| 1. |
- Clatot, Jerome, et al.
(författare)
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A structurally precise mechanism links an epilepsy-associated KCNC2 potassium channel mutation to interneuron dysfunction
- 2024
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 121:3
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Tidskriftsartikel (refereegranskat)abstract
- De novo heterozygous variants in KCNC2 encoding the voltage-gated potassium (K+) channel subunit Kv3.2 are a recently described cause of developmental and epileptic encephalopathy (DEE). A de novo variant in KCNC2 c.374G > A (p.Cys125Tyr) was identified via exome sequencing in a patient with DEE. Relative to wild-type Kv3.2, Kv3.2-p.Cys125Tyr induces K+ currents exhibiting a large hyperpolarizing shift in the voltage dependence of activation, accelerated activation, and delayed deactivation consistent with a relative stabilization of the open conformation, along with increased current density. Leveraging the cryogenic electron microscopy (cryo-EM) structure of Kv3.1, molecular dynamic simulations suggest that a strong π-π stacking interaction between the variant Tyr125 and Tyr156 in the α-6 helix of the T1 domain promotes a relative stabilization of the open conformation of the channel, which underlies the observed gain of function. A multicompartment computational model of a Kv3-expressing parvalbumin-positive cerebral cortex fast-spiking γ-aminobutyric acidergic (GABAergic) interneuron (PV-IN) demonstrates how the Kv3.2-Cys125Tyr variant impairs neuronal excitability and dysregulates inhibition in cerebral cortex circuits to explain the resulting epilepsy.
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| 2. |
- Kang, Po Wei, et al.
(författare)
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Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Calmodulin (CaM) and PIP2 are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the IKs current important for repolarization of cardiac action potentials. Although cryo-EM structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully-activated state allows PIP2 to compete with CaM for binding to VSD, leading to the conformational change that alters the VSD-pore coupling. We identify a motif in the KCNQ1 cytosolic domain which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its function in the heart.
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| 3. |
- Mitrovic, Darko
(författare)
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Combining Evolution and Physics through Machine Learning to Decipher Molecular Mechanisms
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- From E.coli to elephants, the cells of all living organisms are surrounded by a near impenetrable wall of lipids. The windows through the walls are membrane proteins - receptors, transporters and channels that confer communication, information and metabolites through the membrane. Without opening holes in the membrane, it is necessary for these proteins to alter their shapes by cycling between conformational states to transport signals or molecules. Owing to their important role as information bottle-necks, changes in their function can lead to cancer, infectious diseases, or metabolic disorders. Hence, they are important targets for drug discovery, therapeutic research and understanding the human body.Due to the delicate thermodynamic balance of conformational states of these proteins that are modulated by external stimuli, it is difficult to trap them in experimental setups in which their native states are captured. To add to the problematic nature of their molecular mechanisms, they are too fast to kinetically trap in a certain state long enough to observe without breaking the molecular mechanism. Fast moving mechanisms makes them a good target for molecular dynamics (MD) simulations, where the movement of all atoms in the proteins is simulated over time. Although a powerful tool, modern MD simulations are not able to access long enough timescales to accurately measure macroscopic functionally relevant information, leaving a gap between simulations and reality in which many conclusions made with atomic resolution fail to translate into macroscopic phenomena, such as receptor activity, transport efficiency, mutational stability or allosteric signalling.This work presents novel methodology that efficiently discovers and explores functionally relevant conformational states using MD simulations. By combining evolutionary information with physics using machine learning, the methodology accelerates the sampling while retaining the details of the molecular mechanism and the thermodynamic information. Additionally, the work shows how the methodology is capable of bridging the gap in resolution between experiments and simulations through the in-silico measurement of macroscopic phenomena on a microscopic scale. Moreover, it uniquely presents a framework applied to 4 studies on different target proteins of different families in which conformational change occurs, and is able to independently relate them to different types of measurements.
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| 4. |
- Stevens-Sostre, Whitney A., et al.
(författare)
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An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation
- 2024
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 123:14, s. 2024-2037
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Tidskriftsartikel (refereegranskat)abstract
- Slow deactivation is a critical property of voltage-gated K + channels encoded by the human Ether-a`-go-goRelated Gene 1 ( hERG ). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal PerArnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip"(residues 1-12) and an amphipathic a-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap a helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus"is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-a`-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap a helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.
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| 5. |
- Yee, Sook Wah, et al.
(författare)
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The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics
- 2024
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Ingår i: Molecular Cell. - : Cell Press. - 1097-2765 .- 1097-4164. ; 84:10, s. 10-1932
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.
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