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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 Channel ===
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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|300 px|
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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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<StructureSection
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binding and inhibition mechanisms<ref name="m3" />.</b></span>
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load='3j5p'
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</td></tr><tr><td>
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size='340'
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side='right'
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caption='Cryo-EM structure of the human TRPV1 ion channel in the apo state (Liao et al., 2013; ~3.5 Å resolution)'
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scene=''>
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</StructureSection>
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<span style="font-size:120%">
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=== Introduction ===
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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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The transient receptor potential vanilloid 1 (TRPV1) ion channel is a heat- and ligand-gated cation channel essential for nociception, inflammatory pain, and thermal sensitivity. Activated by capsaicin, protons, noxious heat (>42°C), and lipid mediators, TRPV1 serves as a polymodal molecular sensor in the peripheral nervous system. Because of its central role in pain signaling, TRPV1 has been a major therapeutic target for developing next-generation analgesics. Understanding its three-dimensional structure is therefore crucial for elucidating its gating mechanism and ligand recognition.
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https://doi.org/10.1016/j.str.2025.07.019
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=== Structural Highlights ===
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</span>
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</td></tr></table>
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==Structure Tour==
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Using single-particle cryo-electron microscopy, Liao, Cao, Julius, and Cheng (2013) determined the first near-atomic structures of TRPV1 in multiple functional states, including the apo (resting), capsaicin-bound, and toxin-bound conformations. TRPV1 assembles as a homotetramer, with each subunit containing six transmembrane helices (S1–S6), a re-entrant pore loop, and extensive cytosolic ankyrin repeat domains.
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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=''>
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===Background===
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Members of the organic anion transporter (OAT) family, including
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The vanilloid-binding pocket—formed between the S3–S4 helices and the S4–S5 linker—was resolved in detail, explaining how capsaicin stabilizes the open conformation by pulling on the S4–S5 linker and reshaping the S6 helices to widen the pore. Structures bound to the double-knot toxin (DkTx) reveal an even more dilated pore, representing a fully activated gating state. Comparisons across these states demonstrate the sequence of conformational rearrangements that underlie heat and ligand gating in TRPV1.
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OAT1, are expressed on the epithelial membrane of the kidney,
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liver, brain, intestine, and placenta. 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.The organic anion transporter 1 (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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<center>{{Template:Green links zoom}}</center>
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Human
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OAT1 adopts an inward-facing conformation in a membrane. OAT1 consist of structural features
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including intracellular helices domain (ICD),
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extracellular domain (ECD), N-lobe helices
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(TM1-6), and C-lobe helices (TM7-12). (right) The
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border of the binding cavity (described in solvent
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exclude-surface) is formed by residues N35,
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Y230, Y353, Y354 (upper), and M207 and F442
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(lower).
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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 hOAT1 in complex with the high-affinity antihypertensive drug olmesartan provides a detailed blueprint for substrate specificity and binding.
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'''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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''' 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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''' 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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The OmcS monomer has <scene name='83/835223/Secondary_structure/2'>remarkably little secondary structure</scene>.
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<center>
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{{Template:ColorKey_Helix}},
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{{Template:ColorKey_310Helix}},
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{{Template:ColorKey_Strand}},
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{{Template:ColorKey_Loop}}.
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</center>
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The structure assigned by the authors is '''77% loops'''; Jmol objectively assigns '''82%''' loops. The authors assigned 10% alpha helices, 7% 3<sub>10</sub> helices, and 6% beta strands.
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The OmcS structure determined by Filman ''et al.'' <ref name="strauss" />was very similar, with '''80%''' loops assigned by the authors (86% by Jmol), having only 3% beta strand but otherwise very similar. We compared OmcS with three other c-type multi-heme cytochrome crystal structures: [[1ofw]], [[3ucp]], and [[3ov0]] had 45%, 49%, and 60% loops respectively.
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===Hemes===
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Each OmcS monomer <scene name='83/835223/Hemes/10'>contains 6 hemes</scene>:
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{{Template:ColorKey_Element_C}}
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{{Template:ColorKey_Element_O}}
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{{Template:ColorKey_Element_N}}
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{{Template:ColorKey_Element_Fe}}.
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The hemes are arranged in [https://en.wikipedia.org/wiki/Stacking_(chemistry) parallel-displaced] pairs. Each pair is orthogonal to the next pair.
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The <scene name='83/835223/Hemes/11'>hemes at each monomer-monomer interface form a parallel-displaced pair</scene>, which likely contributes to the stability of the filament. More importantly, this produces a <scene name='83/835223/Filament/5'>continuous chain of hemes through the length of the filament</scene>. This continuous chain of hemes is believed to be the basis of the electrical conductivity.
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====Cysteine Anchors====
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Each heme is <scene name='83/835223/Heme_cysteine/4'>covalently anchored to two cysteines</scene>, which form thioether bonds with the heme vinyl groups (opposite the heme carboxyls):
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{{Template:ColorKey_Element_C}}
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{{Template:ColorKey_Element_O}}
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{{Template:ColorKey_Element_N}}
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{{Template:ColorKey_Element_S}}
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{{Template:ColorKey_Element_Fe}}.
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12 '''CxxCH''' motifs in the [https://www.uniprot.org/uniprot/Q74A86#sequences OmcS sequence] anchor the 6 hemes within each OmcS chain.
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====Histidine to Iron====
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Each heme <scene name='83/835223/Histidine-iron/1'>iron atom is coordinated by two histidine sidechain nitrogens</scene>, in addition to the four heme nitrogens. The iron of heme 5 (the next to last heme at the carboxy end of the chain) is bound to His 332 from its own chain (<font color="#6070cf">'''Chain A'''</font>), and '''His 16''' in the N-terminal "bulge" of the '''next protein chain''' (<font color="#40af58">'''Chain B'''</font>) in the filament. This inter-chain histidine-iron bond is undoubtedly important in strengthening the monomer-monomer interfaces in the filament. The histidines bound to hemes 1, 2, 3, 4, and 6 are all in the same protein chain that contains those hemes.
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===Salt Bridges===
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Using a 4.0 Å cutoff, [[6ef8]] has 7 salt bridges between amino acid sidechains (not shown). One of these, <scene name='83/835223/Inter-chain_salt_bridge/2'>Arg176 to Asp432 (2.6 Å)</scene> (<font color="#6070cf">'''Chain A'''</font>, <font color="#40af58">'''Chain B'''</font>,
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{{Template:ColorKey_Element_O}},
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{{Template:ColorKey_Element_N}}),
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is between protein chains, further strengthening the interfaces between monomers in the filament. (These opposing charges are 4.9 Å apart in [[6nef]].)
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The amino-terminal NH<sup>3</sup>+ on Phe 1 forms a salt bridge with one carboxy of heme 2 (HEC503; 3.65 Å; not shown).
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Each heme has close to zero net charge, since the two carboxyls are compenated by Fe<sup>++</sup>. About half of the heme carboxyls are on the surface, exposed to water (not shown). Several of the heme carboxyls form salt bridges with sidechains of arginine or lysine (not shown).
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===Buried Cations===
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The <scene name='83/835223/Buried_cations/1'>sidechain nitrogens of Arg333, Arg344, and Arg375 are buried</scene>. None have anions within 5 Å (not shown). The sidechain nitrogens of Arg333 and Arg344 touch each other (3.0 Å). These characteristics are confirmed in [[6nef]]. The presence of these cations deep within OmcS is plausible, since proteins of this size have, on average, several buried charges<ref name="pace">PMID: 19164280</ref><ref name="kajander">PMID: 11080642</ref>. Moreover, on average from many proteins, more than half of all arginine guanidiniums are buried<ref name="pace" />. Burying charge seems to be an important factor in how evolution regulates protein stability<ref name="pace" /><ref name="kajander" />.
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The buried contact between two usually-cationic sidechains of Arg333 and Arg344 is also plausible because, when buried, the positive charge of the guanidinium group can be greatly diminished due to dehydration and nearby positive charges<ref name="pace" />. Although hydrated guanidinium retains more than half of its charge when the pH is below ~12 (its intrinsic pKa<ref name="pace" />), dehydration due to burial decreases the pKa. Furthermore, the samples for cryo-electron microscopy were prepared at pH 10.5<ref name="m3" /> (despite the pH being incorrectly stated as 7.0 in REMARK 245 of the PDB file).
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===Other Findings & Conclusions===
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<center>''References for the assertions below are cited in the journal publication<ref name="m3" />.
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</center>
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Seamless micrometer-long polymerization of hundreds of cytochromes is without precedent, to the knowledge of the authors. The filaments whose structure was determined here were obtained from electrode-grown cells. However, fumarate-grown cells produced filaments with similar sinusoidal morphology. The purified OmcS filaments have morphology and power spectra similar to cell-attached filaments previously thought to be type IV pili. Direct current electrical conductivity of individual wild type ~4 nm OmcS filaments was confirmed, and was comparable to previously reported filament conductivity values.
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Cells with the ''omcS'' gene deleted (''&Delta;omcS'') produced thinner (~1.7 nm) filaments that were smooth (not sinusoidal) and had electrical conductivity >100-fold lower than the OmcS filaments. ''&Delta;omcS'' cells can produce electrically conductive biofilms, but that conductivity might well depend on filaments of OmcZ, whose expression is known to increase in ''&Delta;omcS'' cells.
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Previous studies showed that PilA is required for export of OmcS. However, PilA was not found in the structure of the OmcS nanowires studied here. Thus, PilA appears to be required for production of OmcS nanowires, but not to be a structural component of those nanowires.
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</StructureSection>
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<br>
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=== Significance ===
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<hr>
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<br>
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==See Also==
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These cryo-EM structures provide a mechanistic blueprint for understanding how TRPV1 integrates thermal, chemical, and lipid-derived signals to regulate ion permeation. They reveal conserved gating transitions and define pharmacologically relevant ligand-binding pockets essential for rational drug design. The ability to visualize TRPV1 in distinct activation states enables development of selective analgesic modulators targeting neuropathic and inflammatory pain while minimizing adverse thermo-sensory effects.
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* [[Malvankar]]: A list of all interactive 3D complements for publications from the Malvankar group.
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==Notes & References==
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=== References ===
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<references />
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* Liao M., Cao E., Julius D., Cheng Y. (2013). Structure of the TRPV1 ion channel determined by electron cryo-microscopy. *Nature*, 504, 107–112.

Current revision

Contents

Cryo-EM Structure of the Human TRPV1 Ion Channel

Cryo-EM structure of the human TRPV1 ion channel in the apo state (Liao et al., 2013; ~3.5 Å resolution)

Drag the structure with the mouse to rotate

Introduction

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a heat- and ligand-gated cation channel essential for nociception, inflammatory pain, and thermal sensitivity. Activated by capsaicin, protons, noxious heat (>42°C), and lipid mediators, TRPV1 serves as a polymodal molecular sensor in the peripheral nervous system. Because of its central role in pain signaling, TRPV1 has been a major therapeutic target for developing next-generation analgesics. Understanding its three-dimensional structure is therefore crucial for elucidating its gating mechanism and ligand recognition.

Structural Highlights

Using single-particle cryo-electron microscopy, Liao, Cao, Julius, and Cheng (2013) determined the first near-atomic structures of TRPV1 in multiple functional states, including the apo (resting), capsaicin-bound, and toxin-bound conformations. TRPV1 assembles as a homotetramer, with each subunit containing six transmembrane helices (S1–S6), a re-entrant pore loop, and extensive cytosolic ankyrin repeat domains.

The vanilloid-binding pocket—formed between the S3–S4 helices and the S4–S5 linker—was resolved in detail, explaining how capsaicin stabilizes the open conformation by pulling on the S4–S5 linker and reshaping the S6 helices to widen the pore. Structures bound to the double-knot toxin (DkTx) reveal an even more dilated pore, representing a fully activated gating state. Comparisons across these states demonstrate the sequence of conformational rearrangements that underlie heat and ligand gating in TRPV1.

Significance

These cryo-EM structures provide a mechanistic blueprint for understanding how TRPV1 integrates thermal, chemical, and lipid-derived signals to regulate ion permeation. They reveal conserved gating transitions and define pharmacologically relevant ligand-binding pockets essential for rational drug design. The ability to visualize TRPV1 in distinct activation states enables development of selective analgesic modulators targeting neuropathic and inflammatory pain while minimizing adverse thermo-sensory effects.

References

  • Liao M., Cao E., Julius D., Cheng Y. (2013). Structure of the TRPV1 ion channel determined by electron cryo-microscopy. *Nature*, 504, 107–112.
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