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[[Interactive_3D_Complement_in_Proteopedia|Interactive 3D Complement in Proteopedia]]<br>
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= Cryo-EM structure of the human TRPV1 ion =
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<table width="95%" border="0"><tr><td>
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{| align="left"
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<imagemap>
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Image:Cell press logo.png|250px|
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default [http://cell.com]
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</imagemap>
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|}
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</td></tr><tr><td>
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<span style="font-size:160%"><b>Cryo-EM structures of human OAT1 reveal drug
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binding and inhibition mechanisms<ref name="m1">Cryo-EM structures of human OAT1 reveal drug
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binding and inhibition mechanisms https://doi.org/10.1016/j.str.2025.07.019</ref>.</b></span>
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</td></tr><tr><td>
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<span style="font-size:120%">
<span style="font-size:120%">
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Hyung-Min Jeon, Jisung Eun, Kelly H. Kim, and Youngjin Kim.
 
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Cell Volume 33, Issue 11, P1856-1866.E5, November 06, 2025
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<StructureSection load='3j5p' size='340' side='right' caption='Cryo-EM structure of the human TRPV1 ion channel in the apo state (resolution ~3.5 Å)' scene=''>
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https://doi.org/10.1016/j.str.2025.07.019
 
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</span>
 
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</td></tr></table>
 
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==Structure Tour==
 
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<StructureSection load='9kkk' size='340' side='right'caption='Cryo-EM structure of human SLC22A6 (OAT1) in the apo-state, [[Resolution|resolution]] 3.85&Aring;' scene=''>
 
===Introduction===
===Introduction===
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Members of the organic anion transporter (OAT) family, including
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The transient receptor potential vanilloid 1 (TRPV1) ion channel is a heat- and ligand-activated cation channel widely recognized for its central role in pain detection and inflammatory hypersensitivity. It responds to noxious temperatures, capsaicin, protons, and endogenous inflammatory mediators, making it a crucial molecular sensor in the peripheral nervous system. TRPV1 is a high-value therapeutic target for analgesic drug development, and therefore, understanding its three-dimensional architecture is essential for elucidating its gating mechanism and ligand recognition.
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OAT1, are expressed on the epithelial membrane of the kidney,
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liver, brain, intestine, and placenta.<ref>Molecular cloning and characterization of a novel liver-specific transport protein https://doi.org/10.1242/jcs.107.4.1065</ref><ref>Molecular Cloning and Characterization of NKT, a Gene Product Related to the Organic Cation Transporter Family That Is Almost Exclusively Expressed in the Kidney https://doi.org/10.1074/jbc.272.10.6471</ref> OAT1 regulates the transport
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of organic anion drugs from the blood into kidney epithelial
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cells by utilizing the α-ketoglutarate (α-KG) gradient across the
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membrane established by the tricarboxylic acid (TCA) cycle.<ref>Ingraham, L., Li, M., Renfro, J.L., Parker, S., Vapurcuyan, A., Hanna, I., and
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Pelis, R.M. (2014). A plasma concentration of α-ketoglutarate influences
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the kinetic interaction of ligands with organic anion transporter 1. Mol.
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Pharmacol. 86, 86–95. https://doi.org/10.1124/mol.114.091777.</ref>OAT1 also plays a key role in excreting waste from organic drug metabolism and
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contributes significantly to drug-drug interactions and drug disposition. However, the structural basis of specific
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substrate and inhibitor transport by human OAT1 (hOAT1) has remained elusive. Here are four
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[[cryo-electron microscopy]] (cryo-EM) structures of hOAT1 in its inward-facing conformation: the apo
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form, the substrate (olmesartan)-bound form with different anions, and the inhibitor (probenecid)-bound
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form.
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===Cryo-EM structure of hOAT1===
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The apo state structure of human Organic Anion Transporter 1 (hOAT1), determined by cryo-EM, reveals the transporter in an inward-facing conformation. This means the central substrate-binding cavity is open toward the intracellular side of the membrane, ready to release a substrate or accept one from the cytoplasm.
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'''Key Structural Characteristics:'''
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*'''Overall Fold:'''
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::*Adopts the classic Major Facilitator Superfamily (MFS) fold.
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::*Comprises 12 transmembrane helices (TMs 1-12).
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::*Exhibits pseudo-two-fold symmetry, divided into an N-lobe (TMs 1-6) and a C-lobe (TMs 7-12).
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*'''Central Binding Cavity:'''
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::*The cavity is located between the N-lobe (formed by TM1, TM2, TM4, TM5) and the C-lobe (formed by TM7, TM8, TM10, TM11).
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::*It possesses a positively charged electrostatic environment, which explains its strong preference for transporting anionic substrates.
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::*The cavity is lined by 29 residues, forming a hydrophobic and aromatic-rich environment.
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*'''Cavity Borders and Cytosolic Gate:'''
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::*The top border (extracellular side) of the cavity is formed by residues including N35, Y230, Y353, and Y354.
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::*The bottom border (cytosolic side) features a narrow "thin bottom gate" formed by residues M207 and F442. The interaction between these two residues splits the cytosolic entrance into two distinct pathways:
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:::*Path A: Located between TM2 and TM11.
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:::*Path B: Located between TM5 and TM8.
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*'''Conformational State:'''
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::*In the apo state, the transporter is in a relaxed, inward-open conformation, providing access for substrates from the cytoplasm.
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::*The structure serves as a baseline for understanding the conformational changes that occur upon substrate or inhibitor binding.
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===Olmesartan recognition by hOAT1===
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The structural and functional analysis of <scene name='85/857155/Olsmartin/1'>hOAT1 in complex with the high-affinity antihypertensive drug olmesartan</scene> provides a detailed blueprint for substrate specificity and binding.
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'''1. Binding Location and Pose'''
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:*Olmesartan binds within the central cavity of hOAT1 in an inward-facing conformation.
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:*It occupies Site 3 of the binding pocket, which is the primary polyspecific site for anionic substrates.
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:*The drug adopts a diagonal orientation relative to the membrane plane, a pose that requires more space than the smaller inhibitor probenecid. This orientation is similar to its conformation when bound to the angiotensin receptor.
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'''2. Key Interacting Residues'''
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Olmesartan is surrounded by residues from multiple transmembrane helices (TM1, TM4, TM5, TM7, TM10, TM11) within a 5 Å distance. The critical interactions involve:
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*'''Aromatic and Hydrophobic Cage:'''
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::*The biphenyl group of olmesartan is nestled near residue F438.
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::*The tetrazole ring is positioned between the bottom-gate residues M207 and F442.
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::*The imidazole moiety is located close to Y354.
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*'''Critical Role of Y230:'''
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::*Upon olmesartan binding, the side chain of Y230 undergoes a vertical rotation to accommodate and interact with the substrate.
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::*Mutagenesis studies confirm its importance: the Y230F mutation increased the IC₅₀ for olmesartan inhibition from 845.3 nM (Wild Type) to 2.36 µM, indicating a reduction in binding affinity.
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*'''The Bottom Gate Residues (M207 and F442):'''
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::*These residues are crucial for high-affinity olmesartan binding.
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::*The M207A mutant caused a 4-fold reduction in affinity (IC₅₀ = 3.78 µM).
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::*The F442A mutant caused a dramatic 12-fold reduction in affinity (IC₅₀ = 10.32 µM).
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::*This suggests these residues not only form a gate but also directly interact with large, transportable substrates like olmesartan.
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'''3. Chloride Ion Coordination is Essential'''
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A key finding is the role of a chloride ion in stabilizing the olmesartan-bound state.
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:*'''The Chloride-Binding Site:''' A chloride ion (or bromide, used for confirmation) is observed coordinated between residues S203, Y230, and R466.
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:*'''Indirect Role of S203:''' While S203 does not directly contact olmesartan, it is critical for chloride coordination. This is a major species-specific difference, as rat OAT1 has an alanine at this position.
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:*'''Functional Evidence of Chloride Dependence:'''
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::*The IC₅₀ of olmesartan is 2.01 µM in chloride-rich conditions but improves to 0.91 µM in chloride-depleted conditions, suggesting a more complex relationship where chloride may facilitate transport.
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::*The S203A mutant shows a severe ~5-fold reduction in olmesartan binding affinity specifically in the presence of chloride (IC₅₀: WT = 2.47 µM; S203A = 29.52 µM).
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::*The S203A-Y230F double mutant has an even more profound effect, increasing the IC₅₀ to 93.30 µM in chloride conditions, highlighting their synergistic role in chloride-dependent substrate binding.
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===Mechanism of OAT1 inhibition by probenecid===
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The cryo-EM structure of <scene name='85/857155/Prob/1'>hOAT1 bound to the classic inhibitor probenecid</scene> reveals a dual-mechanism of action that goes beyond simple competition, effectively arresting the transporter in a restricted state.
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'''1. Binding Mode and Direct Competition'''
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*Probenecid binds at the top of the central cavity, parallel to the membrane plane.
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*Its binding site overlaps with both Site 1 (partially) and Site 3.
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*It engages in specific, high-affinity interactions with key residues:
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:*K382 on TM8 forms a hydrogen bond with the carboxylate group of probenecid.
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:*Y354 on TM7 forms a hydrogen bond with its sulfonyl group.
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:*Crucially, K382 is also the residue that interacts with the counter-substrate α-ketoglutarate (α-KG), establishing a direct competitive inhibition mechanism by blocking α-KG binding.
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'''2. Conformational Arrest and Cytoplasmic Path Blockage'''
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The primary inhibitory mechanism is a probenecid-induced conformational change that physically blocks substrate access and exit.
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*'''Constriction of the Binding Pocket:''' Compared to the apo state, the cytoplasmic opening of the binding pocket narrows from ~15 Å to ~12 Å in the probenecid-bound state.
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*'''Dual-Pathway Blockade:''' The cytosolic entrance is split into two paths. Probenecid binding critically affects both:
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:*'''Path A''' (between TM2 and TM11) is narrowed from ~5 Å to ~4 Å.
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:*'''Path B''' (between TM5 and TM8) is completely blocked.
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This structural rearrangement is caused by a slight inward movement of the cytoplasmic ends of TM5, TM8, TM10, and TM11 toward the binding pocket.
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'''3. Locked Conformation'''
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===Full Mechanism of Binding and Inhibition in hOAT1===
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[[Image:HOAT1mechanism.png | frame | upright= 1.5 |none | alt= | Fig.1. Mechanism of olmesartan binding and conformational inhibition by probenecid. A) When the transporter is in its outward-facing conformation, substrates or inhibitors enter the central binding pocket and undergo structural rearrangement to
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the inward-facing conformation. When olmesartan interacts with the bottom gating residues M207 and F442, the side chains S203, Y230 (not shown here), and
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R466 appear to rearrange to coordinate with a chloride ion and drug compared to the apo structure. Whereas probenecid binding induces an additional
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conformation change for inhibition (apo-like conformation).]]
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'''Overall Transport Cycle & Substrate Binding (e.g., Olmesartan)'''
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'''1. Outward-Facing State (Hypothesized):''' The transport cycle begins with the transporter in an outward-facing conformation, open to the extracellular space. Substrates and inhibitors from the blood enter the central binding pocket at this stage.
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'''2. Transition to Inward-Facing State:''' Upon binding a substrate like olmesartan, the transporter undergoes a conformational change to the inward-facing state, which is the conformation captured in this study.
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'''3. Substrate Binding and Chloride Coordination in the Inward-Open State:'''
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*Olmesartan docks into Site 3, the polyspecific substrate-binding site, engaging a cage of hydrophobic and aromatic residues (e.g., F438, Y354).
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*Its binding induces specific structural rearrangements, most notably a vertical rotation of the Y230 side chain.
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*Crucially, olmesartan binding creates a favorable environment for chloride ion coordination. The chloride ion is stabilized by a network involving S203, the rotated Y230, and R466.
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*This chloride coordination, facilitated by the species-specific residue S203, is essential for high-affinity binding and efficient translocation of olmesartan. The bottom-gate residues M207 and F442 also interact with the drug, potentially playing a role in its final release into the cytoplasm.
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'''4. Substrate Release:''' The inward-facing conformation with its open paths (Path A and Path B) allows the substrate to dissociate into the cytoplasm. The transporter then likely resets to the outward-facing state, driven by the exchange with intracellular α-ketoglutarate (α-KG).
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'''Inhibition Mechanism (e.g., Probenecid)'''
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The inhibitor probenecid exploits the transport cycle but arrests it through a dual mechanism:
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'''1. Binding and Competition:'''
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*Probenecid enters the binding pocket from the extracellular side and binds in the inward-facing conformation.
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*It occupies Site 3 and partially extends into Site 1. In Site 1, it directly competes with the counter-substrate α-KG by forming a key hydrogen bond with K382, a residue critical for α-KG binding.
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'''2. Conformational Arrest and Cytoplasmic Blockade:'''
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*This is the primary inhibitory mechanism. Probenecid binding induces subtle but critical conformational changes in the cytoplasmic regions of TM5, TM8, TM10, and TM11.
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*These helices shift inward, causing a constriction of the entire cytoplasmic opening of the binding pocket.
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*This constriction completely blocks Path B and severely narrows Path A.
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*By physically obstructing these cytosolic paths, probenecid achieves two things:
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:*It prevents intracellular substrates from entering the binding pocket.
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===Structural Highlights===
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:*It traps the transporter in a locked, inward-facing, apo-like conformation, preventing the conformational changes needed to complete the transport cycle.
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Recent high-resolution cryo-electron microscopy studies have revealed the detailed structure of human TRPV1 in multiple functional states, including the apo (inactive), capsaicin-bound, and toxin-bound conformations. TRPV1 forms a homotetramer, with each subunit contributing six transmembrane helices (S1–S6), a pore-forming loop, and large cytoplasmic ankyrin-repeat domains.
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The vanilloid-binding pocket, located between the S3–S4 helices and the S4–S5 linker, was clearly visualized, revealing how capsaicin stabilizes rearrangements in the S4–S5 linker that propagate toward the S6 helices to open the pore. Toxin-bound structures exhibit an even wider pore diameter, representing a fully activated gating state. Comparison across these structural states highlights the sequence of conformational transitions that underlie heat and ligand-activated gating.
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== Significance ==
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These cryo-EM structures provide a mechanistic framework for understanding how TRPV1 integrates chemical and thermal stimuli to control ion permeation. The detailed visualization of ligand-binding pockets and pore conformations enables structure-based development of novel analgesic compounds targeted toward inflammatory and neuropathic pain. Furthermore, these structures offer insights into how lipid environment, toxins, and small molecules differentially modulate TRPV1 activity, establishing a foundation for rational drug design.
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==Notes & References==
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== References ==
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<references />
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* Gao et al., Cryo-EM structures of the TRPV1 ion channel in different activation states. Nature.

Revision as of 15:39, 30 November 2025

Cryo-EM structure of the human TRPV1 ion

Cryo-EM structure of the human TRPV1 ion channel in the apo state (resolution ~3.5 Å)

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