title: Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression.
abstract: A number of mutations in α4β2-containing (α4β2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the β2 subunit called β2V287L. Two α4β2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(β2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(β2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4β2 to form a high-sensitivity α5α4β2 nAChR. Previous studies suggest that the β2V287L mutation suppresses low-sensitivity α4β2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the β2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The β2V287L mutation reduced the EC50 values of high- and low-sensitivity α4β2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4β2 expression. In contrast, it did not affect the EC50 of α5α4β2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, β2V287L increased the α4β2 response to a sub-saturating ACh concentration (1 μM). Finally, imaging SEP-tagged α5, α4, β2, and β2V287L subunits showed that β2V287L reduced total α4β2 nAChR surface expression, increased the number of β2 subunits per α4β2 receptor, and increased surface α5α4β2 nAChR expression. Thus, the β2V287L mutation alters the subunit composition and sensitivity of α4β2 nAChRs, and increases α5α4β2 surface expression.
authors.affiliation: Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Weston A
authors.initials: WA
authors.lastname: Nichols
authors.affiliation: Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Henderson
authors.affiliation: Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marotta
authors.affiliation: Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Caroline Y
authors.initials: CY
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY, United States of America.
authors.forename: Chris
authors.initials: C
authors.lastname: Richards
authors.affiliation: Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
authors.affiliation: Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
authors.affiliation: Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Bruce N
authors.identifier: http://orcid.org/0000-0003-2913-6238
authors.initials: BN
authors.lastname: Cohen
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017034:Epilepsy, Frontal Lobe / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D012333:RNA, Messenger; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C523972:CHRNA5 protein, mouse