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cAMP binding to closed pacemaker ion channels is non-cooperative

Abstract

Electrical activity in the brain and heart depends on rhythmic generation of action potentials by pacemaker ion channels (HCN) whose activity is regulated by cAMP binding1. Previous work has uncovered evidence for both positive and negative cooperativity in cAMP binding2,3, but such bulk measurements suffer from limited parameter resolution. Efforts to eliminate this ambiguity using single-molecule techniques have been hampered by the inability to directly monitor binding of individual ligand molecules to membrane receptors at physiological concentrations. Here we overcome these challenges using nanophotonic zero-mode waveguides4 to directly resolve binding dynamics of individual ligands to multimeric HCN1 and HCN2 ion channels. We show that cAMP binds independently to all four subunits when the pore is closed, despite a subsequent conformational isomerization to a flip state at each site. The different dynamics in binding and isomerization are likely to underlie physiologically distinct responses of each isoform to cAMP5 and provide direct validation of the ligand-induced flip-state model6,7,8,9. This approach for observing stepwise binding in multimeric proteins at physiologically relevant concentrations can directly probe binding allostery at single-molecule resolution in other intact membrane proteins and receptors.

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Fig. 1: fcAMP binding to intact HCN channels in ZMWs.
Fig. 2: fcAMP binds non-cooperatively to both HCN isoforms.
Fig. 3: Ligand binding induces a conformational change at each HCN subunit.
Fig. 4: A revised flip-state model.

Data availability

All experimental data are available upon reasonable request. Source data are provided with this paper.

Code availability

The DISCO software package is available at https://github.com/ChandaLab/DISC and fully described elsewhere36. All additional MATLAB scripts for single-molecule analysis and image processing are available upon reasonable request.

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Acknowledgements

This research was supported by the NIH grants to B.C. (NS-116850, NS-101723, and NS-081293), D.S.W. (T32 fellowship GM007507) and NSF to R.H.G. (CHE-1856518). We thank K. A. Knapper and C. H. Vollbrecht for assistance with cover glass cleaning at the Wisconsin Centers for Nanoscale Technologies; M. P. Goldschen-Ohm and M. Smith for helpful discussions; C. Lingle and L. Anson for their feedback on the manuscript; and M. Jackson for sparking this collaboration. ZMWs were fabricated at the Center for Nanophase Materials Sciences at Oak Ridge National Laboratory, which is a DOE Office of Science User Facility.

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Contributions

D.S.W., S.C., R.H.G. and B.C. conceived and designed the studies; V.I. performed electrophysiology experiments; S.C. performed molecular biology and protein purification; D.S.W. and R.Z. performed single-molecule experiments; D.S.W. analysed single-molecule data. D.S.W. fabricated ZMWs under supervision of S.T.R.; D.S.W., S.C., R.H.G. and B.C. wrote the manuscript with input from others.

Corresponding authors

Correspondence to Randall H. Goldsmith or Baron Chanda.

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The authors declare no competing interests.

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Peer review information Nature thanks the anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of HCN1SM and HCN2SM.

a, Representative electrophysiological recordings (top) of HCN1SM (left) and HCN2SM (right) with voltage protocol. Tail currents (arrow) were collected at −130 mV and were used to generate the activation curves. b, Normalized activation curves of HCN1SM (left) and HCN2SM (right) in the absence or presence of saturating concentrations (500 μM) of internal cAMP with a Boltzmann fit (red). Data points are mean ± s.e.m. (n = 5 patches). V1/2 values for are HCN1SM -71.2 ± 0.4 mV without cAMP and −69.1 ± 0.5 mV with cAMP. V1/2 values for are HCN2SM -105.2 ± 0.6 mV without cAMP and −84.6 ± 0.5 mV with cAMP). c, Size exclusion chromatography (SEC) profiles of HCN1SM (grey) and HCN2SM (orange dashed). Triangles indicate the peak fraction (0.3 ml) used for single-molecule measurements. d, Example fluorescence vs time trajectory of photobleaching eGFP-tagged HCN2SM tetramers via TIRFM. e, Distributions of photobleaching steps overlaid with a maximum likelihood estimate of a zero-truncated binomial distribution (red) for a tetrameric complex with a probability (P) of observing eGFP (HCN1: P = 0.65, 95% CI [0.63, 0.67], n = 752; HCN1: P = 0.67, 95% CI [0.65, 0.69], n = 588).

Source data

Extended Data Fig. 2 Non-specific binding in ZMWs.

a, Bright field image of ZMW array on single-molecule imaging set-up featuring a 512 × 512 pixel EMCCD and a 100× objective. Each white dot (about 1,600 per field of view) is a ZMW. b, c, Test of specific binding of eGFP-tagged HCN2SM to ZMWs (b) and of fcAMP (c) to HCN2SM in ZMWs. For b and c, all images shown are averaged over the first 10 frames (1 s) and background subtracted for visualization. Brightness and contrast were adjusted for clarity. d, Representative and randomly selected fluorescence trajectories of empty (no HCN) and passivated ZMWs with 1,000 nM fcAMP fit with DISC (black). The first 50 frames (grey) were removed from analysis.

Source data

Extended Data Fig. 3 fcAMP binding to HCN1SM in ZMWs.

Representative fluorescence trajectories of fcAMP (100 nM to 900 nM) binding to HCN1SM in ZMWs with idealized fits (black) imaged at 100-ms resolution. The first 50 frames (grey) were removed from analysis.

Source data

Extended Data Fig. 4 fcAMP binding to HCN2SM in ZMWs.

Representative fluorescence trajectories of fcAMP (100 nM to 1,500 nM) binding to HCN2SM in ZMWs with idealized fits (black) imaged at 100-ms resolution. The first 50 frames (grey) were removed from analysis.

Source data

Extended Data Fig. 5 All state occupancy distributions.

a, b, Normalized state occupancy distributions for HCN1SM (a) and HCN2SM (b) across all recorded fcAMP concentrations. Each plot indicates the total number of molecules (n) and data points (that is, frames, m) included in the analysis. P is the success rate of the optimized binomial distribution considering four binding sites. All obtained and expected state occupancies values are in Supplementary Table 2.

Source data

Extended Data Fig. 6 Isolated-B1 Events of HCN1SM.

a, Dwell time distributions of isolated-B1 events for HCN1SM at various fcAMP concentrations overlaid with maximum likelihood estimates for monoexponential (blue dashed) and biexponential (red) distributions (Supplementary Table 3). For inset, error bars are the error of a binomial distribution (Methods). b, c, Coordinates of identified single-molecules in the 512 × 512 pixel field of view superimposed across all ZMW arrays. The colour bars denote the average dwell time (b) and fluorescence (c) of the isolated-B1 state for each molecule (n). d, Correlation of fluorescence intensity and dwell times for each isolated-B1 event (m), where r is the Pearson correlation coefficient. Data are binned for visualization.

Source data

Extended Data Fig. 7 Isolated-B1 Events of HCN2SM.

a, Dwell time distributions of isolated-B1 events for HCN2SM at various fcAMP concentrations overlaid with maximum likelihood estimates for monoexponential (blue dashed) and biexponential (red) distributions (Supplementary Table 3). For inset, error bars are the error of a binomial distribution (Methods). b, c, Coordinates of identified single-molecules in the 512 × 512 pixel field of view superimposed across all ZMW arrays. The colour bars denote the average dwell time (b) and fluorescence (c) of the isolated-B1 state for each molecule (n). d, Correlation of fluorescence intensity and dwell times for each isolated-B1 event (m), where r is the Pearson correlation coefficient. Data are binned for visualization.

Source data

Extended Data Fig. 8 Isolated-B1 events do no exhibit static heterogeneity.

a, e, Average isolated-B1 dwell time of HCN1SM (a) and HCN2SM (e) for each molecule at 100 nM fcAMP. Outliers (diamonds) were identified by three scaled median absolute deviations. Data plotted as mean ± s.d. of exponential distribution. The blue dashed line indicates the average B1 dwell time across all molecules (HCN1SM: n = 176; HCN2SM: n = 77). b, f, Histograms of average isolated-B1 dwell times for each HCN1SM (b) and HCN2SM (f) molecule. c, g, Parameters for a monoexponential fit (τ) to isolated-B1 dwell times of HCN1SM (c) and HCN2SM (g). d, h, Parameters for a biexponential fit (τ1, τ2, A1) to isolated-B1 dwell times of HCN1SM (d) and HCN2SM (h). For c, g, d, h, the ordinate corresponds to the obtained parameter (τ, τ1, τ2, A1) and error bars are 95% confidence intervals. All parameters were obtained using maximum likelihood estimates across all isolated-B1 events in either all data (HCN1SM: n = 8,229; HCN2SM: n = 2,676) or inlier (HCN1SM: n = 7,816; HCN2SM: n = 2,575) groups, as indicated on the abscissa.

Source data

Extended Data Fig. 9 HCN1SM dwell time distributions.

Dwell time distributions of all liganded states of HCN1SM across all fcAMP concentrations overlaid with expectations from the optimized rates in Fig. 4b.

Source data

Extended Data Fig. 10 HCN2SM dwell time distributions.

Dwell time distributions of all liganded states of HCN2SM across all fcAMP concentrations overlaid with expectations from the optimized rates from in Fig. 4b.

Source data

Supplementary information

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This file contains Supplementary Note 1, Supplementary Methods, Supplementary Tables 1-7, and Supplementary Figures 1-2.

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White, D.S., Chowdhury, S., Idikuda, V. et al. cAMP binding to closed pacemaker ion channels is non-cooperative. Nature 595, 606–610 (2021). https://doi.org/10.1038/s41586-021-03686-x

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