INTRODUCTION

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Members of a structurally diverse family of toxins isolated from several species of sea anemone and scorpion have proven useful tools in the functional characterization of mammalian voltage-gated sodium (Na) channels (1, 2). These toxins bind to the extracellular face of the channel at a locus termed site-3 (3) and selectively delay current inactivation (4-6), principally by inhibiting transitions of the channel from the open to the inactivated state (7-9). Several of the sea anemone toxins discriminate between different mammalian isoforms of the Na channel, generally binding to the cardiac isoform with highest affinity (10). Because these toxins are only active when applied extracellularly (5, 6) and because most regions of the channel known to affect inactivation are located intracellularly or within the plasma membrane (11-14), it is of particular interest to characterize the binding interface between toxin and channel.

Several regions of the channel have been implicated in toxin binding. Studies involving covalent attachment of scorpion toxin derivatives (15) and protection against such labeling by site-specific antibodies (16) have implicated regions in the first channel domain in toxin binding, while other studies involving antibodies (16) and chimeric channels (17) have pointed to regions in domain 4. Most recently, mutation of a negative amino acid at the outer end of the third helix in the fourth domain of rat brain sodium channels (Glu-1613) was shown to inhibit the binding of the sea anemone toxin ATX-II (from Anemonia sulcata) by 80-fold (18).

The sea anemone toxin anthopleurin B (ApB)¹ was originally isolated from the Pacific coast sea anemone Anthopleura xanthogrammica (19, 20) and subsequently cloned and expressed heterologously (21). It has seven positive amino acid residues (22), and the ability of a subset of those to affect the binding affinity of the toxin (23-25) suggested the possibility of an electrostatic interaction between toxin and channel. We therefore attempted to determine which of these cationic toxin residues mediate the decrease in affinity observed with the channel mutation at the end of domain IV, helix 3 (18). Specifically, we constructed a series of mutations of residues on ApB and used the formalism of thermodynamic mutant cycle analysis (26, 27) to assess the degree to which these toxin mutations interacted with the mutation on the channel. Taking advantage of the higher affinity of ApB for cardiac sodium channels than for neuronal channels, we performed our experiments in cells transfected with the rat cardiac channel isoform rH1.

	MATERIALS AND METHODS

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Molecular Biology-- The rat heart sodium channel cDNA (rH1) (28, 29) subcloned into the pSP64t vector was generously provided by R. Kallen (University of Pennsylvania). The aspartic acid at amino acid 1612 (corresponding to position 1613 in rBr2a) was mutated to asparagine (D1612N) or arginine (D1612R) using polymerase chain reaction (PCR) in a 4-primer strategy (30). Sequences in this region were as follows, where the capitalized residues denote the position of Glu-1613 (rBr2a) and Asp-1612 (rH1) and asterisks denote the third and fourth helices of channel repeat IV (see Ref. 31). PCR primers included the desired mutations and an additional mutation to introduce a silent KpnI restriction site, which did not alter the amino acid sequence but facilitated identification of mutated PCR products. The final PCR products spanned unique BstEII (base pair 4632) and BspDI (base pair 5069) endonuclease restriction sites in rH1 and were subsequently used in the reconstruction of the full-length rH1 cDNA in pSP64t. The entire BstEII to BspDI PCR-generated region found in the full-length construct was completely sequenced by dideoxy methods (32) to confirm the presence of the desired mutations and the absence of spurious PCR-generated errors. Expression was first tested in Xenopus oocytes, and the entire coding region of rH1 was subsequently shuttled into the HindIII site in the polylinker of pREP9 (Invitrogen, Carlsbad, CA) using HindIII (in pSP64t) to HindIII (base pair 6371 in rH1) endonuclease restriction sites.

Cell Culture-- Channels from the rH1-pREP9 constructs were expressed in tsA201 cells (33) and maintained as described previously (17). Two to three days prior to recording, cells were seeded onto 60-mm culture dishes and transfected with 10 µg of plasmid pREP9-rH1 using a calcium phosphate coprecipitate. Cells were cotransfected with pHook1, and transfectants were visualized with the Capture-Tec bead system (Invitrogen) using supplied protocols. Approximately 30-50% of cells bound beads; virtually all cells binding two or more beads expressed Na⁺ current (I_Na).

Electrophysiological Solutions-- Pipette solution contained (in mM) 140 Cs⁺, 10 Na⁺, 100 F, 50 Cl, 10 HEPES, with pH adjusted to 7.3 with CsOH. Bath solution contained (in mM) 140 Na⁺, 2 Ca²⁺, 144 Cl, 10 HEPES, with pH adjusted to 7.3 with NaOH. Because toxin in solution tends to adhere to glassware and tubing, bath solutions containing toxin were supplemented with 5 mg/ml bovine serum albumin (Sigma) (17). Toxins ApB, ApA, and ApB mutants were produced in Escherichia coli strain BL21(DE3) transformed with the appropriate version of the plasmid pKB-13 as described previously (21, 25, 34) and characterized by amino acid analysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Circular dichroism spectra of the toxin derivatives used in the present study to confirm proper folding of toxins were determined in a Jasco 710 spectropolarimeter (Easton, MD) equilibrated with camphor d₁₀ sulfonic acid, and the spectra deconvoluted using the standard error of the estimate program supplied by the manufacturer.

Recording Techniques-- Recording protocols were generated using Clampex 6.0.2 (Axon Instruments, Foster City, CA) running on a Pentium-based microcomputer and were imposed through a 12-bit DAC controlling an Axopatch 200B amplifier (Axon). The recording chamber was maintained at 16 °C with a Sensortek TS-4 controller (Clifton, NJ). Currents were 4-pole Bessel filtered at 5 kHz and digitized at 12-bit resolution at 100 kHz.

Data Analysis-- Analysis and fitting were performed with locally written programs (GRB, DAH) running under Matlab 5.0 (The MathWorks, Natick MA). Traces were leak corrected based on the resistance calculated from the current at holding potential. The capacity transient was removed through subtraction of summed subthreshold pulses, and traces were digitally refiltered at 5 kHz using a zero-phase fitting algorithm.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

	RESULTS

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Kinetics of Mutant Channels-- We introduced mutations into rH1 at the position identified by Rogers and colleagues (18) in rat brain channels (rBrIIa). Channels rH1 D1612N and D1612R displayed currents similar to those of the wild-type channel (Fig. 1A). The mutations produced little change in voltage-dependence of conductance (Fig. 1B, left); the charge neutralization mutant D1612N was very slightly but significantly shifted in a depolarizing direction (p  0.02, Student's t-test), while the halfpoint of the charge reversal mutation was unchanged. Halfpoints of the conductance curve were as follows: wild-type rH1, 41 ± 1 (n = 14); D1612N, 38 ± 2 (n = 13); D1612R, 43 ± 2 (n = 9). Cells with conductance slope factors <6.0 mV were deemed to be inadequately voltage controlled and were excluded from any further study (38). On the other hand, the charge reversal mutation D1612R displayed a steady-state availability curve 14 mV negative to that of wild-type channels (p  0.0001), whereas that of D1612N was similar to the wild-type channel (Fig. 1B, right). Halfpoints of availability were: wild-type rH1, 100 ± 2 (n = 22); D1612N, 101 ± 2 (n = 14); D1612R, 114 ± 2 (n = 9). Paralleling the shift in availability, the kinetics of recovery from inactivation of D1612R were shifted in a hyperpolarizing direction by 16 ± 3 mV across a range of recovery potentials with respect to rH1 (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Kinetic characterization of wild-type and mutant rH1 channels. tsA201 cells were transfected with wild-type rH1 (); rH1 D1612N (); and rH1 D1612R DNA (). Horizontal scale bar, 5 ms. A, raw currents evoked by depolarizing pulses from a holding potential of 150 mV up to +20 mV at 10 mV intervals. Traces were leak- and capacity-corrected as described under "Materials and Methods." Vertical scale bars, 500 pA. B, normalized steady-state availability (left curves) and conductance (right curves) relations of wild-type and mutant rH1 channels. Insets show voltage protocols. Steady-state availability curves were fit with the Boltzmann equation, and current/voltage relations were fit with the product of the Boltzmann equation and Ohm's law. Average values of fit parameters and n values are given in the text. C, currents evoked by depolarizing pulses to 10 mV before (top traces) and after (bottom traces) modification to steady-state with 100 nM ApB. Traces were leak- and capacity-corrected as described under "Materials and Methods."

Calculation of Toxin/Channel Interaction-- The energies of interaction of the positive residues of ApB with channel residue Asp-1612 were calculated through thermodynamic mutant cycle analysis (26, 27). In short, the effects of point mutations of ApB on toxin/channel affinity were tested both in the context of wild-type rH1 channels and in the context of channels mutated at Asp-1612. Differences in the effects of the toxin mutation were quantified as coupling coefficients , which are related logarithmically to the energy of interaction between the particular toxin and channel residues (see "Materials and Methods").

(Eq. 5)

(Eq. 6)

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Affinities and interactions of toxins with residue Asp-1612. A, sample affinity calculation. A cell expressing rH1 D1612N channels was modified to steady-state with 100 nM ApB H39A toxin. Average current over a window 7.6-8.0 ms after depolarization onsets (dotted lines in insets) is shown during treatment with toxin (, left) and subsequent wash-out (, right). Note the change in scale along the time axis. Smooth curves show fits to data by single exponentials with rate constants kmod and koff as shown. Insets show INa before toxin modification (1), at steady-state (2), before unmodification (3), and after unmodification (4). Vertical scale bar, 500 pA; horizontal scale bar, 2 ms. Cell 97424-5, Rm = 1.5 G, Cm = 23 picofarad. B, values of KD calculated from association and dissociation kinetics, except for the binding of ApA to rH1 D1612N, which was obtained from steady-state measurements. Note logarithmic scale. Black, affinities to rH1; white, affinities to rH1 D1612N. C, coupling coefficients (left axis, logarithmic scale) and energies of interaction (right axis, linear scale) are shown for channel residue Asp-1612 and the indicated toxin residues. (In keeping with convention (26), any values of < 1 are shown as reciprocals.) n values are shown in Table I.

Toxin Interactions with D1612N-- Including its -amino group, ApB possesses six fully positive charges and two histidines (22), any of which might be a candidate for binding to the negative channel residue Asp-1612. Residues Arg-14, His-34, His-39, and Lys-48 have produced only small effects on toxin affinity when neutralized (25, 39) and so were relatively poor candidates for interacting with Asp-1612; similarly, short N-terminal extensions of the toxin have no measurable effect on its affinity (21, 40). We tested toxin ApB H39A as a representative of this group. Residues Arg-12 and Lys-49 are known to have a significant and cooperative effect on toxin affinity (23) but are not conserved among all anemone toxins; in particular, they are neutral in ATX-II, the toxin tested by Rogers and colleagues (18). We tested these residues by using toxin ApA, which has neutral residues at both positions (in addition to several more conservative differences which have been shown to have only minor effects on toxin affinity) (22, 40). The remaining residue, Lys-37, significantly decreased the affinity of ApB when neutralized (25) and is also conserved among a number of anemone toxins. It was, therefore, a strong candidate for the interaction and was tested with toxin ApB K37A. As a positive control, we also tested a toxin mutated at a hydrophobic position, toxin ApB W45F. This toxin has lower affinity for cardiac channels than does wild-type ApB (34) but would not be expected to interact with an anionic channel residue.

Toxin Interactions with D1612R-- Having demonstrated an attraction between cationic toxin residue Lys-37 and anionic channel residue Asp-1612, we attempted to preserve this interaction by transposing the charges of the two residues. To this end, we constructed the charge-reversed toxin ApB K37D and the charge-reversed channel rH1 D1612R. Unfortunately, circular dichroism revealed that ApB K37D possessed a significantly less ordered secondary structure than that of wild-type ApB or the neutralization mutant ApB K37A (25) (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Circular dichroism spectra of wild-type ApB (line), ApB K37D (), and ApB K37A (). Spectra were measured at room temperature and neutral pH in cylindrical quartz cells at protein concentrations of 100-200 µg/ml and deconvoluted by least squares comparison to a data base containing proteins of known structure (cytochrome C, ribonuclease A, myoglobin, hemoglobin, papain, -chymotrypsinogen, trypsin, lysozyme, and alcohol dehydrogenase). Calculated secondary structure was as follows: ApB, 0.8% -helix, 52.3% -sheet, 21.0% -turn; ApB K37A, 0.0% -helix, 50.9% -sheet, 18.8% -turn; ApB K37D, 1.0% -helix, 17.1% -sheet. 8.2% -turn (ApB and ApB K37A, 25).

	DISCUSSION

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We have isolated a specific interaction involved in the binding of the site-3 toxin anthopleurin B to the rat cardiac sodium channel. Channel residue Asp-1612 and toxin residue Lys-37 display an energy of interaction of G = 1.5 kcal/mol. This coupling energy is less than that expected for a hydrogen bond to a charged residue (41) but is well within the range predicted for electrostatic interactions (41). For example, Schreiber and Fersht (42) reported values ranging from a noise level of 0.4 kcal/mol up to values typical of H-bonds. Indeed, while neutralization of Lys-37 in the context of a wild-type channel decreased affinity by 15-fold, the mutant channel D1612N was almost insensitive to a charge at toxin position 37. Thus all of the effects of neutralizing toxin residue Lys-37 on the affinity of the toxin are accountable through the actions of channel residue Asp-1612. A comparison of the sequences of 15 homologous toxins isolated from sea anemone revealed all toxins but one, ATX-I from A. sulcata (43), to have a positive residue at or adjacent to position 37. Interestingly, toxin ATX-I is ineffective when applied to vertebrate sodium channels at 100 µM (5). Further, an anionic residue at channel position 1612 is highly conserved between mammalian sodium channel isoforms (31). These data strongly support the hypothesis that the interaction between Lys-37 and Asp-1612 comprises a conserved component of binding site-3 in mammalian channels.

Site-3 toxins act to delay sodium channel inactivation (4-6). Because the extracellular face of the channel is not typically thought to be associated with the inactivation process, the manner by which these toxins produce their kinetic effect remains unclear. In light of this ambiguity, it is interesting that reversal of the charge at channel residue Asp-1612 produced a shift in the voltage dependence of steady-state availability. Since perturbation of this residue affects inactivation gating, it is possible that Asp-1612 participates in the control of both the affinity and the kinetic effects of toxin binding.

At present, little is known of the structure of the extracellular face of the sodium channel outside of the immediate region of the pore vestibule. The interaction between Asp-1612 and toxin residue Lys-37 provides a defined interaction between the channel and a molecule with a known three-dimensional structure (44). The determination of more such interactions should therefore increase our insight into the structure and function of the exterior of the channel.