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Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components*

  • Jessica Oyola-Cintrón
    Footnotes
    Affiliations
    Departments of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
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  • Daniel Caballero-Rivera
    Footnotes
    Affiliations
    Departments of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931

    Departments of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
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  • Leomar Ballester
    Affiliations
    Departments of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
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  • Carlos A. Baéz-Pagán
    Affiliations
    Departments of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
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  • Hernán L. Martínez
    Affiliations
    the California State University Dominguez Hills, Carson, California 90747
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  • Karla P. Vélez-Arroyo
    Affiliations
    Departments of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
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  • Orestes Quesada
    Affiliations
    Departments of Physical Sciences, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
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  • José A. Lasalde-Dominicci
    Correspondence
    To whom correspondence should be addressed: Dept. of Biology, University of Puerto Rico, Rio Piedras Campus, PO Box 23360, San Juan, PR 00931-3360. Tel.: 787-764-0000 (ext: 4887); Fax: 787-753-3852.
    Affiliations
    Departments of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931

    Departments of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants 1R01GM098343 (to J. A. L. D.) and 1P20GM103642 (to J. R. and J. A. L. D.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Supported by Research Initiative for Scientific Enhancement (RISE) Program, National Institutes of Health Grant 2 R25 GM061151, and the Puerto Rico Industrial Development Company (PRIDCO).
    2 Supported by the Research Initiative for Scientific Enhancement (RISE) Program, National Institutes of Health Grant 2 R25 GM061151, the Puerto Rico Industrial Development Company (PRIDCO), and the UPR Golf Tournament Fellowship.
Open AccessPublished:September 09, 2015DOI:https://doi.org/10.1074/jbc.M115.678573
      Lipid rafts, specialized membrane microdomains in the plasma membrane rich in cholesterol and sphingolipids, are hot spots for a number of important cellular processes. The novel nicotinic acetylcholine receptor (nAChR) mutation αC418W, the first lipid-exposed mutation identified in a patient that causes slow channel congenital myasthenia syndrome was shown to be cholesterol-sensitive and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1. The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. We performed fluorescent recovery after photobleaching (FRAP), quantitative RT-PCR, and whole cell patch clamp recordings of GFP-encoding Mus musculus nAChRs transfected into HEK 293 cells to assess the role of cholesterol and caveolin-1 (CAV-1) in the diffusion, expression, and functionality of the nAChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive nAChR might reside in specialized membrane microdomains that upon cholesterol depletion become disrupted and release the cholesterol-sensitive nAChRs to the pool of activable receptors. In addition, our results in HEK 293 cells show an interdependence between CAV-1 and αC418W that could confer end plates rich in αC418W nAChRs to a susceptibility to changes in cholesterol levels that could cause adverse drug reactions to cholesterol-lowering drugs such as statins. The current work suggests that the interplay between cholesterol and CAV-1 provides the molecular basis for modulating the function and dynamics of the cholesterol-sensitive αC418W nAChR.

      Introduction

      The nicotinic acetylcholine receptor (nAChR)
      The abbreviations used are: nAChR
      nicotinic acetylcholine receptor
      CAV-1
      caveolin-1 protein
      CBM
      caveoline binding motif
      CMS
      congenital myasthenia syndrome
      Dapp
      approximate diffusion coefficient
      FRAP
      fluorescent recovery after photobleaching
      GFP
      green fluorescent protein
      MβCD
      methyl-β-cyclodextrin
      OA
      okadaic acid
      PI3K
      phosphoinositide 3-kinase
      PI(4,5)P2
      phosphatidylinositol 4,5-bisphosphate
      ROI
      region of interest
      SCCMS
      slow channel congenital myasthenic syndrome.
      is part of the Cys-loop family of ligand-gated ion channels that include: γ-aminobutyric acid, glycine, and 5-hydroxytryptamine. It is an allosteric and integral membrane protein composed of four different subunits arranged pseudo-pentamerically in the stoichiometry of 2α1:β1:δ or ϵ:γ to form an ion channel. Each subunit contains a large hydrophilic amino-terminal (NH2) domain that faces the extracellular environment, four transmembrane domains (M1, M2, M3, and M4) made up of 19–25 amino acids, a large cytoplasmic loop between the M3 and M4 domains, and a short extracellular carboxylic terminal (COOH) domain (
      • Karlin A.
      • Akabas M.H.
      Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins.
      ,
      • Hucho F.
      • Tsetlin V.I.
      • Machold J.
      The emerging three-dimensional structure of a receptor: the nicotinic acetylcholine receptor.
      ,
      • Corringer P.J.
      • Le Novère N.
      • Changeux J.P.
      Nicotinic receptors at the amino acid level.
      ).
      Diseases involving nAChRs can be divided into two broad categories: those in which the structure and function of the nAChR are affected (e.g. congenital myasthenic syndromes, and frontal lobe epilepsy) and those involving a reduction in the number of functional nAChRs (e.g. Alzheimer, Parkinson, and schizophrenia) (
      • Léna C.
      • Changeux J.P.
      Pathological mutations of nicotinic receptors and nicotine-based therapies for brain disorders.
      ). Because nAChRs are the major type of receptors at the neuromuscular junction they are directly associated to muscle-skeletal diseases like myasthenia gravis and the congenital myasthenia syndrome (CMS) (
      • Lindstrom J.
      Nicotinic acetylcholine receptors in health and disease.
      ). CMS is characterized by a deficiency or kinetic abnormality of the nAChR at the postsynaptic level (
      • Engel A.G.
      • Ohno K.
      • Sine S.M.
      The spectrum of congenital myasthenic syndromes.
      ). Mutations that produce CMS are found in all nAChR subunits, including all transmembrane domains and the cytoplasmic loop between transmembrane domains 3 and 4. CMS mutations are classified into two categories: slow channel (prolonged receptor activations) and fast channel (brief receptor activations) syndromes (
      • Engel A.G.
      • Ohno K.
      • Sine S.M.
      The spectrum of congenital myasthenic syndromes.
      ). Slow channel congenital myasthenia syndromes (SCCMS) are a group of genetic disorders of neuromuscular transmission characterized by a progressive degeneration of the neuromuscular junction and muscle atrophy leading to fatigability and weakness. The novel SCCMS nAChR mutant αC418W is the first lipid-exposed mutation identified in a patient (
      • Shen X.M.
      • Deymeer F.
      • Sine S.M.
      • Engel A.G.
      Slow-channel mutation in acetylcholine receptor αM4 domain and its efficient knockdown.
      ).
      Lipid rafts are microdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids and have been shown to be insoluble in non-ionic detergents. Caveolae, a subset of lipid rafts, are small plasma-membrane invaginations that are rich on the cholesterol-binding protein caveolin-1. The lipid raft hypothesis postulates that some lipid species can associate to form microdomains that can be involved in protein partition, membrane sorting and trafficking, and signaling (
      • Simons K.
      • Ikonen E.
      Functional rafts in cell membranes.
      ,
      • Simons K.
      • van Meer G.
      Lipid sorting in epithelial cells.
      ). A fraction of nAChRs occurs in raft domains in mammalian cells, as demonstrated in vitro and in vivo (
      • Brusés J.L.
      • Chauvet N.
      • Rutishauser U.
      Membrane lipid rafts are necessary for the maintenance of the α7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons.
      ,
      • Campagna J.A.
      • Fallon J.
      Lipid rafts are involved in C95 (4,8) agrin fragment-induced acetylcholine receptor clustering.
      ,
      • Stetzkowski-Marden F.
      • Gaus K.
      • Recouvreur M.
      • Cartaud A.
      • Cartaud J.
      Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes.
      ,
      • Stetzkowski-Marden F.
      • Recouvreur M.
      • Camus G.
      • Cartaud A.
      • Marchand S.
      • Cartaud J.
      Rafts are required for acetylcholine receptor clustering.
      ,
      • Willmann R.
      • Pun S.
      • Stallmach L.
      • Sadasivam G.
      • Santos A.F.
      • Caroni P.
      • Fuhrer C.
      Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction.
      ).
      As a consequence of the Cys to Trp substitution, the lipid-exposed αC418W nAChR mutation introduces a caveolin-binding motif (CBM) into the αM4 transmembrane domain sequence (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ). These motifs, which are present in most caveolae-associated proteins, have been shown to favor partitioning of proteins into membrane rafts (
      • Okamoto T.
      • Schlegel A.
      • Scherer P.E.
      • Lisanti M.P.
      Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane.
      ). Previous studies performed by Báez-Pagan et al. (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ), Santiago et al. (
      • Santiago J.
      • Guzmàn G.R.
      • Rojas L.V.
      • Marti R.
      • Asmar-Rovira G.A.
      • Santana L.F.
      • McNamee M.
      • Lasalde-Dominicci J.A.
      Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation αC418W in Xenopus oocytes.
      ), and Grajales et al. (
      • Grajales-Reyes G.E.
      • Báez-Pagán C.A.
      • Zhu H.
      • Grajales-Reyes J.G.
      • Delgado-Vélez M.
      • García-Beltrán W.F.
      • Luciano C.A.
      • Quesada O.
      • Ramírez R.
      • Gómez C.M.
      • Lasalde-Dominicci J.A.
      Transgenic mouse model reveals an unsuspected role of the acetylcholine receptor in statin-induced neuromuscular adverse drug reactions.
      ) have shown that the novel αC418W mutant is sensitive to changes in membrane cholesterol levels and that it preferentially accumulates in CAV-1-positive membrane microdomains. These results suggested that upon cholesterol depletion a significant number of αC418W mutants move from a non-functional to a functional pool of nAChRs and display normal αC418W channel kinetics (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ).
      A question remains as to the molecular basis for cholesterol regulation of nAChR function and dynamics. Previous studies have postulated two possible mechanisms: 1) that modulation of nAChR function by cholesterol might be associated with lipid bilayer fluidity (
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor.
      ,
      • Sunshine C.
      • McNamee M.G.
      Lipid modulation of nicotinic acetylcholine receptor function: the role of membrane lipid composition and fluidity.
      ,
      • Fong T.M.
      • McNamee M.G.
      Correlation between acetylcholine receptor function and structural properties of membranes.
      ) and that 2) cholesterol may act as an “allosteric effector” at some binding sites located within the protein that are distinct from the lipid-protein interface (
      • Antollini S.S.
      • Barrantes F.J.
      Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane.
      ,
      • Fernandez-Ballester G.
      • Castresana J.
      • Fernandez A.M.
      • Arrondo J.L.
      • Ferragut J.A.
      • Gonzalez-Ros J.M.
      Role of cholesterol as a structural and functional effector of the nicotinic acetylcholine receptor.
      ,
      • Narayanaswami V.
      • McNamee M.G.
      Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function: 2. Membrane fluidity and ligand-mediated alteration in the accessibility of γ subunit cysteine residues to cholesterol.
      ,
      • Jones O.T.
      • McNamee M.G.
      Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor.
      ). Corbing et al. (
      • Corbin J.
      • Wang H.H.
      • Blanton M.P.
      Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol.
      ) mapped the binding sites for cholesterol at the lipid-protein interface of the Torpedo nAChR to the αM4, αM1, and γM4 transmembrane domains. However, Hamouda et al. (
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor.
      ) demonstrated that the cholesterol-binding domain fully overlaps the Torpedo nAChR lipid-protein interface as cholesterol-binding sites were found in the M4, M3, and M1 transmembrane domains of each subunit. In addition, molecular dynamic simulations have shown that the structure of the nAChR includes internal sites capable of containing cholesterol whose occupation stabilizes protein structure (
      • Brannigan G.
      • Hénin J.
      • Law R.
      • Eckenhoff R.
      • Klein M.L.
      Embedded cholesterol in the nicotinic acetylcholine receptor.
      ). Hence, nAChR-cholesterol interactions are known to regulate the function, dynamics, and number of activable nAChRs; however, the underlying mechanisms are poorly understood (
      • Barrantes F.J.
      Structural basis for lipid modulation of nicotinic acetylcholine receptor function.
      ). Thus, there is a critical need for identifying and gaining insight into the mechanism through which lipid-protein interactions regulate nAChR function and dynamics.
      The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. We performed fluorescent recovery after photobleaching (FRAP) experiments and whole cell patch clamp recordings of GFP-encoding Mus musculus nAChRs transfected into HEK 293 cells under cholesterol enrichment and depletion conditions to assess the role of cholesterol levels in the diffusion and functionality of the nAChR (WT and αC418W). Lateral diffusion and mobile fraction are modified by either cholesterol enrichment or depletion differently in the αC418W mutant when compared with the WT, further demonstrating the cholesterol-sensitive nature of the αC418W mutant. The low mobile fraction (<14%) displayed by the nAChR provides additional evidence of its trafficking to membrane microdomains. Our findings support the hypothesis that a cholesterol-sensitive nAChR might reside in a specialized membrane microdomain; however, when cholesterol is depleted in vitro or in vivo, the membrane microdomains disrupt and the cholesterol-sensitive nAChRs are released to the pool of activable receptors. Furthermore, we show a relationship between expression levels of CAV-1 and the αC418W nAChR, a result that has implications on statin treatment of patients expressing this mutation.

      Discussion

      The mobile fraction of nAChRs expressed in HEK 293 cells was determined to be ≤14% (Table 1, Fig. 2A), which is consistent with a primarily immobile membrane protein. This immobility might be due to receptor clustering mediated by inter-molecular receptor-receptor associations, interactions with non-receptor scaffolding or cytoskeleton proteins, and/or protein-lipid interactions (
      • Barrantes F.J.
      Cell-surface translational dynamics of nicotinic acetylcholine receptors.
      ). A recent study combined FRAP and confocal fluorescence correlation spectroscopy to examine the mobility of the AChR and its dependence on cholesterol levels at the cell surface of a mammalian CHO-K1/A5 cell line (
      • Baier C.J.
      • Gallegos C.E.
      • Levi V.
      • Barrantes F.J.
      Cholesterol modulation of nicotinic acetylcholine receptor surface mobility.
      ). This minimalist mammalian expression model produces heterologous adult murine muscle-type acetylcholine receptors, and lacks rapsyn and other receptor-anchoring proteins. Depletion of membrane cholesterol by mβCD strongly affected the mobility of the AChR at the plasma membrane, reducing the mobile fraction by 35% in cholesterol-depleted cells, whereas cholesterol enrichment did not affect receptor mobility at the cell surface (
      • Baier C.J.
      • Gallegos C.E.
      • Levi V.
      • Barrantes F.J.
      Cholesterol modulation of nicotinic acetylcholine receptor surface mobility.
      ). These results were confirmed by scanning fluorescence correlation spectroscopy experiments that showed that the diffusion coefficient of the AChR was ∼30% lower upon cholesterol depletion. That study suggested that membrane cholesterol modulates AChR mobility at the plasma membrane through a cholesterol-dependent mechanism sensitive to cortical actin (
      • Baier C.J.
      • Gallegos C.E.
      • Levi V.
      • Barrantes F.J.
      Cholesterol modulation of nicotinic acetylcholine receptor surface mobility.
      ).
      The αC418W nAChR from T. californica has been previously shown to be sensitive to changes in membrane cholesterol levels and that a substantial fraction of these mutant nAChRs accumulates in CAV-1-positive membrane microdomains in the oocyte surface membrane, where they are trapped in a non-activable state (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ). FRAP experiments performed on HEK 293 cells showed that the mobile fraction of the αC418W mutant nAChR was significantly reduced when compared with the WT nAChR. These results correlate with the phenotype associated to the αC418W nAChR in SCCMS in which a 30% reduction in the number of αC418W nAChRs in HEK cells is reported when compared with the WT nAChR (
      • Shen X.M.
      • Deymeer F.
      • Sine S.M.
      • Engel A.G.
      Slow-channel mutation in acetylcholine receptor αM4 domain and its efficient knockdown.
      ). In addition, several studies have reported that CAV-1 associated to caveolae has a very low mobile fraction (
      • Thomsen P.
      • Roepstorff K.
      • Stahlhut M.
      • van Deurs B.
      Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking.
      ). Based on the introduction of a CBM upon tryptophan substitution at position Cys-418, we propose that the αC418W mutant nAChR is less mobile than the WT nAChR due to favorable association with CAV-1-positive membrane microdomains.
      Báez-Pagan et al. (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ) demonstrated that the increase in macroscopic peak currents observed upon cholesterol depletion was not due to changes in αC418W nAChR kinetics or conductance, but rather an increase in the number of receptors in the oocyte surface membrane as a consequence of cholesterol depletion. To determine the mechanism that regulates the activable pool of nAChRs in a mammalian expression system we modulated cholesterol levels in the surface membrane of HEK 293 cells. Membrane cholesterol enrichment by cholesterol-loaded MβCD produced a statistically significant reduction in both the mobile fraction and macroscopic ACh-induced currents of the αC418W mutant nAChR. This result was expected as it has been previously demonstrated that cholesterol enrichment affects nAChR trafficking through the endocytic pathway, decreasing cell-surface expression by promoting internalization of nAChRs (
      • St. John P.A.
      Cellular trafficking of nicotinic acetylcholine receptors.
      ). However, cholesterol depletion in HEK 293 cells expressing the αC418W mutant nAChR produced a remarkable increase (∼2-fold) in macroscopic ACh-induced currents, suggesting that when lipid rafts are disrupted the αC418W mutant nAChRs are redistributed in the membrane surface where they become activable and contribute to the overall macroscopic current. These results demonstrate that in HEK 293 cells the regulation of the αC418W mutation by membrane cholesterol and CAV-1 is consistent with the results described by Báez-Pagan et al. (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ) when the mutant is expressed in X. laevis oocytes.
      Treatment with OA did not result in a reduction of αC418W whole cell currents (Fig. 3B) and a pre-treatment with OA before cholesterol depletion inhibits the increase in macroscopic whole cell current that is produced by cholesterol depletion in cells expressing the αC418W nAChR (Fig. 3B). These results suggest that αC418W mutant nAChRs expressed in CAV-1-positive domains are trapped in a non-activable state and thus precluded from contributing to the overall macroscopic current observed upon ACh activation due to their favorable interaction with CAV-1. Taken together these results suggest there is no apparent correlation between the nAChR mobile fraction and whole cell currents for either the WT or αC418W mutant nAChR under cholesterol-depleted conditions.
      Fluorescence recovery curves provide information on two parameters: the diffusion coefficient provides a measure of the kinetics of translational mobility, whereas the mobile fraction reports on the proportion of fluorescent molecules in the membrane surface that are able to laterally diffuse back into the bleached area over time. Recently, Báez-Pagan (
      • Báez-Pagán C.A.
      • Martínez-Ortiz Y.
      • Otero-Cruz J.D.
      • Salgado-Villanueva I.K.
      • Velázquez G.
      • Ortiz-Acevedo A.
      • Quesada O.
      • Silva W.I.
      • Lasalde-Dominicci J.A.
      Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome.
      ) showed an increase in the number of αC418W mutant nAChRs available to be activated in the membrane surface upon cholesterol depletion and how that effect translated into higher whole cell currents. However, as is the case in the current study a higher number of receptors in the membrane surface does not necessarily translate to a higher mobile fraction. Previous studies have shown that the mobility of raft- and non-raft resident proteins decreases when cholesterol is depleted from the membrane surface (
      • Kenworthy A.K.
      • Nichols B.J.
      • Remmert C.L.
      • Hendrix G.M.
      • Kumar M.
      • Zimmerberg J.
      • Lippincott-Schwartz J.
      Dynamics of putative raft-associated proteins at the cell surface.
      ,
      • O'Connell K.M.
      • Tamkun M.M.
      Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains.
      ). Other studies have postulated that restricted diffusion of membrane proteins upon cholesterol depletion stems from the formation of solid-like clusters in the membrane (
      • Nishimura S.Y.
      • Vrljic M.
      • Klein L.O.
      • McConnell H.M.
      • Moerner W.E.
      Cholesterol depletion induces solid-like regions in the plasma membrane.
      ,
      • Vrljic M.
      • Nishimura S.Y.
      • Moerner W.E.
      • McConnell H.M.
      Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane.
      ). Lowering membrane cholesterol levels with MβCD alters membrane viscosity and has been shown to hinder membrane protein diffusion (
      • Shvartsman D.E.
      • Gutman O.
      • Tietz A.
      • Henis Y.I.
      Cyclodextrins but not compactin inhibit the lateral diffusion of membrane proteins independent of cholesterol.
      ). In addition, it has been proposed that changes in cholesterol levels affect the mechanical properties of plasma membrane through the underlying cytoskeleton (
      • Sun M.
      • Northup N.
      • Marga F.
      • Huber T.
      • Byfield F.J.
      • Levitan I.
      • Forgacs G.
      The effect of cellular cholesterol on membrane-cytoskeleton adhesion.
      ). Fernandes et al. (
      • Fernandes C.C.
      • Berg D.K.
      • Gómez-Varela D.
      Lateral mobility of nicotinic acetylcholine receptors on neurons is determined by receptor composition, local domain, and cell type.
      ) showed that nAChR mobility is subtype specific as cholesterol depletion with MβCD increased the mobility of neuronal α7 nAChRs but not that of α3 nAChRs in the central nervous system synapses.
      Recently, a transgenic mouse model expressing the SCCMS αC418W mutant nAChR was used to demonstrate in vivo that the single-nucleotide polymorphism rs137852808 (αC418W) was sensitive to changes in membrane cholesterol levels (
      • Grajales-Reyes G.E.
      • Báez-Pagán C.A.
      • Zhu H.
      • Grajales-Reyes J.G.
      • Delgado-Vélez M.
      • García-Beltrán W.F.
      • Luciano C.A.
      • Quesada O.
      • Ramírez R.
      • Gómez C.M.
      • Lasalde-Dominicci J.A.
      Transgenic mouse model reveals an unsuspected role of the acetylcholine receptor in statin-induced neuromuscular adverse drug reactions.
      ), a result in agreement with what we have observed in HEK 293 cells. Furthermore, this mutation produced in mice a myopathy-like picture after statin treatment similar to statin-induced adverse drug reactions. Mice expressing this allele showed a remarkable contamination of end plates with CAV-1 and developed signs of neuromuscular degeneration upon statin treatment as the percentage of CAV-1-positive neuromuscular junctions was significantly reduced. The reduction in the percentage of end plates displaying co-localization of αC418W and CAV-1 in statin-treated neuromuscular junctions suggested that these end plates are sensitive to cholesterol concentration. Our results in HEK 293 cells supports the notion of an interdependence between CAV-1 and the αC418W nAChR that is observed in the neuromuscular junction, and that confers the αC418W nAChR end plate a susceptibility to changes in cholesterol levels that can lead to adverse drug reactions due to modifications in end plate plasticity.
      PI(4,5)P2 is an abundant phospholipid inside lipid rafts that has been linked to the regulation of a wide diversity of ion channels. This led us to hypothesize that it could be a potential candidate for a αC418W mutant nAChR modulator. The reduction of PI(4,5)P2 levels caused by treatment of HEK 293 cells with wortmannin, a PI3K inhibitor, produced a statistically significant reduction in whole cell currents for the αC418W mutant nAChR, an effect not observed for the WT nAChR. However, this reduction in whole cell currents was not observed when using the PI(4,5)P2 sequestering agent neomycin. In addition, enrichment with PI(4,5)P2 did not produce the hypothesized increase in whole cell currents. Additional studies will be needed to clarify the role, if any, that PI(4,5)P2 might play on the regulation of the αC418W mutant nAChR.

      Author Contributions

      J. O. C., D. C. R., and J. A. L. designed the study. J. O. C., D. C. R., C. A. B., and J. A. L. wrote the paper. J. O. C., K. P. V., and D. C. R. designed the plasmids, prepared DNAs and RNAs, and cultivated and treated cells. D. C. R. designed siRNAs and PCR primers. J. O. C. and D. C. R. performed FRAP and gene knockdown experiments. L. B., D. C. R., and J. O. C. performed whole cell electrophysiology experiments. O. Q. performed cholesterol content determinations. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank the UPR Confocal Imaging Facility and the UPR Cell Culture Facility. In addition, we thank Anthony Auerbach for the M. musculus (muscle-type) AChR subunit cDNAs and Veitz Witzemann for the M. musculus ϵ-GFP and γ-GFP AChR subunit cDNAs.

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