{"doi":"10.1101/522581","title":"The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization","abstract":"<jats:p>\n                  Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body\n                  <jats:sup>1</jats:sup>\n                  . They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells\n                  <jats:sup>1</jats:sup>\n                  . In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration\n                  <jats:sup>2</jats:sup>\n                  . VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore\n                  <jats:sup>3–6</jats:sup>\n                  . These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)\n                  <jats:sup>3</jats:sup>\n                  and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization\n                  <jats:sup>7</jats:sup>\n                  . HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization\n                  <jats:sup>8–10</jats:sup>\n                  . The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels\n                  <jats:sup>11</jats:sup>\n                  , no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.\n                </jats:p>","journal":null,"year":null,"id":685401,"datarank":0.24141568686511508,"base_score":1.6094379124341003,"endowment":1.6094379124341003,"self_citation_contribution":0.24141568686511508,"citation_network_contribution":0.0,"self_endowment_contribution":0.24141568686511508,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":694685,"name":"Teresa K. Aman","orcid":null,"position":1,"is_corresponding":false},{"id":246362,"name":"Frank DiMaio","orcid":"0000-0002-7524-8938","position":2,"is_corresponding":false},{"id":323094,"name":"William N. Zagotta","orcid":"0000-0002-7631-8168","position":3,"is_corresponding":false},{"id":576182,"name":"Gucan Dai","orcid":"0000-0002-4692-4060","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization","abstract":"<jats:p>\n                  Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body\n                  <jats:sup>1</jats:sup>\n                  . They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells\n                  <jats:sup>1</jats:sup>\n                  . In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration\n                  <jats:sup>2</jats:sup>\n                  . VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore\n                  <jats:sup>3–6</jats:sup>\n                  . These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)\n                  <jats:sup>3</jats:sup>\n                  and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization\n                  <jats:sup>7</jats:sup>\n                  . HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization\n                  <jats:sup>8–10</jats:sup>\n                  . The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels\n                  <jats:sup>11</jats:sup>\n                  , no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.\n                </jats:p>","is_dataset_classified":null,"base_score":1.6094379124341003,"endowment":1.6094379124341003,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"26207759","pmcid":null,"openalex_id":"https://openalex.org/W2909552347","authors":[],"funders":[{"funder_name":"National Institutes of Health","grant_id":"5R01MH102378-04","title":"Regulation of KCNH ion channels"},{"funder_name":"National Institutes of Health","grant_id":"5R01EY010329-18","title":"Gating Mechanisms of Retinal Rod cGMP Activated Channels"},{"funder_name":"National Institutes of Health","grant_id":"1F32NS077622-01A1","title":"Protein kinase A modulation of NMDA receptor gating and permeability"},{"funder_name":"National Institutes of Health","grant_id":"3R01GM125351-01A1S1","title":"Supplement to Multimodal Gating Mechanisms of TRPV1 Ion Channels"}],"total_grants":4,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[{"year":2021,"count":1},{"year":2024,"count":2},{"year":2025,"count":1}],"oa_status":"green","license":"cc-by","oa_locations":[{"url":"https://www.biorxiv.org/content/biorxiv/early/2019/01/16/522581.full.pdf","host_type":"repository"},{"url":"https://www.biorxiv.org/content/biorxiv/early/2019/01/16/522581.full.pdf","host_type":"repository"},{"url":"https://syndication.highwire.org/content/doi/10.1101/522581","host_type":"publisher"},{"url":"https://doi.org/10.1101/522581","host_type":"repository"},{"url":"https://doi.org/10.1038/s41594-019-0259-1","host_type":""},{"url":"https://europepmc.org/articles/pmc6692172?pdf=render","host_type":""},{"url":"https://dx.doi.org/10.5281/zenodo.3066430","host_type":""},{"url":"https://zenodo.org/records/3066430","host_type":""},{"url":"http://dx.doi.org/10.5281/zenodo.3066430","host_type":""},{"url":"https://dx.doi.org/10.5281/zenodo.3066494","host_type":""},{"url":"https://zenodo.org/records/3066494","host_type":""},{"url":"http://dx.doi.org/10.5281/zenodo.3066494","host_type":""},{"url":"https://dx.doi.org/10.5281/zenodo.3066429","host_type":""},{"url":"https://pubmed.ncbi.nlm.nih.gov/31285608","host_type":""},{"url":"http://dx.doi.org/10.1038/s41594-019-0259-1","host_type":""},{"url":"https://dx.doi.org/10.1101/522581","host_type":""},{"url":"https://dx.doi.org/10.1038/s41594-019-0259-1","host_type":""},{"url":"http://dx.doi.org/10.1101/522581","host_type":""}],"fields_of_study":["Ion channel regulation and function","Neuroscience and Neuropharmacology Research","Cardiac electrophysiology and arrhythmias"],"mesh_terms":[],"keywords":["Hyperpolarization (physics)","Membrane potential","Depolarization","Gating","Chemistry","Biophysics","Ion channel","Transmembrane domain","Voltage-gated ion channel","HCN channel","Transmembrane protein","Ion","Membrane","Resting potential","Helix (gastropod)","Stereochemistry","Biochemistry","Biology","Nuclear magnetic resonance spectroscopy","Models, Molecular","Patch-Clamp Techniques","Protein Conformation","Article","Recombinant Proteins","Membrane Potentials","Motion","Protein Domains","Cyclic AMP","Fluorescence Resonance Energy Transfer","Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels","Potassium","Animals","Point Mutation","Amino Acid Sequence","Ion Channel Gating","Strongylocentrotus purpuratus"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Affordable and clean energy"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-18T16:34:50.924582Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}