Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels

1) For a non-expert, the effect of PcTx1 on membrane partitioning using Trp fluorescence seems non-significant (Figure 2B). Can the authors elaborate on why they think this is significant in the corresponding manuscript section? Maybe it is worth showing fluorescence emission spectra for PcTx1 at higher lipid concentrations? We do understand that ensuing experiments attempt to verify the effect shown in Figure 2B but readers may get confused here. Also, do the authors expect to see a difference in membrane partitioning when adding in other lipids such as sphingomyelin and cholesterol to make the vesicles more mammalian-like (e.g. Cao et al., Neuron. 2013 77(4):667-79)? Finally, can the authors refer to (or perform) a control experiment under similar conditions with a peptide that is know not to partition in the membrane at all (if this exists)?

The blue shifts we observed in Trp Fluorescence for PcTx1 as liposomes are added to aqueous solutions of the toxin are indeed very small. However, we think these blue shifts are significant and suggestive of an interaction between the toxin and membranes because they are reproducible and titrate with the amount of lipid added to the cuvette, as shown in the data plotted in Figure 2D. Having said that, we also thought that it was crucial to verify this initial suggestion of an interaction between PcTx1 and membranes by asking whether membranes protect the toxin from quenching by acrylamide, as shown in Figure 2F and Figure 3. We have revised this section of the manuscript to clarify our thinking and we have removed any quantitative analysis of these data due to the very small blue shifts observed with PcTx1 (see Results, third paragraph).

We have not undertaken experiments with PcTx1 and more mammalian-like vesicles. It would be interesting, but we do not have a clear expectation of what we would find. In the case of the voltage-sensor toxins SGTx1, we can detect an interaction between the toxin and Xenopus oocyte membranes (containing both cholesterol and sphingomyelin), but these are relatively weak and difficult to quantify (see Milescu et al., 2007 in the reference list). The weak nature of the interaction of SGTx1 with more mammalian-like membranes led us to conclude that membrane partitioning will not lead to large increases in the toxin concentration within the membrane, but would certainly be sufficient for the toxin to access its receptor (the voltage-sensing domains) within the membrane.

It is an interesting point about whether any small protein toxin would interact with membranes with properties similar to what we observed with PcTx1. In response to the final point above, we examined the interaction between membrane vesicles and charybdotoxin, a pore-blocking scorpion toxin that contains one Trp residue surrounded by basic residues, which we suspected might not interact with membranes. However, we observed measurable (but small) blue shifts in Trp fluorescence, suggesting that charybdotoxin does interact with membranes. Moreover, a previous study investigated the interaction of radiolabeled charybdotoxin with membrane vesicles and the toxin was found to interact with membranes through an electrostatic mechanism (Ben-Tal et al., 1997, Biophysical J. 73, 1717-1727.) We now cite this paper and discuss the relevance to our findings with PcTx1 on the second paragraph of the Discussion.

2) The Kdx value of PcTx1 obtained using the acrylamide experiment seems very different than that obtained using blue-shifts in Trp fuorescence whereas that of GxTx-1E is in reasonable agreement with the value obtained in Figure 2. Is there an explanation for this discrepancy? Is it possible that the two toxins present different surfaces to lipid vesicles which may alter the Trp that is presented to the lipid membrane?

We attribute this difference to the very small blue shifts observed with PcTx1, as discussed above. We have now removed the fits to the titration experiment in Figure 1D to avoid confusion. The blue shifts in Trp fluorescence for PcTx1 can only be taken as a qualitative measure of a membrane interaction (which itself required validation), and not as a quantitative measure of the strength of membrane interaction.

Although we have not determined the orientation of either GxTx-1E or PcTx1 with membranes, we think that it is likely that they present different surfaces to membranes as their amphipathic character in their surfaces overlap, but are clearly distinct. This can be appreciated by comparing Figure 1E and F, and appreciating that GxTx-1E in panel F has been rotated by 90° relative to the backbone superposed orientations shown in panels C, D and H.

3) Have the authors tried to insert longer S3b-S4 segments of ASIC1a into Kv2.1 to make the channel sensitive to PcTx1? From their previous publications on sodium channel constructs, it seems that transferring regions between channels is not always straightforward and may require some ingenuity (e.g. alignment gaps are frequently observed).

We have not tried to make larger chimeras (beyond transferring helix 5) because most of the buried surface of PcTx1 when bound to ASIC involves interactions with residues in helix 5. PcTx1 does contain an arginine finger motif composed of R26, R27 and R28, that inserts into a crevice situated at the subunit interface in ASIC housing the protonated residues responsible for proton activation (Baconguis and Gouaux 2012). However, the residues of the channel interacting with the Arg finger motif are located in regions discontinuous with helix 5, and thus would not be amenable to making chimeras. We have added several sentences to the Results section to explain these structural issues to the reader.

4) If GxTx-1E can partition deeply into the membrane to interact with the voltage sensor in Kv2.1 and PcTx1 cannot, but both toxins share a mechanism to latch onto alpha-helices, is it unreasonable to expect an effect of GxTx-1E on ASIC channels? Have the authors tested the effect of (maybe higher concentrations) GxTx-1E on ASICs?

It is possible that GxTx-1E might be able to bind to ASICs at high concentrations, but it would not be able to activate the channel because it lacks the Arg finger of PcTx1 that is required for activation of the channel by protons.