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 model6–9. 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.
Original language | English |
---|---|
Pages (from-to) | 606-610 |
Number of pages | 5 |
Journal | Nature |
Volume | 595 |
Issue number | 7868 |
DOIs | |
State | Published - Jul 22 2021 |
Funding
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.