Dynamic landscape of the intracellular termini of acid-sensing ion channel 1a

Reviewer #1 (Public Review):

Summary: Cullinan et al. explore the hypothesis that the cytoplasmic N- and C-termini of ASIC1a, not resolved in x-ray or cryo-EM structures, form a dynamic complex that breaks apart at low pH, exposing a C-terminal binding site for RIPK1, a regulator of necrotic cell death. They expressed channels tagged at their N- and C-termini with the fluorescent, non-canonical amino acid ANAP in CHO cells using amber stop-codon suppression. Interaction between the termini was assessed by FRET between ANAP and colored transition metal ions bound either to a cysteine reactive chelator attached to the channel (TETAC) or metal-chelating lipids (C18-NTA). A key advantage to using metal ions is that they are very poor FRET acceptors, i.e. they must be very close to the donor for FRET to occur. This is ideal for measuring small distances/changes in distance on the scales expected from the initial hypothesis. In order to apply chelated metal ions, CHO cells were mechanically unroofed, providing access to the inner leaflet of the plasma membrane. At high pH, the N- and C- termini are close enough for FRET to be measured, but apparently too far apart to be explained by a direct binding interaction. At low pH, there was an apparent increase in FRET between the termini. FRET between ANAP on the N-and C-termini and metal ions bound to the plasma membrane suggests that both termini move away from the plasma membrane at low pH. The authors propose an alternative hypothesis whereby close association with the plasma membrane precludes RIPK1 binding to the C-terminus of ASIC1a.

Strengths: The findings presented here are certainly valuable for the ion channel/signaling field and the technical approach only increases the significance of the work. The choice of techniques is appropriate for this study and the results are clear and high quality. Sufficient evidence is presented against the starting hypothesis.

Weaknesses: I have a few questions about certain controls and assumptions that I would like to see discussed more explicitly in the manuscript.

--My biggest concern is with the C-terminal citrine tag. Might this prevent the hypothesized interaction between the N- and C-termini? What about the serine to cysteine mutations? The authors might consider a control experiment in channels lacking the C-terminal FP tag.

--Figure 2 supplement 1 shows apparent read-through of the N-terminal stop codons. Given that most of the paper uses N-terminal ANAP tags, this figure should be moved out of the supplement. Do N-terminally truncated subunits form functional channels? Do the authors expect N-terminally truncated subunits to co-assemble in trimers with full-length subunits? The authors should include a more explicit discussion regarding the effect of truncated channels on their FRET signal in the case of such co-assembly.

--As the epitope used for the western blots in Figure 2 and supplements is part of the C-terminal tag, these blots do not provide an estimate of the fraction of C-terminally truncated channels (those that failed to incorporate ANAP at the stop codon). What effect would C-terminally truncated channels have on the FRET signal if incorporated into trimers with full-length subunits?

--Some general discussion of these results in the context of trimeric channels would be helpful. Is the putative interaction of the termini within or between subunits? Are the distances between subunits large enough to preclude FRET between donors on one subunit and acceptor ions bound on multiple subunits?

--The authors conclude that the relatively small amount of FRET between the cytoplasmic termini suggests that the interaction previously modeled in Rosetta is unlikely. Is it possible that the proposed structure is correct, but labile? For example, could it be that the FRET signal is the time average of a state in which the termini directly interact (as in the Rosetta model) and one in which they do not?

Reviewer #2 (Public Review):

Summary:
The authors use previously characterised FRET methods to measure distances between intracellular segments of ASIC and with the membrane. The distances are measured across different conditions and at multiple positions in a very complete study. The picture that emerges is that the N- and C-termini do not associate.

Strengths:
Good controls, good range of measurements, advanced, well-chosen and carefully performed FRET measurements. The paper is a technical triumph. Particularly, given the weak fluorescence of ANAP, the extent of measurements and the combination with TETAC is noteworthy.

The distance measurements are largely coherent and favour the interpretation that the N and C terminus are not close together as previously claimed.

Weaknesses:
One difficulty is that we do not have a positive control for what binding of something to either N- or C-terminus would look like (either in FRET or otherwise).

One limitation that is not mentioned is the unroofing. The concept of interaction with intracellular domains is being examined. But the authors use unroofing to measure the positions, fully disrupting the cytoplasm. Thus it is not excluded that the unroofing disrupts that interaction. This should be mentioned as a possible (if unlikely) limitation.

Reviewer #3 (Public Review):

Summary: The manuscript by Cullinan et al., uses ANAP-tmFRET to test the hypothesis that the NTD and CTD form a complex at rest and to probe these domains for acid-induced conformational changes. They find convincing evidence that the NTD and CTD do not have a propensity to form a complex. They also report these domains are parallel to the membrane and that the NTD moves towards, and the CTD away, from the membrane upon acidification.

Strengths:
The major strength of the paper is the use of tmFRET, which excels at measuring short distances and is insensitive to orientation effects. The donor-acceptor pairs here are also great choices as they are minimally disruptive to the structure being studied.

Furthermore, they conduct these measurements over several positions with the N and C tails, both between the tails and to the membrane. Finally, to support their main point, MST is conducted to measure the association of recombinant N and C peptides, finding no evidence of association or complex formation.

Weaknesses:
While tmFRET is a strength, using ANAP as a donor requires the cells to be unroofed to eliminate background signal. This causes two problems. First, it removes any possible low affinity interacting proteins such as actinin (PMID 19028690). Second, the pH changes now occur to both 'extracellular' and 'intracellular' lipid planes. Thus, it is unclear if any conformational changes in the N and CTDs arise from desensitization of the receptor or protonation of specific amino acids in the N or CTDs or even protonation of certain phospholipid groups such as in phosphatidylserine. The authors do comment that prolonged extracellular acidification leads to intracellular acidification as well. But the concerns over disruption by unroofing/washing and relevance of the changes remain.

The distances calculated depend on the R0 between donor and acceptor. In turn, this depends on the donor's emission spectrum and quantum yield. The spectrum and yield of ANAP is very sensitive to local environment. It is a useful fluorophore for patch fluorometry for precisely this reason, and gating-induced conformational changes in the CTD have been reported just from changes in ANAP emission alone (PMID 29425514). Therefore, using a single R0 value for all positions (and both pHs at a single position) is inappropriate. The authors should either include this caveat and give some estimate of how big an impact changes spectrum and yield might have, or actually measure the emission spectra at all positions tested.

Overall, the writing and presentation of figures could be much improved with specific points mentioned in the recommendations for authors section.

The authors argue that the CTD is largely parallel to the plasma membrane, yet appear to base this conclusion on ANAP to membrane FRET of positions S464 and M505. Two positions is insufficient evidence to support such a claim. Some intermediate positions are needed.

Upon acidification, NTD position Q14 moves towards the plasma membrane (Figure 8B). Q14 also gets closer to C515 or doesn't change relative to 505 (Figures 7C and B) upon acidification. Yet position 505 moves away from the membrane (Figure 8D). How can the NTD move closer to the membrane, and to the CTD but yet the CTD move further from the membrane? Some comment or clarification is needed.