HOAT1: Difference between revisions

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==Structure Tour==
==Structure Tour==
<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=''>
<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=''>
Classification: MEMBRANE PROTEIN
Classification: MEMBRANE PROTEIN


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Funding Organization(s): National Research Foundation (NRF, Korea)
Funding Organization(s): National Research Foundation (NRF, Korea)


'''Experimental Data Snapshot'''
'''Experimental Data Snapshot'''
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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.
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.


'''1. Binding Location and Pose'''
*Olmesartan binds within the central cavity of hOAT1 in an inward-facing conformation where it occupies Site 3 of the binding pocket. The drug adopts a diagonal orientation relative to the membrane plane, a pose that requires more space than the smaller inhibitor probenecid.
 
:*Olmesartan binds within the central cavity of hOAT1 in an inward-facing conformation.
 
:*It occupies Site 3 of the binding pocket, which is the primary polyspecific site for anionic substrates.
 
:*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.
 
'''2. Key Interacting Residues'''
 
:*Olmesartan occupies Site 3 of the binding pocket and is located within 5A˚ distance of residues of TM1, TM4, TM5, TM7, TM10, and TM11, namely N35, M207, G227, Y230, W346, Y353, Y354, F438, F442, S462, and R466.
 
:*The biphenyl group and tetrazole ring of olmesartan rely on interactions with hydrophobic residues close to the bottom gate in the binding pocket.
 
:*Upon olmesartan binding, the side chain of Y230 undergoes a vertical rotation to accommodate and interact with the substrate.
 
:*M207 and F442 residues forming the bottom gate of the binding pocket affect olmesartan interactions
 


*Olmesartan occupies Site 3 of the binding pocket and is located within 5A˚ distance of residues of TM1, TM4, TM5, TM7, TM10, and TM11, namely N35, M207, G227, Y230, W346, Y353, Y354, F438, F442, S462, and R466.


===Mechanism of OAT1 inhibition by probenecid===
===Mechanism of OAT1 inhibition by probenecid===
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'''1. Binding Mode and Direct Competition'''
'''1. Binding Mode and Direct Competition'''


*Probenecid binds at the top of the central cavity, parallel to the membrane plane.
*Probenecid binds at the top of the central cavity, parallel to the membrane plane. Its binding site overlaps with both Site 1 (partially) and Site 3.
 
*Its binding site overlaps with both Site 1 (partially) and Site 3.


*In the binding pocket of Site 1, surrounded by 16 residues located within a 5 A ˚ (M31, N35, M142, V145, G227, Y230, W346, Y353, Y354, K382, D378, F438, S462, A465, R466, and S469).
*In the binding pocket of Site 1, surrounded by 16 residues located within a 5 A ˚ (M31, N35, M142, V145, G227, Y230, W346, Y353, Y354, K382, D378, F438, S462, A465, R466, and S469).
*It engages in specific, high-affinity interactions with key residues:
:*K382 on TM8 forms a hydrogen bond with the carboxylate group of probenecid.
:*Y354 on TM7 forms a hydrogen bond with its sulfonyl group.
:*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.


'''2. Conformational Arrest and Cytoplasmic Path Blockage'''
'''2. Conformational Arrest and Cytoplasmic Path Blockage'''


The primary inhibitory mechanism is a probenecid-induced conformational change that physically blocks substrate access and exit.
The primary inhibitory mechanism is a probenecid-induced conformational change that physically blocks substrate access and exit. Compared to the apo state, the cytoplasmic opening of the binding pocket narrows from ~15 Å to ~12 Å in the probenecid-bound state. Probenecid binding narrows Path A and completely blocks Path B. Restriction of the access route to path B likely limits the entry of substrates to Site 1 and the exit of substrates from the binding pocket.
 
*'''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.
 
*'''Dual-Pathway Blockade:''' The cytosolic entrance is split into two paths. Probenecid binding critically affects both:
 
:*'''Path A''' (between TM2 and TM11) is narrowed from ~5 Å to ~4 Å.
 
:*'''Path B''' (between TM5 and TM8) is completely blocked. Restriction of the access route to path B likely limits the entry of substrates to Site 1 and the exit of substrates from the binding pocket.


This structural rearrangement is caused by a slight inward movement of the cytoplasmic ends of TM5, TM8, TM10, and TM11 toward the binding pocket.
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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By constricting the cytoplasmic access routes, probenecid does not just compete for the substrate-binding site; it stabilizes the transporter in an apo-like, inward-facing conformation that is inaccessible to cytosolic substrates. This prevents the entry of new substrates and likely traps the transporter in this non-functional state, effectively "locking" it and preventing the conformational changes necessary for the transport cycle.
By constricting the cytoplasmic access routes, probenecid does not just compete for the substrate-binding site; it stabilizes the transporter in an apo-like, inward-facing conformation that is inaccessible to cytosolic substrates. This prevents the entry of new substrates and likely traps the transporter in this non-functional state, effectively "locking" it and preventing the conformational changes necessary for the transport cycle.


===Mechanistic Insights into hOAT1 Function and Inhibition===
===Mechanistic Insights into hOAT1 Function and Inhibition===
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conformation change for inhibition (apo-like conformation).]]
conformation change for inhibition (apo-like conformation).]]


'''1. A Dual-Mechanism for Potent Inhibition by Probenecid'''
''' A Dual-Mechanism for Potent Inhibition by Probenecid'''


The study reveals that the classic inhibitor probenecid employs a sophisticated, dual-mechanism to arrest OAT1 function, moving beyond simple competition.
The study reveals that the classic inhibitor probenecid employs a sophisticated, dual-mechanism to arrest OAT1 function, moving beyond simple competition.
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'''Conformational Arrest:''' More significantly, probenecid binding induces subtle conformational changes in the cytoplasmic ends of transmembrane helices (TM5, TM8, TM10, TM11). This leads to a constriction of the cytosolic opening, completely blocking one access path (Path B) and narrowing the other (Path A). This physically prevents substrates from entering or exiting the binding site from the cytoplasm, effectively "locking" the transporter in an inactive, inward-facing state. This mechanism is reminiscent of inhibition seen in other transporters like hURAT1, suggesting it may be a general strategy for effective transport arrest.
'''Conformational Arrest:''' More significantly, probenecid binding induces subtle conformational changes in the cytoplasmic ends of transmembrane helices (TM5, TM8, TM10, TM11). This leads to a constriction of the cytosolic opening, completely blocking one access path (Path B) and narrowing the other (Path A). This physically prevents substrates from entering or exiting the binding site from the cytoplasm, effectively "locking" the transporter in an inactive, inward-facing state. This mechanism is reminiscent of inhibition seen in other transporters like hURAT1, suggesting it may be a general strategy for effective transport arrest.


'''2. Structural Basis for Species-Specific Drug Transport'''
===Conclusion===
 
rOAT1 structures with probenecid have been reported previously, <ref>Parker, J.L., Kato, T., Kuteyi, G., Sitsel, O., and Newstead, S. (2023).
A major advancement of this work is the structural explanation for long-observed differences in drug handling between human OAT1 and animal orthologs.
Molecular basis for selective uptake and elimination of organic anions in
 
the kidney by OAT1. Nat. Struct. Mol. Biol. 30, 1786–1793. https://doi.
'''The Critical Role of S203:''' The residue S203 in hOAT1 (which is an alanine, A203, in rat OAT1) is identified as a key species-specific determinant. It does not contact substrates like olmesartan directly. Instead, its hydroxyl group is crucial for coordinating a chloride ion along with residues Y230 and R466.
org/10.1038/s41594-023-01039-y.</ref> and our hOAT1 structures align with findings for rOAT1 and provide new insights into the mechanism by which probenecid inhibits transport activity. Additionally, this study reveals the structure of hOAT1 with olmesartan, offering mechanistic insights into species-specific differences in OAT1 transport of specific substrates.
 
'''Chloride Coordination Enhances Substrate Affinity:''' This chloride coordination network stabilizes the binding of certain substrates. Functional data confirms that the S203A mutation drastically reduces olmesartan affinity specifically in the presence of chloride. This explains why drugs like olmesartan and tenofovir show different transport kinetics between species; the human transporter, with its S203, has a enhanced, chloride-dependent mechanism for high-affinity binding that the rat ortholog lacks.
 
 
 
===Author===
Kaushki Sharma
 
Indian Institute of Science Education and Research, Pune, India
 
BI3323-Aug2025
 




==Notes & References==
==Notes & References==
<references />
<references />
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