Peer review process
Consolidated peer review report (23 July 2021)
GENERAL ASSESSMENT
This manuscript reports the development of a single molecule (SM) approach for studying individual ligand binding events for a membrane protein in a native lipid membrane environment. The authors express the eukaryotic TAX-4 cyclic nucleotide-gated channel in mammalian cells tagged on their cytoplasmic N-terminus with EGFP, form nanovesicles using nitrogen cavitation, separate the plasma membrane fraction from ER vesicles using gradient ultracentrifugation and then purify the fraction of vesicles containing TAX-4 oriented with its intracellular domains outward using GFP nanobodies immobilized onto cover slips. To visualize binding and unbinding of an agonist at the SM level, the authors utilize low concentrations of the fluorescent cGMP analog fcGMP along with TIRF microscopy. The authors develop the approach in a nuanced and cautious fashion, with nice controls to demonstrate that the preparation works for the TAX-4 channel, but also for a more complex assembly of GABA receptors comprised of alpha1, beta2 and gamma subunits. Adsorption of both TAX-4 and GABA receptors appears to be specific as fluorescent puncta are not observed without the GFP nanobody. In the case of TAX-4, many EGFP-positive puncta are observed that do not bind fcGMP, indicating that not all channels remain functional. fcGMP puncta are also observed that do not contain EGFP fluorescence, indicating that fcGMP can adhere to coverslips non-specifically. However, single binding events of fcGMP to EGFP-positive puncta can be readily observed, they can be competed out using cGMP, and both binding and unbinding events were analyzed quantitatively. The authors demonstrate that bound lifetimes are independent of agonist concentration, whereas unbound lifetimes decrease as agonist concentration increases, as would be expected if they are able to resolve individual binding and unbinding events. Bound lifetimes are poorly described by single exponential functions over a range of fcGMP concentrations, suggesting that TAX-4 channels have at least two distinct bound conformations. The authors explore a range of binding models to account for their results and find that their results are consistent with a model in which the nucleotide binding domain alternates between an open conformation that can bind and unbind agonist, and a closed conformation that hinders both binding and unbinding. The authors hypothesize that the conformational change correspond to an early step in the activation process, agreeing with cryo-EM structures of TAX-4 in the absence and presence of agonist. The modeling and interpretation of results is nuanced and presented in an open and objective fashion, although more details on the modeling would be helpful. The authors also study double-bound events and uncover evidence that unbinding of the second ligand is slower than the first, suggesting some positive cooperativity between the first and second agonist binding events. Overall, the methodology is well described, and the controls certify the quality of the data shown. The authors explain the limitations of the technique and restrict their focus to the two initial binding event and do not draw conclusions on the nature of the conformational change or whether the conformational change correspond to individual subunits or all subunits at the same time. Despite these limitations, the manuscript beautifully describes an elegant approach for studying ligand binding dynamics at the SM level and the possible relationship with conformational changes while the molecule is embedded in the physiological membrane. The following are suggestions the authors should consider when revising the manuscript.
RECOMMENDATIONS
Revisions essential for endorsement:
The authors should provide statistics on the number of colocalized spots compared to the number of non-colocalized GFP and fcGMP spots in GABA vs TAX-4. As seen in the GABA negative controls, colocalization does occasionally occur spuriously. It would be ideal to show this type of quantitation for experiments done on different days and with different vesicle preps to give the reader a sense of the frequency of binding competent channel proteins are observed and whether there is day to day variability. 60 hours of binding dynamics is also not a very useful statistic. Please provide the number of traces/replicates/individual vesicle preparations in the figure legends.
The authors prefer a model wherein a conformation change prevents the binding and unbinding of agonist. The authors explanation of their thinking and the exclusion of model M1.F wasn’t always easy to follow. It would helpful if the authors provided a somewhat more complete explanation for why the model with the lower BIC is not the preferred and what constants are significantly lower to make the model M1.F worse than M1.E. This is important because this is used to discard binding and unbinding after the conformational change and support the hypothesized conformational change. Have the authors explored the possibility that the conformational changes detected are intermediate states different to the final state with the four binding sites occupied?
Additional suggestions for the authors to consider:
Do the authors have any additional data to inform on how well gradient ultracentrifugation enriches the PM fraction? Have the authors probed fractions with antibodies against ER resident proteins or those in other intracellular organelles?
Quantifying fcGMP photobleaching rates based on those that are nonspecifically adsorbed to the surface probably isn’t the most robust method. Dyes stuck to the surface will likely encounter higher intensities in the evanescent field due to their proximity to the surface and will often also have distorted photophysical properties as is observed in the traces shown. A better method would be to encapsulate fcGMP in vesicles and measure bleaching rates.
The distribution in individual fcGMP intensities observed could be caused by irregularities in the laser illumination spot or the emission pathway though this is limited in mmTIRF setups generally. The authors should comment on this in the methods.
Have the authors considered whether it might be possible to do experiments with APT-cGMP or another analog for covalent ligand attachment to two of the CNBDs while the others are activated by fcGMP? This might be a useful way to examine agonist binding steps beyond the first two given the limitation of having to use low agonist concentrations.
The authors don’t comment on whether the conformational change can occur in absence of ligand binding, a possibility included in the models. This event cannot be detected with this methodology but are relevant for the relationship of the conformational changes and gating of the channel and can modify the binding kinetics detected but not the unbinding kinetics. It would be helpful if the authors discuss uncertainties created by not knowing the extent to which the conformational change does or does not occur in the absence of agonist.
The inside-out orientation appears to work robustly for the TAX-4 channel. Can the authors comment in the discussion on potential intracellular mechanisms known to regulate TAX-4 or other cyclic nucleotide-gate channels that might be disrupted in this orientation? Many channels are regulated by PIP2, which would be depleted in the inside-out orientation.
REVIEWING TEAM
Reviewed by:
Gabriel Fitzgerald, Postdoctoral Fellow (J.A. Mindell lab, NINDS, USA): membrane protein mechanisms, single molecule spectroscopy
Pablo Miranda, Staff Scientist (M. Holmgren lab, NINDS, USA): ion channel mechanisms, electrophysiology, fluorescence spectroscopy
Kenton J. Swartz, Senior Investigator, NINDS, USA: ion channel structure and mechanisms, chemical biology and biophysics, electrophysiology and fluorescence spectroscopy
Curated by:
Kenton J. Swartz, Senior Investigator, NINDS, USA
(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)