A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism
Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of fo...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-04-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2021.644720/full |
| _version_ | 1818617657328402432 |
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| author | Alessandro Crnjar Susanne M. Mesoy Sarah C. R. Lummis Carla Molteni |
| author_facet | Alessandro Crnjar Susanne M. Mesoy Sarah C. R. Lummis Carla Molteni |
| author_sort | Alessandro Crnjar |
| collection | DOAJ |
| description | Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening. |
| first_indexed | 2024-12-16T17:09:11Z |
| format | Article |
| id | doaj.art-510b7435334741cea2c5c6ec00efd983 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-16T17:09:11Z |
| publishDate | 2021-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-510b7435334741cea2c5c6ec00efd9832022-12-21T22:23:27ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2021-04-01810.3389/fmolb.2021.644720644720A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating MechanismAlessandro Crnjar0Susanne M. Mesoy1Sarah C. R. Lummis2Carla Molteni3Physics Department, King's College London, London, United KingdomDepartment of Biochemistry, University of Cambridge, Cambridge, United KingdomDepartment of Biochemistry, University of Cambridge, Cambridge, United KingdomPhysics Department, King's College London, London, United KingdomPentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.https://www.frontiersin.org/articles/10.3389/fmolb.2021.644720/fullpentameric ligand-gated ion channels5-HT3 receptorsCys-loop receptorsmutagenesismolecular dynamicsM4 helix |
| spellingShingle | Alessandro Crnjar Susanne M. Mesoy Sarah C. R. Lummis Carla Molteni A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism Frontiers in Molecular Biosciences pentameric ligand-gated ion channels 5-HT3 receptors Cys-loop receptors mutagenesis molecular dynamics M4 helix |
| title | A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism |
| title_full | A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism |
| title_fullStr | A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism |
| title_full_unstemmed | A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism |
| title_short | A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism |
| title_sort | single mutation in the outer lipid facing helix of a pentameric ligand gated ion channel affects channel function through a radially propagating mechanism |
| topic | pentameric ligand-gated ion channels 5-HT3 receptors Cys-loop receptors mutagenesis molecular dynamics M4 helix |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2021.644720/full |
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