Mutations linked to autosomal dominant nocturnal frontal lobe epilepsy affect allosteric Ca2+ activation of the alpha 4 beta 2 nicotinic acetylcholine receptor.
Extracellular Ca(2+) robustly potentiates the acetylcholine response of alpha4beta2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-alpha4(S252F), alpha4(S256L), alpha4(+L264), beta2(V262L), and beta2(V262M)-reduced 2 mM Ca(2+) potentiation of the alpha4beta2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca(2+) activation or enhanced Ca(2+) block caused this reduction, we coexpressed the rat ADNFLE mutations with an alpha4 N-terminal mutation, alpha4(E180Q), that abolished alpha4beta2 allosteric Ca(2+) activation. In each case, Ca(2+) inhibition of the double mutants was less than that expected from a Ca(2+) blocking mechanism. In fact, the effects of Ca(2+) on the ADNFLE mutations near the intracellular end of the M2 region-alpha4(S252F) and alpha4(S256L)-were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca(2+) on the ADNFLE mutations near the extracellular end of the M2 region-alpha4(+L264)beta2, beta2(V262L), and beta2(V262M)-were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca(2+) on the alpha4beta2, alpha4(+L264)beta2, and alpha4beta2(V262L) single-channel conductances, the effects of membrane potential on the beta2(V262L)-mediated reduction in Ca(2+) 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 Ca(2+) block. Moreover, analyses of the alpha4beta2, alpha4(S256L), and alpha4(+L264) Ca(2+) concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca(2+) potentiation of the alpha4beta2 acetylcholine response by altering allosteric activation rather than by enhancing block.
Rodrigues-Pinguet
Nivalda O
NO
Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
Pinguet
Thierry J
TJ
Figl
Antonio
A
Lester
Henry A
HA
Cohen
Bruce N
BN
D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D017034:Epilepsy, Frontal Lobe / Q000235:genetics* / Q000378:metabolism; D005260:Female; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis
D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D002118:Calcium