Synergistic and antagonistic interactions between tetrodotoxin and mu-conotoxin in blocking voltage-gated sodium channels

Abstract

Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, mu-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered mu-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain NaV1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded NaV complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in NaV pharmacology.

Figure 1

Steady state dose-response curves reveal a residual Na current (rI_Na) at saturating peptide concentrations. Data (KIIIA, closed circles; KIIIA[K7A], open circles; TTX, closed squares) were obtained with oocytes expressing Na_V1.2 as described in Methods. Small k_on prevented acquisition of steady-state data at [peptide] in the vicinity of K_d, represented by asterisk, obtained from k_off/k_on (Table 1). Solid lines represent fit of data, including asterisks, to the Langmuir isotherm, Y = 100 • plateau/[1 + (IC₅₀/[toxin])]. Plateau (i.e., Y-axis value at saturating [toxin]) was 95 ± 3% for KIIIA and 77 ± 1% for KIIIA[K7A] and 100% for TTX. Insets, representative current recordings in the absence (control, gray traces) and presence (black traces) of saturating concentrations of KIIIA (10 μM, left) and KIIIA[K7A] (100 μM, right).

Figure 2

TTX blocks residual I_Na that persists in saturating concentrations of KIIIA[K7A] or KIIIA and accelerates the rate of peptide-washout. Example plots of peak I_Na recorded from oocytes during exposure to: TTX (a); KIIIA[K7A] (b); KIIIA[K7A] followed by KIIIA[K7A]+TTX (c); TTX followed by TTX+KIIIA[K7A] (d); KIIIA (e); KIIIA followed by KIIIA+TTX (f); and TTX followed by TTX+KIIIA (g). Different oocyte was used in each of panels b to g, where previous exposure to 10 μM TTX obliterated I_Na (not shown, but as in a). Presence of 10 or 100 μM TTX is indicated by black bar, 30 μM KIIIA[K7A] by hatched bar, and 10 μM KIIIA by gray bar. In all cases, except e, recovery of I_Na during toxin-washout could be fit by a single exponential time course (k_off values are listed in Table 1). Test pulses were applied at 20 s intervals (see Methods).

Figure 3

Rate of TTX-block of control I_Na (a), residual I_Na in 30 μM KIIIA[K7A] (b), and in 10 μM KIIIA(c) differ radically from each other (note different time scales). Time course of block of rI_Na by various [TTX], each fit to a single exponential (not illustrated), provided the observed rate constants (k_obs). Plots show k_obs for different [TTX]. Relative timing of the exposure to toxin was essentially as in Fig. 2a for Na_V, Fig. 2c for Na_V•KIIIA[K7A], and Fig. 2f for Na_V•KIIIA. Linear regression slope, providing apparent k_on, was 58 ± 5.3 μM^-1min^-1 (a), 0.56 ± 0.08 μM^-1min^-1 (b), and 0.003 ± 0.0004 μM^-1min^-1 (c).

Figure 4

Apparent on-rate of peptide binding in the presence of TTX was only slightly slower than the rate of block by peptide in the absence of TTX. Level of peptide bound to Na_V1.2 in the face of 10 μM TTX was determined by the amount of persistent-block following washout of both toxins. (a) Protocol used to determine level of persistent-block by peptide (i.e., formation of Na_V•TTX•peptide). Results from series of three hypothetical experiments are superimposed: oocyte was exposed to TTX for 2 min. followed by exposure to TTX + peptide for 2, 4, or 8 min before washing out both toxins (black bars represent presence of TTX, and gray bars, TTX + peptide). In each case, the washout curve was fit to a double exponential, of which the span and k_off of the faster and slower components represent the recovery from block of Na_V•TTX and Na_V•TTX•peptide, respectively. The closed circles represent the span of the slower component and correspond to the closed circles in c and d. (b) Representative results showing peak I_Na during exposure to, and following washout of, TTX and KIIIA (black and gray bars, respectively). (c, d) Time course for the formation of Na_V • TTX•peptide (closed circles), estimated by the persistent-block produced by 1 μM peptide as described in a for different durations of simultaneous-exposure to TTX+peptide (cf., closed circles in a), compared with the formation of Na_V • peptide (open circles) simply measured by block produced by 1 μM peptide alone. Data were fit to single exponential curves (solid lines). k_obs for formation of Na_V • KIIIA[K7A] and Na_V • TTX • KIIIA[K7A] was 0.17 ± 0.006 and 0.13 ± 0.006 min^-1, respectively (c). k_obs for formation of Na_V • KIIIA and Na_V •TTX • KIIIA was 0.27 ± 0.01 and 0.20 ± 0.008 min^-1, respectively (d).

Figure 5

Single-channel records (a) and proposed reaction scheme (b). The conductance states “R”, “A”, “B”, and “C” in (a) correspond to the 4 configurations in (b). All putative transitions except “B” to “C” are illustrated in (a); the “B” to “C” transition would not result in a change in the current level due to complete block by TTX. (a) Recordings from a single rat brain sodium channel incorporated into a planar bilayer membrane were obtained as described in Methods. Upper trace: Begins in the “R” level (open level, unblocked) in the presence of KIIIA[K7A] (2 μM), and then steps to the partially blocked level “A” indicating binding of the peptide. After KIIIA[K7A] binding, TTX concentration was increased to 10 μM, and ∼1 minute later, a transition was seen to a fully blocked level “C”, which we interpret as the bi-liganded state (Na_V•TTX•KIIIA[K7A]). Lower trace, channel block in the presence of TTX (50 nM), without peptide, clearly defines the unblocked open level and fully blocked/closed level “B”, which shows the same conductance as the bi-liganded state “C” shown in the upper trace. (b) Reaction scheme consistent with both single-channel and macroscopic results. “R” represents ligand-free (fully functional) channel; “A”, peptide-blocked channel (where residual I_Na is indicated by dashed arrow); “B”, TTX-blocked channel; and “C”, the ternary (bi-liganded Na_V) complex.