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Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the 42 Nicotinic Acetylcholine Receptor

Division of Biology, California Institute of Technology, Pasadena, California (N.O.R.-P., H.A.L., B.N.C.); Luxtera, Inc., Carlsbad, California (T.J.P.); and Molecular Devices Corp., Union City, California (A.F.)

Extracellular Ca²⁺ robustly potentiates the acetylcholine response of 42 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)—4(S252F), 4(S256L), 4(+L264), 2(V262L), and 2(V262M)—reduced 2 mM Ca²⁺ potentiation of the 42 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 4 N-terminal mutation, 4(E180Q), that abolished 42 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—4(S252F) and 4(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—4(+L264)2, 2(V262L), and 2(V262M)—were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca²⁺ on the 42, 4(+L264)2, and 42(V262L) single-channel conductances, the effects of membrane potential on the 2(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 42, 4(S256L), and 4(+L264) Ca²⁺ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca²⁺ potentiation of the 42 acetylcholine response by altering allosteric activation rather than by enhancing block.

Address correspondence to: Bruce N. Cohen, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. E-mail: bncohen{at}caltech.edu

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