Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild‐type (WT) subunit (α4 or β2), and studied the Ca²⁺ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca²⁺‐induced increases in the 30 μM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca²⁺‐activated Cl⁻ currents. The S256L mutation also abolished 2 mM Ba²⁺ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration‐response relationship but all three mutations shifted the concentration‐response relationship to the left at [ACh]⩾ 30 μM. Co‐expression of the V262M mutation with a mutation (E180Q) that abolished Ca²⁺ potentiation resulted in 2 mM Ca²⁺ block, rather than potentiation, of the 30 μM ACh response, suggesting that the ADNFLE mutations reduce Ca²⁺ potentiation by enhancing Ca²⁺ block of the α4β2 nAChR. Ca²⁺ modulation may prevent presynaptic α4β2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca²⁺ dependence of the ACh response could trigger seizures by increasing α4β2‐mediated glutamate release during such bouts.

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