Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Abstract

Extracellular Ca²⁺ robustly potentiates the acetylcholine response of α4β2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)—α4(S252F), α4(S256L), α4(+L264), β2(V262L), and β2(V262M)—reduced 2 mM Ca²⁺ potentiation of the α4β2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca²⁺ activation or enhanced Ca²⁺ block caused this reduction, we coexpressed the rat ADNFLE mutations with an α4 N-terminal mutation, α4(E180Q), that abolished α4β2 allosteric Ca²⁺ activation. In each case, Ca²⁺ inhibition of the double mutants was less than that expected from a Ca²⁺ blocking mechanism. In fact, the effects of Ca²⁺ on the ADNFLE mutations near the intracellular end of the M2 region—α4(S252F) and α4(S256L)—were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca²⁺ on the ADNFLE mutations near the extracellular end of the M2 region—α4(+L264)β2, β2(V262L), and β2(V262M)—were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca²⁺ on the α4β2, α4(+L264)β2, and α4β2(V262L) single-channel conductances, the effects of membrane potential on the β2(V262L)-mediated reduction in Ca²⁺ potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca²⁺ block. Moreover, analyses of the α4β2, α4(S256L), and α4(+L264) Ca²⁺ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca²⁺ potentiation of the α4β2 acetylcholine response by altering allosteric activation rather than by enhancing block.