μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

Abstract

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na(V)1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na(V)1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 μ-conopeptides for their ability to block rodent Na(V)1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked Na(V)1.8, a TTX-resistant isoform, the resulting "activity matrix" revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A- and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A- and C-fibers were Na(V)1.6 and 1.7, respectively. Ruled out as major players in both fiber types were Na(V)1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive Na(V)1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of μ-conopeptides should be useful in identifying the functional contributions of Na(V)1 isoforms in other preparations.

Fig. 1.

Effect of SmIIIA on Na_V1.1 through 1.8 expressed in oocytes. Oocytes, each expressing a different Na_V1 isoform, were voltage-clamped, and I_Na measurements were recorded as described in Materials and Methods. (A) Representative currents traces of Na_V1.1 through 1.8 before (gray trace) and during exposure to SmIIIA (1 μM for all except 10 μM for Na_V1.8; bold trace) and after its washout (gray trace). (B) Representative time courses of block by SmIIIA and recovery during washout. Oocytes were voltage clamped as in A. Plotted are peak I_Na obtained every 20 s before, during, and after an approximately 3- to 10-min exposure to SmIIIA (concentrations as in A, presence of peptide indicated by bar above each plot). Note biphasic recovery from block of Na_V1.6. Data are quantified in Table 1 and Table S2.

Fig. 2.

Dose–response curves for all peptides and all Na_V1 isoforms tested. Na_V1 isoform numbers are listed above each plot in the order of appearance (left to right) of their respective curves. Data points for KIIIA with Na_V1.2 were previously published; note that 100% block was not observed at saturating [KIIIA] because its blocking efficacy is 95% (16, 17)—a similar phenomenon appears to occur with KIIIA block of Na_V1.7, with a blocking efficacy of approximately 90%. Asterisks indicate K_d values for those instances in which block was too slow to reach steady state within the experimental time frame at peptide concentrations near expected IC₅₀ values (Materials and Methods). Table 1 provides a tabulated summary. Data points are mean ± SD (n ≥ 3 oocytes).

Fig. 3.

Susceptibility of A- and C-CAPs of rat sciatic nerve to μ-conopeptides. CAPs were evoked by electrical stimulation and recorded as described in Materials and Methods. Upper two panels in each group of four panels show time course of normalized peak-to-peak amplitudes of A-CAPs (Left) and C-CAPs (Right) simultaneously recorded from sciatic nerve, where the bar above each plot indicates when toxin was present; horizontal calibration (time, in min; Left) applies to all. Data points represent mean ± SE (n ≥ 4). Discontinuities in plots occurred when solutions in wells were refreshed (SI Materials and Methods). Note delay before response starts to recover following toxin washout for A-CAPs in A and F and for C-CAPs in F. Lower two panels of each group of four show representative traces of A-CAPs (Left) and C-CAPs (Right). Traces obtained in control solution are gray and in those the presence of 10 μM μ-conopeptide (A–E) or 1 μM TTX (F) are black. A- and C-CAPs were captured in the same sweep, but for clarity, the displayed durations of their respectively traces differ. Note stimulus artifact at the start of each A-CAP trace. C-CAP traces are illustrated with a gap between the end of a given A-CAP trace and the beginning of the corresponding C-CAP trace, the actual time represented by the gap varied depending on length of nerve used.