Lateral diffusion, function, and expression of the slow channel congenital myasthenia syndrome αC418W nicotinic receptor mutation with changes in lipid raft components.
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.
From the Departments of Chemistry.
Jessica
J
Oyola-Cintrón
From the Departments of Chemistry, Biology, and.
Daniel
D
Caballero-Rivera
Biology, and.
Leomar
L
Ballester
Biology, and.
Carlos A
CA
Baéz-Pagán
the California State University Dominguez Hills, Carson, California 90747.
Hernán L
HL
Martínez
From the Departments of Chemistry.
Karla P
KP
Vélez-Arroyo
Physical Sciences, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, 00931 and.
Orestes
O
Quesada
From the Departments of Chemistry, Biology, and jlasalde@gmail.com.
José A
JA
Lasalde-Dominicci
D000818:Animals; D051242:Caveolin 1 / Q000235:genetics* / Q000378:metabolism; D002784:Cholesterol / Q000172:deficiency; D004058:Diffusion; D004705:Endocytosis / Q000187:drug effects; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019319:Okadaic Acid / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
C495540:CAV1 protein, human; D051242:Caveolin 1; D011978:Receptors, Nicotinic; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; D002784:Cholesterol
electrophysiology; fluorescence recovery after photobleaching (FRAP); lipid raft; lipid-protein interaction; nicotinic acetylcholine receptors (nAChR); phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2); slow channel congenital myasthenia syndrome; whole cell patch clamp