1.4. Wild-type primers F1 and R1 (described above) were used together with multiple mutagenic primers (Table 1) to introduce the respective substitutions.

Transfection of HEK 293 Cells. Transfections were carried out using the calcium phosphate precipitation technique, as described previously (Cummins et al., 1998). HEK 293 cells are grown under standard tissue culture conditions (5% CO₂, 37°C) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The calcium phosphate-DNA mixture (channel constructs and a green fluorescent protein reporter plasmid) was added to the cell culture medium and left for 1 to 2 h, after which time the cells were washed with fresh medium. Cells with green fluorescent protein fluorescence were selected for whole-cell patch-clamp recordings after 1 to 2 days in culture.

Whole-Cell Patch-Clamp Recordings. Whole-cell patch-clamp recordings were conducted at room temperature (~21°C) using an EPC-9 amplifier (HEKA, Lambrecht, Germany). Data were acquired on a Pentium III computer using the Pulse program (version 8.31; HEKA). Fire-polished electrodes (0.8-1.5 M) were fabricated from 1.7-mm capillary glass (VWR, West Chester, PA) using a P-97 puller (Sutter, Novato, CA). Cells were not considered for analysis if the initial seal resistance was less than 5 G or if they had high leakage currents (holding current >0.2 nA at 100 mV), membrane blebs, or an access resistance greater than 4 M. The average access resistance was 1.5 ± 0.1 M (mean ± S.E., n = 208). Voltage errors were minimized using 80 to 85% series resistance compensation and the capacitance artifact was canceled using the computer-controlled circuitry of the patch-clamp amplifier. The average current amplitude was 11.6 ± 0.8 nA (n = 208) and the average maximum theoretical voltage-clamp error was 3.5 ± 0.2 mV. Linear leak subtraction, based on resistance estimates from four to five hyperpolarizing pulses applied before the depolarizing test potential, was used for all voltage-clamp recordings. Membrane currents were usually filtered at 2.5 kHz and sampled at 10 kHz. The pipette solution contained 140 mM CsF, 1 mM EGTA, 10 mM NaCl, and 10 mM HEPES, pH 7.3. The standard bathing solution was 140 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 10 mM HEPES, pH 7.3. The liquid junction potential for these solutions was <8 mV; data were not corrected to account for this offset. The osmolarity of all solutions was adjusted to 310 mOsM with sucrose (5500 osmometer; Wescor, Logan, UT).

Toxin Solutions and Bath Application. GIIIB was obtained from Alamone Laboratories (Jerusalem, Israel) and Sigma-Aldrich (St. Louis, MO). Similar results were obtained for rNa_v1.4 currents with GIIIB from both sources. GIIIA was obtained from Sigma-Aldrich. Stock solutions (20 µM for GIIIA and GIIIB) were made using extracellular recording solution and aliquots were stored at 20°C. Toxins were diluted into the recording chamber (volume of 500 µl) and mixed by repeatedly pipetting 50 µl to achieve the specified final concentration. The chamber was not perfused during toxin application. The mixing procedure typically took ~5 s. Toxin was applied for 5 to 25 min to allow the peak current amplitude to reach a steady-state level. Toxins were washed off in specific experiments using a gravity-fed perfusion system, with the delivery tube (0.5 mm i.d., 0.5 ml/min flow rate) placed 1 mm from the cell. This directly bathed the cell in toxin-free solution and allowed a rapid solution change (<1 s) for measuring off rates. Toxin wash-off was continued for 10 to 60 min to allow peak current amplitude to reach a steady-state level. Toxin application and wash-off protocols were validated using tetrodotoxin (TTX) and rNa_v1.4 channels. A _on of 8 ± 0.2 s and a _off of 26.7 ± 4.1 s was measured using 25 nM TTX, which results in a calculated K_D of 9.1 nM (n = 3). The K_D closely matches the IC₅₀ measured using 25 nM TTX (8.0 ± 0.2 nM, n = 3), and demonstrates that this system is sufficient for measuring the "on" and "off" kinetics of µ-conotoxins.

Data Analysis. Data were analyzed using the Pulsefit (HEKA) and Origin (MicroCal Software, Northampton, MA) software programs. Unless otherwise noted, statistical significance was determined at p < 0.05 using an unpaired t test. The half-blocking concentration (IC₅₀) was calculated based on the single-site Langmuir inhibition isotherm using the following function: IC₅₀ = (I_toxin / I₀) × [toxin] / (1  I_toxin / I₀), where I₀ and I_toxin are the peak sodium currents measured before and after application of toxin, respectively, and [toxin] is the concentration of toxin. Unless otherwise noted, the IC₅₀ was calculated using a toxin concentration of 300 nM. Time course data for toxin block and wash-off were fitted with single exponential functions to estimate the time constants _on and _off. The "on" and "off" rate constants for block (k_on and k_off) were calculated using the following equations: k_off = 1 / _off and k_on = [(1 / _on)  (1 / _off)] / [toxin]. The kinetically derived toxin equilibrium constant (K_D) was calculated using the equation K_D = k_off + k_on. Results are presented as mean ± S.E.M. and error bars in the figures represent S.E..

	Results

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Human Na_v1.4 Sodium Channel Is Resistant to GIIIB µ-Conotoxin. The skeletal muscle sodium channel cloned from rat (rNa_v1.4) is blocked by GIIIA/B µ-conotoxins at nanomolar concentrations (Cruz et al., 1985; Moczydlowski et al., 1986; Yanagawa et al., 1987; Trimmer et al., 1989; Chen et al., 1992). As Fig. 1A illustrates, most of the current through rNa_v1.4 expressed in HEK 293 cells is blocked by 300 nM GIIIB. Although rat and human Na_v1.4 channels exhibit more than 90% identity at the amino acid level, the current through hNa_v1.4 expressed in HEK 293 cells is relatively insensitive to GIIIB (Fig. 1B). Based on fits to the relationship between percentage of current inhibition versus toxin concentration, the IC₅₀ for GIIIB block of hNa_v1.4 channels is 1065 nM (18 measurements from 12 cells), compared with 49 nM (14 measurements from 8 cells) for rNa_v1.4 channels (Fig. 1C). Based on the percentage of current inhibition measured with just a single concentration (300 nM) of GIIIB, the estimated IC₅₀ for hNa_v1.4 channels is 1357 ± 314 nM (n = 6), compared with 49 ± 5 nM (n = 12) for rNa_v1.4 channels. The sensitivity of hNa_v1.4 channels to GIIIB is similar to that previously reported for µ-conotoxin GIIIA (Chahine et al., 1994a).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   GIIIB differentially blocks rat and human Nav1.4 sodium channels. Cells expressing rNav1.4 (A) or hNav1.4 (B) sodium channels were exposed to 300 nM µ-conotoxin GIIIB. The cells were held at 120 mV and stepped to 10 mV for 50 ms every 10 s. GIIIB was applied in the bath solution after the fourth pulse. Data were fitted with single exponential functions to estimate the time constants for toxin binding (on is 257 s in A and 93 s in B). Insets show representative current traces before and after application of 300 nM GIIIB. Horizontal scale bars, 2 ms. Vertical scale bars, 10 nA. C, dose-response relationship for µ-conotoxin GIIIB inhibition of rNav1.4 () and hNav1.4 () channels expressed in HEK 293 cells. The rNav1.4 channels are ~20-fold more sensitive to GIIIB inhibition than hNav1.4 channels.

Sensitivity to GIIIB Is Determined by Residues in S5-S6 Linker of Domain 2. Chahine et al. (1999b) reported that mutations in the S5-S6 linker of domain 2 (D2/S5-S6) altered rNa_v1.4 sensitivity to GIIIA by ~5- to 6-fold. Therefore, we compared the sequences of rNa_v1.4 and hNa_v1.4 in this region to identify residues that might contribute to the differential sensitivity of the two channels to µ-conotoxins. The residues that Chahine et al. (1998b) mutated in rNa_v1.4 (A728 and D730) are conserved in hNa_v1.4; however, the D2/S5-S6 linker in these two channels differs by two amino acid residues (Fig. 2). A serine at position 729 (S729) in rNa_v1.4 is replaced by leucine in hNa_v1.4; and an asparagine at position 739 (N739) of rNa_v1.4 is replaced by histidine (H) in hNa_v1.4 (numbers are based on rNa_v1.4 sequence). We reasoned that one or both of these substitutions might be critical for the selectivity of GIIIA/B to rNa_v1.4. To examine the effect of these residues on GIIIB sensitivity, we exchanged the D2/S5-S6 linker between rNa_v1.4 and hNa_v1.4. The effect of swapping this linker on the toxin block was studied in transiently transfected HEK 293 cells. Cells transfected with parental and chimera constructs produced currents with indistinguishable kinetic and voltage-dependent properties. We used a toxin concentration of 300 nM (unless otherwise noted) to estimate the sensitivity of the channels to the toxin. As can be seen in Fig. 3A, hNa_v1.4 channels with the rat D2/S5-S6 linker (hRhh) had a GIIIB sensitivity (IC₅₀ = 80 ± 7, n = 6) close to that of rNa_v1.4 channels. In contrast, rNa_v1.4 channels with the human D2/S5-S6 linker (rHrr) had a GIIIB sensitivity (IC₅₀ = 1304 ± 287, n = 5) similar to that of hNa_v1.4 channels (Fig. 3B). Thus, the D2/S5-S6 linker of rNa_v1.4 channels is crucial in determining sensitivity to GIIIB conotoxins.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Alignment of the S5-S6 linker of domain 2 containing pore-lining and extra-pore residues of select mammalian sodium channels. Residues that are predicted to line the pore are underlined and designated SS-1 and SS-2. The 21-amino acid residues that are N-terminal to SS-1 are designated extra-pore residues. The amino acid numbers are those of rNav1.4. Compared with hNav1.4, rNav1.1a, rNav1.2a, and rNav1.7a, rNav1.4 is different at one, three, and four extra-pore residues, respectively. The E722D replacement in rNav1.1a, rNav1.2a, and rNav1.7a may not have a functional consequence. The GenBank accession numbers of these sodium channel -subunits are as follows: rNav1.4, NM_013178; hNav1.4, NM_000334; rNav1.1a, X03638; hNav1.1 AY043484; rNav1.2a, NM_012647; rNav1.7, U79568; rNav1.5, NM_013125; and hNav1.5, NM_000335.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   GIIIB inhibition of Nav1.4 channels is determined by residues in the D2/S5-S6 linker. A, human Nav1.4 sodium channel with the rat D2/S5-S6 linker (hRhh) is sensitive to GIIIB. B, rat Nav1.4 sodium channel with the human D2/S5-S6 linker (rHrr) is relatively insensitive to GIIIB. C, N739H mutation does not alter the sensitivity of rNav1.4 channel to GIIIB. D, S729L mutation decreases the sensitivity of rNav1.4 channels to GIIIB by 20-fold. E, S729T mutation has little effect on the GIIIB sensitivity of rNav1.4 channels. F, S729A mutation also has little effect on the GIIIB sensitivity of rNav1.4 channels. Cells expressing mutant sodium channels were exposed to 300 nM GIIIB. The cells were held at 120 mV and stepped to 10 mV for 50 ms every 10 s. GIIIB was applied in the bath solution after the fourth pulse. Data were fitted with single exponential functions to estimate the time constants for toxin binding (on is 146 s in A, 24 s in B, 126 s in C, 44 s in D, 135 s in E, and 202 s in F). Insets show representative current traces before and after application of GIIIB. Horizontal scale bars, 2 ms. Vertical scale bars, 4 nA (A, E, and F) and 2 nA (B, C, and D).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   GIIIB inhibition of rNav1.4 channels is significantly reduced by replacing another residue in the external pore region of the D2/S5-S6 linker. A, rat Nav1.4 sodium channel with the N732K mutation exhibits reduced sensitivity to GIIIB inhibition. B, N732Q mutation does not reduce GIIIB sensitivity. C, rat Nav1.4 sodium channels with the N732E mutation also exhibit a GIIIB sensitivity similar to that of rNav1.4. D, S729L/N732K mutation reduces the GIIIB sensitivity of rNav1.4 channels by more than 50-fold. Cells expressing mutant sodium channels were exposed to 300 nM (A-C) or 1200 nM (D) µ-conotoxin GIIIB. The cells were held at 120 mV and stepped to 10 mV for 50 ms every 10 s. GIIIB was applied in the bath solution after the fourth pulse. Data were fitted with single exponential functions to estimate the time constants for toxin binding (on is 533 s in A, 168 s in B, 142 s in C, and 74 s in D). Insets show representative current traces before and after application of GIIIB. Horizontal scale bars, 2 ms. Vertical scale bars, 5 nA (A, B, and D) and 3 nA (C).

Sensitivity of Mutant Na⁺ Channels to GIIIA µ-Conotoxin. Rat Na_v1.4 channels are also more sensitive to µ-conotoxin GIIIA (IC₅₀ = 58 ± 5, n = 14; Fig. 5A) than are hNa_v1.4 channels (IC₅₀ = 1228 ± 139 nM, n = 11; Fig. 5B) (Chahine et al., 1994a). Although these GIIIA and GIIIB are closely related and often considered indistinguishable, they do have slightly different sequences (Sato et al., 1983). Therefore, we investigated whether the residues that altered µ-conotoxin GIIIB sensitivity also altered µ-conotoxin GIIIA sensitivity. Replacing the D2/S5-S6 linker of hNa_v1.4 channels with the D2/S5-S6 linker of rNa_v1.4 channels caused a significant increase (p < 0.002) in the sensitivity of hNa_v1.4 to µ-conotoxin GIIIA (IC₅₀ = 139 ± 16 nM, n = 5; Fig. 5C). Similar to its effect on sensitivity to GIIIB, the S729L substitution significantly decreased (p < 0.002) rNa_v1.4 sensitivity to µ-conotoxin GIIIA (IC₅₀ = 778 ± 74 nM, n = 9; Fig. 5D). However, the GIIIA sensitivity of rNa_v1.4 channels was differentially affected by replacements of N732. Both the rNa_v1.4-N732E and rNa_v1.4-N732Q replacements, which had no effect on GIIIB sensitivity, slightly increased the sensitivity of rNa_v1.4 channels to GIIIA (IC₅₀ = 32 ± 4 nM, n = 6 and IC₅₀ = 37 ± 5 nM, n = 6; p < 0.01). In addition, although GIIIB sensitivity was reduced 20-fold by the N732K mutation, rNa_v1.4-N732K channels were only 4-fold less sensitive to GIIIA (IC₅₀ = 228 ± 37 nM, n = 5; Fig. 5E) compared with wild-type rNa_v1.4 channels. The double mutant S729L/N732K also had less of an effect on GIIIA sensitivity than on GIIIB sensitivity. The GIIIA sensitivity of rNa_v1.4-S729L/N732K channels (IC₅₀ = 906 ± 110 nM, n = 5; Fig. 5F) was only reduced by 16-fold compared with wild-type rNa_v1.4 channels, and was similar to that of the single substitution S729L. Therefore, although rNa_v1.4 channels and hNa_v1.4 channels show similar IC₅₀ values for GIIIA and GIIIB and these µ-conotoxins have often been considered indistinguishable, inhibition of rNa_v1.4 channels by these two closely related µ-conotoxins was differentially affected by a single amino acid substitution in the D2/S5-S6 linker (Fig. 6).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   GIIIA differentially blocks rat and human Nav1.4 sodium channels, and this block is sensitive to changes in the D2/S5-S6 linker. Cells expressing rNav1.4 (A) or hNav1.4 (B) sodium channels were exposed to 300 nM µ-conotoxin GIIIA. C, hNav1.4 sodium channel with the rat D2/S5-S6 linker (hRhh) is sensitive to GIIIA. D, S729L mutation decreases the sensitivity of rNav1.4 to GIIIA by ~15-fold. E, N732K mutation, on the other hand, only reduces the sensitivity of rNav1.4 to GIIIA inhibition by ~4-fold. F, S729L/N732K double mutation reduces the GIIIA sensitivity of rNav1.4 by slightly more than 15-fold. The cells were held at 120 mV and stepped to 10 mV for 50 ms every 10 s. GIIIA (300 nM) was applied in the bath solution after the fourth pulse. Data were fitted with single exponential functions to estimate the time constants for toxin binding (on is 67 s in A, 9 s in B, 40 s in C, 28 s in D, 293 s in E, and 35 s in F). Insets show representative current traces before and after application of GIIIA. Horizontal scale bars, 2 ms. Vertical scale bars, 3 nA (A, B, and D) and 5 nA (C, E, and F).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Bar graph comparing the half-blocking concentration (IC50) for µ-conotoxin GIIIB and GIIIA inhibition of rat, human, and mutant Nav1.4 channels. *, significant difference (p < 0.05) between GIIIA and GIIIB IC50. **, significant difference (p < 0.005) between GIIIA and GIIIB IC50. The D2/S5-S6 mutations have a reduced effect on GIIIA sensitivity of rNav1.4.

Effect of rNa_v1.4 Mutations on k_on and k_off. The S729L and the N732K mutations individually altered the GIIIB IC₅₀ of rNa_v1.4 channels by ~20-fold, and the S729L/N732K double mutation altered the IC₅₀ by ~50-fold (Fig. 7). To further characterize the mechanisms by which these mutations alter toxin sensitivity, we examined the on (k_on) and off (k_off) rates for GIIIB block of rNa_v1.4 and hNa_v1.4 channels using 600 nM µ-conotoxin (unless otherwise noted). GIIIB was difficult to wash off rNa_v1.4 channels, and for some cells little or no recovery was seen after 30 min (data not shown). In those cells in which wash-off did seem to occur, the time constant for wash-off (_off) was 1326 ± 311 s (n = 4), which is similar to that reported by Li et al. (2000). In contrast, GIIIB washed off hNa_v1.4 channels relatively easily (_off = 47.6 ± 8 s, n = 5). Although the k_on for GIIIB was slightly increased for hNa_v1.4 channels compared with rNa_v1.4 channels, the k_off was increased by at least 20-fold for hNa_v1.4 channels (Table 2). GIIIB also washed-off rNa_v1.4-S729L mutant channels relatively easily (_off = 46.6 ± 2.8 s, n = 4). The S729L mutation increased the k_off of GIIIB by ~20-fold but only increased k_on by ~2-fold (Table 2), suggesting that this mutation destabilizes the toxin-channel complex.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Bar graph summarizing the half-blocking concentration (IC50) for µ-conotoxin GIIIB inhibition of Nav1.4 channels. The S729L and N732K single mutants reduced GIIIB sensitivity of rNav1.4 channels by ~20-fold. These two mutants had a partially additive effect, and the rNav1.4 S729L/N732K double mutant showed a 50-fold reduction in the GIIIB sensitivity of rNav1.4. **, significant difference from the IC50 of WT rNav1.4 channels (p < 0.005).

	Discussion

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Our data demonstrate that the part of the D2/S5-6 linker that is N-terminal to the SS-1 segment contains critical determinants of rNa_v1.4 sensitivity to GIIIB, and may further define contact points between the channel and the toxin. We show that exchanging the D2/S5-S6 linker between rNa_v1.4 channels, which are GIIIB-sensitive, and hNa_v1.4 channels, which are GIIIB-resistant, switches their respective sensitivity to GIIIB. We have identified a single amino acid substitution in this linker, S729L, that produces ~20-fold reduction in the sensitivity of rNa_v1.4 to GIIIB and accounts for the difference between rNa_v1.4 and hNa_v1.4 GIIIB sensitivity. We provide evidence that additional residues in the D2/S5-S6 linker might also be important determinants of the GIIIB resistance of neuronal channels. Both rNa_v1.1a and rNa_v1.7, which are GIIIB-resistant (Safo et al., 2000), have a lysine at the position corresponding to N732 in rNa_v1.4. We found that the N732K substitution also produced ~20-fold reduction in rNa_v1.4 GIIIB sensitivity and the double mutant S729L/N732K decreased rNa_v1.4 GIIIB sensitivity by ~50-fold (Fig. 6). Thus, we have identified naturally occurring polymorphic residues in the D2/S5-S6 linker of sodium channels that are critical determinants of µ-conotoxin GIIIB sensitivity.

Several studies have identified other residues in the D2/S5-S6 linker of rNa_v1.4 channels that can influence the affinity for GIIIA/B. Neutralization of the negatively charged residues E758, E765, and D762 decreased µ-conotoxin affinity by 10- to 50-fold (Dudley et al., 1995; Li et al., 2000). These residues are thought to line the pore, indicating that GIIIA/B block conductance by binding in the pore. However, these pore-lining residues are conserved between Na_v1.4 channels, TTX-sensitive (TTX-S) neuronal channels, and the cardiac (Na_v1.5) channel (Fig. 2), and therefore cannot be the primary determinants of the selectivity of GIIIA/B to rNa_v1.4. Chahine et al. (1998b) found that mutations A728L and D730Q, near S729 and N732, caused a small decrease (5- to 6-fold) in GIIIB inhibition of rNa_v1.4 channels. The difference in the effect of the replacement of the adjacent residues A728 and S729 by leucine (A728L and S729L cause 6- and 20-fold reduction in IC₅₀, respectively) strongly suggests a more direct role of S729 in defining the sensitivity of rNa_v1.4 channels to the GIIIB toxin.

Does D2/S5-S6 Linker Determine GIIIB Sensitivity? Comparison of the D2/S5-S6 linker from rNa_v1.4 with those of hNa_v1.4 and TTX-S neuronal channels reveals a limited number of amino acid substitutions in the D2/S5-S6 linker. Most of these changes are located in the region N-terminal to SS-1 (Fig. 2), which includes S729 and N732 of rNa_v1.4. Based on our results, we propose that one or more of these naturally occurring variants play a critical role in determining the toxin sensitivity of the respective channel. The TTX-S channels rNa_v1.2a and rNa_v1.7, for example, differ from rNa_v1.4 at the residues corresponding to 728, 729, and 730 (Fig. 2). The partially additive effect of two mutations, S729L and N732K, on increasing the resistance to toxin block suggests that multiple variant residues in the D2/S5-S6 linker, compared with rNa_v1.4, may contribute to the increased resistance of some neuronal channels. Interestingly, the N732Q mutation did not decrease GIIIB binding, and, therefore it is possible that the human Na_v1.1 channel might be sensitive to GIIIB. It should be noted, however, that contributions by S5-S6 linkers in other domains of the sodium channels to differences in toxin binding affinity could not be ruled out at this time.

How Does D2/S5-S6 Linker Alter GIIIB Sensitivity? The S729L and N732K mutations each individually altered the GIIIB IC₅₀ of rNa_v1.4 channels by ~20-fold. The effects of these two mutations on binding were only partially additive, suggesting that these two residues could facilitate the same aspect of the toxin-channel interaction (Mildvan et al., 1992). However, the two mutations altered different aspects of the binding of the toxin. Whereas the S729L mutation primarily increased the off rate of GIIIB, the N732K mutation significantly decreased the on rate. In the double mutant S729L/N732K both the on and off rates were significantly altered compared with wild-type rNa_v1.4, suggesting that the D2/S5-S6 linker affects both the accessibility of the pore to GIIIB and the stabilization of the toxin-channel complex. This indicates that although the effects of the S729L and N732K mutations are interdependent, S729 and N732 might facilitate related nonrate-limiting aspects of the toxin-channel interaction (Mildvan et al., 1992). Recently, Feng et al. (2001) reported that a single residue in the D3/S5-S6 linker of the N-type calcium channel _1B subunit dramatically increased both the on and off rates for -conotoxin GVIA block, and proposed that this residue, which is N-terminal to the P loop, acts as a barrier that controls both the accessibility of GVIA to the binding site and the reversibility of GVIA block. Our data indicate that the D2/S5-S6 residues S729 and N732 may play an analogous role in µ-conotoxin GIIIB block of rNa_v1.4 channels. The S729L substitution, which enhanced the toxin off rate, is likely to destabilize the toxin-channel complex and enhance reversibility of toxin block due to steric hindrance. In contrast, the N732K mutation decreased the on rate of toxin binding, suggesting that it interfered with the formation of an initial toxin-channel complex, and that N732 might control access of GIIIB to the high-affinity binding site.

Are µ-Conotoxins GIIIA and GIIIB Indistinguishable? GIIIA and GIIIB are often considered indistinguishable. The two peptides share a similar folded structure and a predicted manner of interaction with their sodium channel target (Hill et al., 1996), and they seem to have a comparable effect on rNa_v1.4 expressed in HEK 293 cells (Fig. 6). However, GIIIA is reported to be less potent than GIIIB in vivo (Sato et al., 1983). In addition, although D762Q and E765Q substitutions rendered rNa_v1.4 resistant to GIIIB (Li et al., 2000), GIIIA binding was not affected by these same substitutions (Chahine et al., 1998b). Recently, Li et al. (2001b) also showed that although the GIIIB sensitivity of rNa_v1.4-D762K and rNa_v1.4-E765K mutant channels was greatly reduced (~200-fold), the GIIIA sensitivity of these channels was only slightly reduced (~3-fold) compared with wild type rNa_v1.4 channels. Thus, residues at various positions in the D2/S5-S6 linker differentially interact with GIIIA and GIIIB.