Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition
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Evaluation Summary:
This work provides direct evidence that PcTx1, a modulator commonly used to study acid-sensing ion channels, induces a conformational change that persists long after an effect on the channel activity has dissipated. The data support this central claim of the paper and invite future investigation of the precise mechanism. The work is of general interest to those studying ion channel biophysics and pharmacology and is a fine example of the power of combined functional and fluorescence 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 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition, remain unknown. Here, we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveals that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.
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Author Response:
Reviewer #3 (Public Review):
In this study Borg et al. explore the mechanism of PcTx1 inhibition of ASIC1a using TEVC fluorometry. They detected a robust change of a fluorescence signal when PcTx1 was added, and based on this finding, propose that the toxin has three different binding modes: 'loos', 'global' and 'ECDonly'. In addition, using concatamers they conclude that damage of a single PcTx1 binding site out of the three sites present in ASIC1a destabilizes the conformational changes but disruption of two or three binding sites is required to prevent PcTx1-mediated inhibition.
The main weakness of the study is the lack of additional experiments to confirm that the proposed three PcTx1 binding modes are actually happening.
We thank the reviewer for the constructive feedback on our work. While PcTx1 modulation …
Author Response:
Reviewer #3 (Public Review):
In this study Borg et al. explore the mechanism of PcTx1 inhibition of ASIC1a using TEVC fluorometry. They detected a robust change of a fluorescence signal when PcTx1 was added, and based on this finding, propose that the toxin has three different binding modes: 'loos', 'global' and 'ECDonly'. In addition, using concatamers they conclude that damage of a single PcTx1 binding site out of the three sites present in ASIC1a destabilizes the conformational changes but disruption of two or three binding sites is required to prevent PcTx1-mediated inhibition.
The main weakness of the study is the lack of additional experiments to confirm that the proposed three PcTx1 binding modes are actually happening.
We thank the reviewer for the constructive feedback on our work. While PcTx1 modulation of ASIC is no doubt complex and other methods might reveal additional binding modes in the future, we believe that our contribution has indeed provided insights that go beyond the knowledge gained from functional experiments using only electrophysiology experiments, as well as structural efforts. The strength of the VCF method lies in the simultaneous measurement of function and conformation. Here, we have used VCF to uncover three distinct conformational states, of which only one was previously known. We have now included several new experiments, along with changes to the text that hopefully alleviate some of the concerns regarding the existence of the binding modes. Further, we have included additional text in the discussion to acknowledge that other methods might uncover additional or distinct binding modes in the future.
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Evaluation Summary:
This work provides direct evidence that PcTx1, a modulator commonly used to study acid-sensing ion channels, induces a conformational change that persists long after an effect on the channel activity has dissipated. The data support this central claim of the paper and invite future investigation of the precise mechanism. The work is of general interest to those studying ion channel biophysics and pharmacology and is a fine example of the power of combined functional and fluorescence 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 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This research presents compelling evidence that PcTx1 induces a conformational change of the extracellular domain of mASICa that is distinct from PcTx1 binding and channel pore opening. The data, analysis, and transparent presentation are all of high quality. Strengths of this work are the use of fluorescence detection of conformational change coupled with electrophysiology that enable PcTX1 binding to be distinguished from multiple conformational changes. The use of fluorescently labeled concatemers with appropriate controls allows the stoichiometry of PcTx1 effects to be determined. The use of a binding site mutation F350L to further probe the coupling between binding and conformational changes adds mechanistic depth to the study, although there is some question concerning the mechanism underlying effects …
Reviewer #1 (Public Review):
This research presents compelling evidence that PcTx1 induces a conformational change of the extracellular domain of mASICa that is distinct from PcTx1 binding and channel pore opening. The data, analysis, and transparent presentation are all of high quality. Strengths of this work are the use of fluorescence detection of conformational change coupled with electrophysiology that enable PcTX1 binding to be distinguished from multiple conformational changes. The use of fluorescently labeled concatemers with appropriate controls allows the stoichiometry of PcTx1 effects to be determined. The use of a binding site mutation F350L to further probe the coupling between binding and conformational changes adds mechanistic depth to the study, although there is some question concerning the mechanism underlying effects of PcTx1 on F350L. This study succeeds in identifying conformational changes that are coupled to, yet distinct from channel conductance that impact ASIC function. The interpretation of results is carefully nuanced and well supported by the data. Overall this study is an inspiring conceptually-driven mechanistic characterization of coupling between protein conformational changes. This work is likely to be valuable to the field of ASIC research, with the advances in methodology of interest to a broader community studying coupling between protein conformational changes.
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Reviewer #2 (Public Review):
Borg and colleagues set out to study the mechanism and stoichiometry of acid-sensing ion channel modulation by PcTx1. They do this using voltage clamp fluorometry in conjunction with concatenated subunits. The principle finding is that PcTx1 induces a conformational change, as reported by a delta F, which persists beyond the toxins' apparent effect on channel gating. The authors explain this striking observation by proposing that PcTx1 binds in three distinct modes: a low-affinity non-modulatory 'Loose' mode, a high affinity modulatory 'Global' mode and a high affinity non-modulatory 'ECDonly' mode. The 'Global' mode is the one typically studied using electrophysiology. They argue that given time and some pH-stimulus, the channels accumulate into the 'ECDonly' mode where the influence of PcTx1 is uncoupled …
Reviewer #2 (Public Review):
Borg and colleagues set out to study the mechanism and stoichiometry of acid-sensing ion channel modulation by PcTx1. They do this using voltage clamp fluorometry in conjunction with concatenated subunits. The principle finding is that PcTx1 induces a conformational change, as reported by a delta F, which persists beyond the toxins' apparent effect on channel gating. The authors explain this striking observation by proposing that PcTx1 binds in three distinct modes: a low-affinity non-modulatory 'Loose' mode, a high affinity modulatory 'Global' mode and a high affinity non-modulatory 'ECDonly' mode. The 'Global' mode is the one typically studied using electrophysiology. They argue that given time and some pH-stimulus, the channels accumulate into the 'ECDonly' mode where the influence of PcTx1 is uncoupled from the pore but not the ECD. They further explore these observations using a prior mutation, F350L, which reduces the channel's sensitivity to PcTx1. By pairing this mutation with VCF in concatenated ASICs, they show that a single F350L does not detectably alter PcTx1 inhibition as measured by electrophysiology but does destabilize the ECDonly state, further arguing for a distinction between these conformations.
The paper is well-written with high quality data and generally excellent figures. The persistent PcTx1 effect itself is quite striking and will undoubtedly motivate subsequent study. A further strength of the work is the authors take care to measure the pH responses of their mutants and calibrate the conditioning and test stimuli accordingly. This is critical for ASICs and it's great to see such consideration become more common place. However, an inherent weakness in the VCF approach is the fluorescence signal reflects the occupancy weighted sum of signals from all states. A small fraction of channels moving to one state with a big delta F could occlude signal from a larger fraction with a smaller delta F. VCF is further complicated in this case by the use of pH as a stimulus (which can alter local environment by protonation instead of motion) and a Trp-containing toxin as a ligand. The authors do an excellent job mitigated these latter concerns by reproducing all data at structurally distinct labelling positions. Moreover, they are fairly conservative in their interpretation of data. As a result, the most innovative conclusions (that PcTx1 induces a novel long-lived state and it produces inhibition with two intact binding sites) are well supported by the data.
Overall, this is a deeply interesting study with the characteristic high quality of the Pless group. The stoichiometry experiments are a great addition to the literature as is the observation that PcTx1 induces a novel, long-lived conformation. However, in the present form the more intricate assertions (that PcTx1 is bound in a ECDonly state with weak ECD/pore coupling) require more evidence. Nevertheless, this paper provokes a reassessment of PcTx1 effects on ASICs, motivating much more in-depth examinations of PcTx1 specifically, and other ASIC modulators in general.
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Reviewer #3 (Public Review):
In this study Borg et al. explore the mechanism of PcTx1 inhibition of ASIC1a using TEVC fluorometry. They detected a robust change of a fluorescence signal when PcTx1 was added, and based on this finding, propose that the toxin has three different binding modes: 'loos', 'global' and 'ECDonly'. In addition, using concatamers they conclude that damage of a single PcTx1 binding site out of the three sites present in ASIC1a destabilizes the conformational changes but disruption of two or three binding sites is required to prevent PcTx1-mediated inhibition.
The main weakness of the study is the lack of additional experiments to confirm that the proposed three PcTx1 binding modes are actually happening.
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