1. Evaluation Summary:

    This is a rigorous and clearly written paper that provides quantitative data for the scope of intracellular signalling by ASIC channels. These channels are involved in pain signalling and other processes, and apparently can couple to intracellular pathways independent of ion flow. Here the authors measure the movements of the unstructured intracellular parts of ASIC using fluorescence spectroscopy coupled to functional measurements.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, #2, and #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    ASIC channels are important physiological mediators of pain and have other functions as sensory transducers. Although several high-resolution structures of these channels exist, the terminal domains have not been resolved. Given that they likely mediate important molecular interactions, it is important to obtain at least a coarse-grained approximation to their positions and interactions within the channel.
    Fluorescence methods such as FRET allow these kind of experiments and are used by Couch et at. to provide such an approximation in the ASIC1 channel.
    The authors have made use of the ability of the DPA (dipicryl-amine) molecule to quench the fluorescence of fluorescent proteins (FPs) via FRET, providing a convenient molecular ruler.

    The authors insert FPs in several positions in the N and C terminal regions of the channel and place DPA in the membrane. Estimation of the degree quenching allows to asses the relative position and eventually motions of the FPS with respect to the membrane.

    Using this approach the authors have succeeded in estimating that the N terminus is very close to the membrane and the C-terminus hangs like a tail into the cytoplasm.
    The attempt to measure relative motions of the FPs normal to the membrane, have, in my opinion, failed. The observed changes are to small and seem to be buried in statistical noise.

    The paper however, is a nice collection of well executed experiments that provide interesting information and should be the basis of future, finer resolution, experiments aimed at understanding the dynamics of these intracellular regions.

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  3. Reviewer #2 (Public Review):

    The authors set out to measure intracellular movements of ASIC channels using fluorescence techniques, and they have provided a detailed and thorough account. The manuscript is clearly and concisely written.

    The authors measure proximity between the N and C terminals of individual subunits and the axial displacement of the same parts. They use previous work and exemplary control measurements to bolster their work.

    This work outlines the scope of the movements of the intracellular termini, and provides a benchmark for future studies of its type. The manuscript represents the very best of careful, intelligent biophysics and cell biology.

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  4. Reviewer #3 (Public Review):

    The structure of the acid-sensing ion channel ASIC1a, a proton-gated cation channel, has been determined in resting, desensitized, and toxin-bound open states. However, the intracellular N- and C-termini are not resolved in any of these structures. Couch et al. sought to outline their structures and any conformational changes associated with ASIC gating using FRET coupled with patch-clamp electrophysiology. The authors inserted fluorescent protein tags at the N-terminus and various positions on the C-terminus of ASIC1 and measured the distance between these tags and the plasma membrane using the membrane-embedded FRET acceptor dipicrylamine (DPA). Using fluorescence lifetime measurements, the authors demonstrated that the N- and C-termini of a given subunit are in close proximity to one another. By observing FRET between fluorescent proteins on N- and C-termini of the same ASIC subunit, the authors demonstrate that there is no substantial rearrangement between the intracellular termini during pH gating. However, they did observe that the N- and proximal C-termini do move relative to plasma membrane during the transition from the resting to the pH-desensitized state.

    This paper should be of interest to those working on acid-sensing ion channels and of broader interest to those working on ion channels, receptors, and membrane protein structural biology. The study was well designed and the data are of high quality. The authors took great care to provide a conservative interpretation of their data. I have only minor concerns regarding sources of error, particularly with respect to interpretation of the small effects the authors observe in many of their FRET experiments.

    • Figure 2D shows rather small changes in ΔF/F-15 mV between fluorescent protein labels inserted at different positions in the ASIC sequence, particularly for the YFP constructs. As this metric is determined from the top and bottom asymptotes for the Boltzmann fits shown in Figure 2C, it would be useful to have some estimate as to the error associated with the fits at extreme values. Perhaps the authors could provide fits to their data (as in Figure 2C), including confidence intervals, or some similar estimate as to the size of the expected error compared to the effect size in Figure 2D.

    • Along those same lines, the authors use an interesting (and potentially generalizable) approach to reducing background from intracellular proteins in their experiments: co-transfecting their channels with empty plasmid DNA. What percentage of the remaining fluorescence signal is the result of intracellular background? How would that affect the data in Figure 2 and 3? Is the ΔF/Fnorm curve for YFP labeled positions in Figure 2-figure supplement 4 so flat because of contaminating background fluorescence?

    • In Figure 3D, the FRET efficiency between CFP-cA1-cA1 and N YFP at a 1:15 ratio of the two plasmids is higher than the FRET efficiency between CFP and YFP in the same subunit, even though the authors conclude that fluorescent proteins on the same subunit show considerably more FRET than fluorescent proteins on neighboring subunits. Could this indicate that the N-termini of adjacent subunits are closer together than the N- and C-termini of a single subunit? If, on the other hand, this effect were entirely the result of crowding in the membrane why is FRET efficiency substantially lower when CFP-cA1-cA1 is co-expressed with C4 YFP? Wouldn't this construct produce a similar crowding effect?

    • On page 23, the authors state that they detected no pH-dependent changes in FRET between their GFP tag on the N-terminus of ASIC1 and an RFP tag on the channel's C-terminus. However, Figure 4 shows a small, but significant change in fluorescence between pH 8 and pH 7.

    • The interpretation of distances between various tagged position on ASIC and the plasma membrane in Figure 2 is based on using two different colored tags with two different distance dependences. However, the interpretation of the data from Figure 5 provided on page 25 is less clear. For example, the reduction in fluorescence from the N-terminal tag is interpreted as the tag moving closer to the plasma membrane. Without similar data from a YFP tag to verify, it seems equally likely that the reduction in fluorescence (at steady state) could result from a movement away from the plasma membrane.

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  5. Author Response:

    Reviewer #3 (Public Review):

    [...] I have only minor concerns regarding sources of error, particularly with respect to interpretation of the small effects the authors observe in many of their FRET experiments.

    • Figure 2D shows rather small changes in ΔF/F-15 mV between fluorescent protein labels inserted at different positions in the ASIC sequence, particularly for the YFP constructs. As this metric is determined from the top and bottom asymptotes for the Boltzmann fits shown in Figure 2C, it would be useful to have some estimate as to the error associated with the fits at extreme values. Perhaps the authors could provide fits to their data (as in Figure 2C), including confidence intervals, or some similar estimate as to the size of the expected error compared to the effect size in Figure 2D.

    Thank you for this point. We did use Boltzman’s fits to get the asymptotes for each cell and calculate a ΔF/F. However, we could also use a ‘fit free’ approach of simply taking the difference between fluorescence values measured at -180 mV and that at +120 mV, divided by that at -15 mV to normalize for each cell. This approach completely avoids any error associated with fitting the data or imposing any model at all. Using this approach results in slightly different ΔF/F values but the pattern of statistical significance is identical. This new analysis is included in Figure 2 figure supplement 4. It has also been corrected for multiple comparisons.

    • Along those same lines, the authors use an interesting (and potentially generalizable) approach to reducing background from intracellular proteins in their experiments: co-transfecting their channels with empty plasmid DNA. What percentage of the remaining fluorescence signal is the result of intracellular background? How would that affect the data in Figure 2 and 3? Is the ΔF/Fnorm curve for YFP labeled positions in Figure 2-figure supplement 4 so flat because of contaminating background fluorescence?

    This is a great question. We originally hoped that the CFP and YFP quenching data from different positions could be used to triangulate both a distance from the membrane and a value for background fluorescence assuming that CFP and YFP would yield similar background fluorescences. An analogous approach was used in Zachariassen et al. Proc Natl Acad Sci, 2016 where an equal background was assumed between conformational states within a recording. In the end, the YFP quenching appeared to have a greater background than CFP. We speculate that this may be because the YFP variant we used matures faster than the CFP (mVenus, 17.6 min verses mTurquiose2, 33.5 min; FPbase.org) and hence the YFP matures faster than the ‘new’ channels get to the plasma membrane. However, at present we are uncertain how much of the background fluorescence signal to confidently attribute to this intracellular FP issue.

    • In Figure 3D, the FRET efficiency between CFP-cA1-cA1 and N YFP at a 1:15 ratio of the two plasmids is higher than the FRET efficiency between CFP and YFP in the same subunit, even though the authors conclude that fluorescent proteins on the same subunit show considerably more FRET than fluorescent proteins on neighboring subunits. Could this indicate that the N-termini of adjacent subunits are closer together than the N- and C-termini of a single subunit? If, on the other hand, this effect were entirely the result of crowding in the membrane why is FRET efficiency substantially lower when CFP-cA1-cA1 is co-expressed with C4 YFP? Wouldn't this construct produce a similar crowding effect?

    We strongly suspect the N termini of adjacent subunits are closer to each other than N and C of single subunit simply because the N FPs would all be at the same ‘height’ or same depth with respect to the plasma membrane. Thus the measured FRET in this case primarily reflects distances in the x-y plane. This contrasts with the N and C FPs on the same or different subunits where both x-y distances and axial distances come into play.

    • On page 23, the authors state that they detected no pH-dependent changes in FRET between their GFP tag on the N-terminus of ASIC1 and an RFP tag on the channel's C-terminus. However, Figure 4 shows a small, but significant change in fluorescence between pH 8 and pH 7.

    We have corrected for multiple comparisons within a figure. As a result, this effect is no longer statistically significant (adjusted p value is 0.063).

    • The interpretation of distances between various tagged position on ASIC and the plasma membrane in Figure 2 is based on using two different colored tags with two different distance dependences. However, the interpretation of the data from Figure 5 provided on page 25 is less clear. For example, the reduction in fluorescence from the N-terminal tag is interpreted as the tag moving closer to the plasma membrane. Without similar data from a YFP tag to verify, it seems equally likely that the reduction in fluorescence (at steady state) could result from a movement away from the plasma membrane.

    This is a very good point. We tried to perform DPA quenching of YFP-containing constructs at pH 6.0, but the acidification resulted in proton-quenching of the YFP fluorescence (Figure 4). We didn’t feel confident in measuring DPA quenching with the concomitant loss of YFP fluorescence due to acidification. Therefore, we relied on the pH 8.0 CFP and YFP data as a starting point (Figure 2). Given the C1 insertion gives the greatest extent of CFP quenching, it is reasonable to place it around the top of the curve. The N position could then be on the left or right side of the hump or peak in the CFP distance curve. The N quenching is comparable to the C2 insertion quenching (Figure 2D, left) yet the N FP is ~ 16 amino acids from the pore-forming membrane helices while the C2 insertions is ~ 40 amino acids away. For reference, the C1 is ~ 24 amino acids. Thus we are reasonably confident the N insertion is on the left side of the hump or peak. A reduction in ΔF/F would indicate movement closer to the plasma membrane. While technically possible that the N position could move further away from the membrane, this would have to be a >25 Å movement. Given there are only 16 amino acids between the CFP and the beginning of TM1 of the channel, we do not think such a dramatic movement outward could occur.

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