Nichols_2016_PLoS.One_11_e0158032

A number of mutations in alpha4beta2-containing (alpha4beta2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the beta2 subunit called beta2V287L. Two alpha4beta2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (alpha4)2(beta2)3 subunit stoichiometry and a low-sensitivity subtype with (alpha4)3(beta2)2 stoichiometry. The alpha5 nicotinic subunit also co-assembles with alpha4beta2 to form a high-sensitivity alpha5alpha4beta2 nAChR. Previous studies suggest that the beta2V287L mutation suppresses low-sensitivity alpha4beta2* nAChR expression in a knock-in mouse model and also that alpha5 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 beta2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The beta2V287L mutation reduced the EC50 values of high- and low-sensitivity alpha4beta2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity alpha4beta2 expression. In contrast, it did not affect the EC50 of alpha5alpha4beta2 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, beta2V287L increased the alpha4beta2 response to a sub-saturating ACh concentration (1 muM). Finally, imaging SEP-tagged alpha5, alpha4, beta2, and beta2V287L subunits showed that beta2V287L reduced total alpha4beta2 nAChR surface expression, increased the number of beta2 subunits per alpha4beta2 receptor, and increased surface alpha5alpha4beta2 nAChR expression. Thus, the beta2V287L mutation alters the subunit composition and sensitivity of alpha4beta2 nAChRs, and increases alpha5alpha4beta2 surface expression.