Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression

A number of mutations in alpha 4 beta 2-containing (alpha 4 beta 2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the beta 2 subunit called beta 2V287L. Two alpha 4 beta 2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (alpha 4)(2)(beta 2)(3) subunit stoichiometry and a low-sensitivity subtype with (alpha 4)(3)(beta 2)(2) stoichiometry. The a5 nicotinic subunit also co-assembles with alpha 4 beta 2 to form a high-sensitivity a5 alpha 4 beta 2 nAChR. Previous studies suggest that the beta 2V287L mutation suppresses low-sensitivity alpha 4 beta 2* nAChR expression in a knock-in mouse model and also that alpha 5 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 beta 2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The beta 2V287L mutation reduced the EC50 values of high-and low-sensitivity alpha 4 beta 2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity alpha 4 beta 2 expression. In contrast, it did not affect the EC50 of alpha 5 alpha 4 beta 2 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, beta 2V287L increased the alpha 4 beta 2 response to a sub-saturating ACh concentration (1 mu M). Finally, imaging SEP-tagged alpha 5, alpha 4, beta 2, and beta 2V287L subunits showed that beta 2V287L reduced total alpha 4 beta 2 nAChR surface expression, increased the number of beta 2 subunits per alpha 4 beta 2 receptor, and increased surface alpha 5 alpha 4 beta 2 nAChR expression. Thus, the beta 2V287L mutation alters the subunit composition and sensitivity of alpha 4 beta 2 nAChRs, and increases alpha 5 alpha 4 beta 2 surface expression.