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Polyamine regulation of ion channel assembly and implications for nicotinic acetylcholine receptor pharmacology.

ABSTRACT: Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing ?7 or ?4?2, but not ?6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.

SUBMITTER: Dhara M

PROVIDER: S-EPMC7271128 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Polyamine regulation of ion channel assembly and implications for nicotinic acetylcholine receptor pharmacology.

Dhara Madhurima M Matta Jose A JA Lei Min M Knowland Daniel D Yu Hong H Gu Shenyan S Bredt David S DS

Nature communications 20200603 1

Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4β2, but not α6 subunits. Polyami ...[more]

PMID: 32493979

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Project description:The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O <--> C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.

Project description:The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-beta-D-maltoside), DDM (n-dodecyl-beta-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-beta-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C(12)E(9 )(polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N'-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non-lipid-analogue detergents.

Project description:ObjectiveThe central nervous system can regulate peripheral inflammation, but the efferent neuronal routes and the mediators remain poorly defined. One candidate is the cholinergic pathway, which releases acetylcholine (ACh). This neurotransmitter can bind to the alpha7 cholinergic receptor (alpha7R) expressed by nonneuronal cells and reduce inflammation. To test this possibility, we evaluated the expression of alpha7R and its potential role as a target in rheumatoid arthritis (RA).MethodsThe expression of alpha7R in human synovium and fibroblast-like synoviocytes (FLS) was determined using immunohistochemical, Western blot, and quantitative polymerase chain reaction (PCR) analyses. The effects of ACh in vitro were determined in interleukin-1 (IL-1)-stimulated FLS using immunoassays for protein, quantitative PCR for messenger RNA (mRNA), luciferase reporter constructs for IL-6 and NF-kappaB promoter activity, and electrophoretic mobility shift assays. Expression of alpha7R was knocked down with small interfering RNA (siRNA) or was inhibited with the selective alpha7R antagonist methyllycaconitine (MLA).ResultsProtein and mRNA for alpha7R were demonstrated in RA and osteoarthritis synovium and cultured synoviocytes. Expression in synovium was mainly in the intimal lining. ACh significantly reduced the production of IL-6, CXCL8, CCL2, CCL3, CCL5, and granulocyte colony-stimulating factor by IL-1-stimulated FLS. This effect was blocked by the alpha7R antagonist MLA or by using alpha7R siRNA to knock down receptor expression. The selective alpha7R agonist PNU-282,987 decreased the production of IL-6 by IL-1-stimulated FLS. ACh did not reduce IL-6 transcription, but it decreased IL-6 mRNA half-life and reduced IL-6 mRNA steady-state levels.ConclusionThe alpha7 receptor is expressed in the synovium and by synoviocytes. Receptor ligation inhibits cytokine expression in FLS through a posttranscriptional mechanism. Therefore, alpha7R is a potential therapeutic target for inflammatory diseases.

Project description:Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (?7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of ?7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the ?7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. ?7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, glutamic acid decarboxylase 65/67 (GAD65/67) and the ?1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of ?7 nAChR null mice and ?7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking ?7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in ?7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in ?7 nAChR gene deletion models. This implicates crucial roles of ?7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.

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Polyamine regulation of ion channel assembly and implications for nicotinic acetylcholine receptor pharmacology.

Publications

Polyamine regulation of ion channel assembly and implications for nicotinic acetylcholine receptor pharmacology.

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