Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4ß2, and Increases α5α4ß2, Nicotinic Receptor Surface Expression.

ABSTRACT

A number of mutations in α4ß2-containing (α4ß2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the ß2 subunit called ß2V287L. Two α4ß2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(ß2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(ß2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4ß2 to form a high-sensitivity α5α4ß2 nAChR. Previous studies suggest that the ß2V287L mutation suppresses low-sensitivity α4ß2* 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 ß2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The ß2V287L mutation reduced the EC50 values of high- and low-sensitivity α4ß2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4ß2 expression. In contrast, it did not affect the EC50 of α5α4ß2 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, ß2V287L increased the α4ß2 response to a sub-saturating ACh concentration (1 µM). Finally, imaging SEP-tagged α5, α4, ß2, and ß2V287L subunits showed that ß2V287L reduced total α4ß2 nAChR surface expression, increased the number of ß2 subunits per α4ß2 receptor, and increased surface α5α4ß2 nAChR expression. Thus, the ß2V287L mutation alters the subunit composition and sensitivity of α4ß2 nAChRs, and increases α5α4ß2 surface expression.