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Μ-Conotoxins That Differentially Block Sodium Channels Nav1.1 Through 1.8 Identify Those Responsible for Action Potentials in Sciatic Nerve

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Μ-Conotoxins That Differentially Block Sodium Channels Nav1.1 Through 1.8 Identify Those Responsible for Action Potentials in Sciatic Nerve μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve Michael J. Wilsona, Doju Yoshikamia, Layla Azama, Joanna Gajewiaka, Baldomero M. Oliveraa,1, Grzegorz Bulajb, and Min-Min Zhanga,1 Departments of aBiology and bMedicinal Chemistry, University of Utah, Salt Lake City, UT 84112 Contributed by Baldomero M. Olivera, May 12, 2011 (sent for review October 14, 2010) Voltage-gated sodium channels (VGSCs) are important for action (or μ-conotoxins). Other conopeptides have been effective potentials. There are seven major isoforms of the pore-forming pharmacological agents for distinguishing among subtypes of and gate-bearing α-subunit (NaV1) of VGSCs in mammalian neu- various ion channel families in nervous systems, in particular, rons, and a given neuron can express more than one isoform. Five voltage-gated Ca channels and nicotinic receptors. Unfortuna- of the neuronal isoforms, NaV1.1, 1.2, 1.3, 1.6, and 1.7, are exqui- tely, thus far, no venom component from cone snails has proven sitely sensitive to tetrodotoxin (TTX), and a functional differenti- to be as highly subtype-selective for the various NaV1 isoforms as ation of these presents a serious challenge. Here, we examined some of the peptides that target Ca channels (e.g., ω-conotoxin N a panel of 11 μ-conopeptides for their ability to block rodent GVIA and MVIIA for the -type Ca channel) (14). Nevertheless, as we demonstrate in this report, members of the μ-conotoxin NaV1.1 through 1.8 expressed in Xenopus oocytes. Although none family possess sufficient selectivity for the different neuronal blocked NaV1.8, a TTX-resistant isoform, the resulting “activity matrix” revealed that the panel could readily discriminate be- TTX-sensitive VGSCs to provide a panel of antagonists that can be used to assess which NaV1 isoforms are functionally important tween the members of all pair-wise combinations of the tested μ isoforms. To examine the identities of endogenous VGSCs, a subset in a neuronal preparation (Table S1 shows -conopeptide seq- of the panel was tested on A- and C-compound action potentials uences and primary references). Panel members were described recorded from isolated preparations of rat sciatic nerve. The results previously in varying detail; however, in this report, we greatly expand the database concerning their ability to block Na 1.1 show that the major subtypes in the corresponding A- and C-fibers V through 1.8 expressed in Xenopus oocytes. We then use the were Na 1.6 and 1.7, respectively. Ruled out as major players in V panel to identify the major Na 1 isoforms responsible for action both fiber types were Na 1.1, 1.2, and 1.3. These results are consis- V V potentials in rat sciatic nerve. tent with immunohistochemical findings of others. To our aware- ness this is the first report describing a qualitative pharmacological Results survey of TTX-sensitive Na 1 isoforms responsible for propagating V Sodium Currents in Xenopus Oocytes Expressing Na 1.1 Through 1.8 action potentials in peripheral nerve. The panel of μ-conopeptides V and Their Sensitivities to μ-Conopeptides. Voltage-activated sodium should be useful in identifying the functional contributions of NaV1 currents (I ) of two-electrode voltage-clamped oocytes, each isoforms in other preparations. Na expressing a single NaV1 isoform, were recorded as described in Materials and Methods. All NaV1 isoforms tested were from rat odgkin and Huxley developed a quantitative theory for the except NaV1.6, which was from mouse. INa of NaV1.1, 1.2, 1.3, — μ Hionic basis of the action potential (1) the molecular cor- 1.4, 1.6, and 1.7 were totally obliterated by 1 M TTX, INa of relates of their pioneering efforts are the voltage-gated sodium NaV1.5 was blocked only approximately 40% by 1 μM TTX, and channel (VGSC) and the voltage-gated potassium channel. only approximately 15% of the INa of NaV1.8 was blocked by 10 Critical for the appreciation of the discrete biochemical nature μM TTX. These TTX susceptibilities are as expected (7). of VGSCs were the investigations of the mechanism of action of None of the μ-conopeptides in the panel blocked rNaV1.8; the alkaloids tetrodotoxin (TTX) and saxitoxin (2–4). In the half however, the other channels were susceptible to varying extent. century since these classic experiments, our understanding of the For example, 1 μM SmIIIA blocked the INa of NaV1.1 through molecular structure of VGSCs has advanced dramatically (5). 1.7 by approximately 40% or more but did not affect the TTX- We now recognize that mammals have nine isoforms of the resistant I of Na 1.8 (Fig. 1A). α Na V -subunit of VGSCs, or NaV1, the subunit containing both the The time course of recovery of I of the different Na 1 + Na V Na -conducting pore and voltage-sensing gate (5–7). Mean- isoforms during the washout of μ-conopeptide fell into three while, progress in the characterization of ligands that target categories: (i) recovery was monophasic with kinetics that fol- VGSCs has also progressed (8, 9), although TTX remains a ma- lowed a single-exponential time course from which koff was de- jor pharmacological investigative tool for VGSCs. termined by curve fitting; (ii) recovery from block was very slow, The nine mammalian NaV1 isoforms can be categorized into less than 50% in 20 min, in which case single-exponential block those that are TTX-sensitive versus those that are resistant, with was assumed and koff estimated from the level of recovery after K or IC values in the nM versus μM ranges, respectively (7). −1 d 50 20 min of wash (these involved koff values < 0.04 min ); or (iii) Two, NaV1.8 and NaV1.9, are found in primary sensory neurons the recovery occurred with two distinct phases: the first phase and are highly resistant to TTX (10–12). One, NaV1.5, found in cardiac muscle, is moderately TTX resistant (13), and the remaining six Na 1 isoforms are exquisitely sensitive to TTX. V Author contributions: M.J.W., D.Y., and M.-M.Z. designed research; M.J.W. and M.-M.Z. One of these, NaV1.4 is found exclusively in skeletal muscle. performed research; L.A. and J.G. contributed new reagents/analytic tools; M.J.W., D.Y., Presently, the five neuronal subtypes that are TTX-sensitive (i.e., and M.-M.Z. analyzed data; and M.J.W., D.Y., L.A., J.G., B.M.O., G.B., and M.-M.Z. wrote NaV1.1, 1.2, 1.3, 1.6, and 1.7) cannot be readily functionally the paper. differentiated in native systems. The authors declare no conflict of interest. In this work, we initiate a pharmacology for differentiating the 1To whom correspondence may be addressed. E-mail: [email protected] or TTX-sensitive NaV1 isoforms found in neurons. The biochemical [email protected]. basis for this incipient pharmacology is a family of peptide toxins This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. found in the venoms of fish-hunting cone snails, the μ-conopeptides 1073/pnas.1107027108/-/DCSupplemental. 10302–10307 | PNAS | June 21, 2011 | vol. 108 | no. 25 www.pnas.org/cgi/doi/10.1073/pnas.1107027108 Downloaded by guest on September 28, 2021 KIIIA blocks NaV1.2 with 95% efficacy, as previously described in some detail (16, 17; also see ref. 18). The block of NaV1.7 by KIIIA appears to have approximately a 90% efficacy. We pre- sume KIIIA’s “leaky” block of NaV1.7 occurs by a mechanism similar to that hypothesized for KIIIA’s incomplete block of NaV1.2 (16, 17), and studies are under way to investigate this further. Effect of β1-Subunit Coexpression with NaV1.1 Through 1.7 on KIIIA Activity. In addition to NaV1 isoforms, four isoforms of β-subunits (NaVβ), which can associate with NaV1 and modulate its function, have been identified (19, 20). To examine the possible influence of an NaVβ subunit on μ-conopeptide activity, the effect of the coexpression of NaVβ1 with each of the seven NaV1isoformsonthe affinity of KIIIA was examined. As expected (21, 22), for each NaV isoform except NaV1.5, coexpression with β1 increased the rate of inactivation of INa. Minimal, if any, changes in the kinetics of block by KIIIA were induced by β1coexpression(Table S3). NaV1 Isoform Selectivities of the μ-Conopeptides Quantified by Discrimination Index. The discrimination index (DI), the log of the ratio of the affinity constants of a peptide for two channels (Materials and Methods), served as a simple quantitative measure of a μ-conopeptide’s ability to distinguish among NaV1isoforms. Table 2 displays the peptides with the best DI values distilled from pair-wise comparisons of all NaV1 isoforms tested, save NaV1.8. A DI of 1.0 corresponds to a situation where the peptide’s affinity constant (either IC50 or Kd) for one channel is 10-fold lower than that for the other; thus, it can be surmised from the curves in Fig. 2 (which all fit the Langmuir equation), a concen- tration of the peptide that blocks 90% of one channel would block 50% of the other. If a peptide had a DI of 1.5, its con- centration that blocks 90% of one channel would block only approximately 20% of the other. The peptides with a DI of at Fig. 1. Effect of SmIIIA on NaV1.1 through 1.8 expressed in oocytes. least 1.5 in Table 2 are marked with asterisks.
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