Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization
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Evaluation Summary:
This study will be of broad interest to ion channel researchers interested in understanding the fundamental mechanisms of voltage-sensing. The researchers use a novel approach to determine the mechanism of voltage-sensing in a channel that lacks a canonical voltage-sensing domain.
(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 #3 agreed to share their names with the authors.)
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Abstract
Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.
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Reviewer #3 (Public Review):
P2X2 receptor channels do not have a canonical voltage-sensor, yet they display profound voltage-dependence especially when activated by physiologically relevant low ATP concentrations. Understanding the mechanisms of this voltage dependence is not an easy undertaking because there are neither similar proteins as precedent nor clear indications from available structures. In this manuscript, Andriani and Kubo incorporated Anap into 96 residues (separately) in P2X2 receptor channels and performed a comprehensive scanning using voltage-clamp fluorometry technique to probe structural changes during ATP- and voltage-dependent gating. Out of the 96 residues, the authors only observed voltage-dependent fluorescence intensity (F) changes at A337 and I341 in the TM2 domain. The changes are fast and linear, consistent …
Reviewer #3 (Public Review):
P2X2 receptor channels do not have a canonical voltage-sensor, yet they display profound voltage-dependence especially when activated by physiologically relevant low ATP concentrations. Understanding the mechanisms of this voltage dependence is not an easy undertaking because there are neither similar proteins as precedent nor clear indications from available structures. In this manuscript, Andriani and Kubo incorporated Anap into 96 residues (separately) in P2X2 receptor channels and performed a comprehensive scanning using voltage-clamp fluorometry technique to probe structural changes during ATP- and voltage-dependent gating. Out of the 96 residues, the authors only observed voltage-dependent fluorescence intensity (F) changes at A337 and I341 in the TM2 domain. The changes are fast and linear, consistent with them being electrochromic effect. When an additional mutant K308R is introduced, the authors were able to detect a small slow and voltage-dependent F change at A337, which could potentially result from structural rearrangements at this position. With a P2X2 model built upon the hP2X3 open state structure, they also proposed that A337 interacts with F44 in TM1, and this interaction is important for activation. The amount of work involved in this study is impressive. The data presented are of good quality. Most conclusions drawn from the results are reasonable and backed with good evidence.
Overall, the identification of a converged electric field around A337 and I341 is new and intriguing. Previously reported functional results and available high resolution P2X receptor structures all suggest that residues A337 and I341 are facing TM1 and they are accessible to Ag+ when mutated to Cys. It is conceivable that the "voltage-sensor" in P2X2 receptor channels involve ion filled crevices between TM1 and TM2 in the membrane. This work is of great value for understanding how membrane proteins sense voltages.
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Reviewer #2 (Public Review):
P2X2 activation depends on both ATP binding and voltage. However, the voltage sensor of P2X2 is not elucidated. This manuscript describes the study of voltage dependent conformational changes of P2X2 using voltage clamp fluorometry of the fluorescent unnatural amino acid Anap that substituted P2X2 amino acid residues. 96 positions in different structural domains were scanned by substituting with Anap, and voltage dependent fluorescence signals were detected only at two positions, A337 and I341 in the TM2 domain. A fast and linear voltage dependence of fluorescence suggested that the membrane voltage converged at and around these two positions. With a mutation K308R that was supposed to enhance voltage dependent conformational changes, Anap at the A337 position showed a time and voltage dependent …
Reviewer #2 (Public Review):
P2X2 activation depends on both ATP binding and voltage. However, the voltage sensor of P2X2 is not elucidated. This manuscript describes the study of voltage dependent conformational changes of P2X2 using voltage clamp fluorometry of the fluorescent unnatural amino acid Anap that substituted P2X2 amino acid residues. 96 positions in different structural domains were scanned by substituting with Anap, and voltage dependent fluorescence signals were detected only at two positions, A337 and I341 in the TM2 domain. A fast and linear voltage dependence of fluorescence suggested that the membrane voltage converged at and around these two positions. With a mutation K308R that was supposed to enhance voltage dependent conformational changes, Anap at the A337 position showed a time and voltage dependent fluorescence. The authors concluded that this result indicated a voltage dependent conformational change. Structure guided mutations suggested that F44 in TM1 might move to interact with A337 in response to voltage. In this study the fluorescence signals were small, but the authors made a great effort and managed to obtain the data that are convincing. The experiments were well designed and the manuscript is clearly reasoned. Considering that among all the positions that were tested only at the two positions in the TM2 segment Anap showed voltage dependent fluorescence, and that the F44 mutations abolished voltage dependence of the P2X2 currents, the conclusion that voltage converges at the A337/I341/F44 and induces a conformational change seems to be well supported.
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Reviewer #1 (Public Review):
The study aims to determine the mechanism of voltage-sensing in P2X2 receptor. These receptors are primarily activated by ligand, ATP but their activity is also regulated to some extent by voltage even though they lack a canonical voltage-sensing domain. To address this question, the authors introduce unnatural fluorescent amino acid throughout the structure of the P2X2 receptor. The interaction between excited state dipole and electric fields can cause shift in the fluorescence emission and excitation spectra. For a given probe, the extent of these shifts are directly proportional to the strength of the electric field. The authors exploit this phenomenon to determine the strength of the electric field in the various regions of the P2X2 receptor. The underlying premise is that the regions which sense the …
Reviewer #1 (Public Review):
The study aims to determine the mechanism of voltage-sensing in P2X2 receptor. These receptors are primarily activated by ligand, ATP but their activity is also regulated to some extent by voltage even though they lack a canonical voltage-sensing domain. To address this question, the authors introduce unnatural fluorescent amino acid throughout the structure of the P2X2 receptor. The interaction between excited state dipole and electric fields can cause shift in the fluorescence emission and excitation spectra. For a given probe, the extent of these shifts are directly proportional to the strength of the electric field. The authors exploit this phenomenon to determine the strength of the electric field in the various regions of the P2X2 receptor. The underlying premise is that the regions which sense the largest electric field are likely to be the primary sensors of membrane voltage.
Strengths:
The approach to localize the putative voltage-sensing region is novel and maybe broadly applicable to other voltage-regulated channels which lack canonical voltage-sensors.
Unnatural amino acid, ANAP was introduced and tested at 96 positions in the structure of P2X2 receptor. This is an insane amount of work and has to be a tour de force.
Weakness:
The main limitation of this approach is that ANAP is not going to be incorporated with equal efficiency at all sites and therefore, it is likely that some of the potential where the electric field is strong may remain undetected.
Overall, using ANAP scanning approach, they were able to identify couple of sites in TM2 helix which exhibits large electrochromic signals. Furthermore, they find that the interaction between Ala 337 and Phe44 is critical for voltage-dependent response. These studies lay the groundwork for further investigations of the mechanism of voltage-sensing these physiologically important ion channels.
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Evaluation Summary:
This study will be of broad interest to ion channel researchers interested in understanding the fundamental mechanisms of voltage-sensing. The researchers use a novel approach to determine the mechanism of voltage-sensing in a channel that lacks a canonical voltage-sensing domain.
(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 #3 agreed to share their names with the authors.)
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