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

Weston A Nichols,Chris Richards,Henry A Lester,Caroline Y Yu,Brandon J Henderson,Christopher B Marotta,Bruce N Cohen,Dennis A Dougherty

doi:10.1371/journal.pone.0158032

Weston A Nichols, Chris Richards + Show 6 more

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https://doi.org/10.1371/journal.pone.0158032

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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.

Highlights

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a familial partial epilepsy linked to mutations in the α2, α4, and β2 nicotinic acetylcholine (ACh) receptor subunits [1, 2], and the Na+-gated K+ channel, KCNT1 [3, 4]
Our major findings are that the β2V287L ADNFLE mutation increases the ACh sensitivities of both HS and LS α4β2 nicotinic acetylcholine (ACh) receptors (nAChRs), but reduces the proportion of LS α4β2 nAChRs expressed on the plasma membrane (PM)
Previous experiments with knock-in mice show that β2V287L increases the ACh sensitivities of both HS and LS α4β2Ã nAChRs in brain synaptosomes and suppresses the functional expression of α4β2Ã LS receptors [18]

Summary

Introduction

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a familial partial epilepsy linked to mutations in the α2, α4, and β2 nicotinic acetylcholine (ACh) receptor (nAChR) subunits [1, 2], and the Na+-gated K+ channel, KCNT1 [3, 4]. The onset of ADNFLE seizures coincides with a shift in the location of maximal slow-wave activity (SWA) during sleep from the occipital to frontal lobe This shift occurs around age 10 and persists throughout adult life [9]. Mice with targeted β2V287L gene insertions (β2V287L knock-in mice) display increased mortality and behavioral abnormalities, but apparently not spontaneous epileptiform EEG activity [18, 19]. Undetected epileptiform activity could occur in the β2V287L knock-in mice, in light of their increased mortality Seizures in these mice may be a rare event or occur primarily in older animals, which are not typically used for EEG recordings. Β2V287L reduces the total number of α4β2Ã nAChRs in knock-in mice but increases their ACh sensitivity. Our data provide new information about how ADNFLE mutations alter the function and expression of brain nicotinic receptors

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