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Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

ABSTRACT: Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ? W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKC? and PKC?. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

SUBMITTER: Das J

PROVIDER: S-EPMC6008642 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

Das Joydip J Kedei Noemi N Kelsey Jessica S JS You Youngki Y Pany Satyabrata S Mitchell Gary A GA Lewin Nancy E NE Blumberg Peter M PM

Biochemistry 20180105 5

Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the ...[more]

PMID: 29244485

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Project description:The human hepatic organic cation transporter 1 (hOCT1) is a well-known transporter of both xenobiotic and endogenous cations. The substrates and inhibitors of hOCT1 are structurally and physiochemically diverse and include some widely prescribed drugs (metformin and imatinib), vitamins (thiamine), and neurotransmitters (serotonin). It has been demonstrated that the closely related renal isoform, hOCT2, is subject to ligand-dependent modulation, wherein one ligand may enhance or inhibit transport of a second, chemically unrelated, ligand. This phenomenon has important implications for drug-drug interactions due to the ubiquity of polypharmacy and the large number of drugs that are present as cations under physiological conditions. Therefore, the objective of this study was to determine if hOCT1 is subject to the same ligand-dependent modulation as hOCT2, and to identify unique putative ligand binding sites in the translocation channel for a sub-set of ligands using computational modeling. The competitive counter flow (CCF) assay was employed to examine ligand-dependent effects by utilizing four different radiolabeled probe substrates: MPP+, serotonin, metformin, and TEA. We identified 20 ligands that modulated the transport of the four test substrates examined. One of the putative ligands identified, BSP, is an anion at physiological pH. Direct uptake studies of radiolabeled BSP suggested that it is a hOCT1 substrate with a Km of 13.6?±?2.6?µM and Vmax of 55.1?±?4.1?pmol/mg protein/min. Each ligand identified was computationally docked into a homology model of hOCT1 using the UCSF DOCK software package. The docking study revealed three separate ligand binding pockets within the hOCT1 translocation pathway, defined by their interactions with three prototypical substrates: MPP+, TEA, and acyclovir. Our results suggest that hOCT1 is not only subject to ligand-dependent modulation, but also that individual ligand binding occurs at discrete sites within the hOCT1 translocation pathway which may influence ligand binding at the other sites.

Dataset Information

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

Publications

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

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