Rodrigues-Pinguet Nivalda O, Pinguet Thierry J, Figl Antonio, Lester Henry A, Cohen Bruce N
Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
Mol Pharmacol. 2005 Aug;68(2):487-501. doi: 10.1124/mol.105.011155. Epub 2005 May 18.
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.
细胞外钙离子能显著增强α4β2烟碱型受体对乙酰胆碱的反应。与常染色体显性夜间额叶癫痫（ADNFLE）相关的五个突变的大鼠直系同源基因——α4（S252F）、α4（S256L）、α4（+L264）、β2（V262L）和β2（V262M）——使α4β2对1 mM乙酰胆碱反应的2 mM钙离子增强作用降低了55%至74%。为了确定变构钙离子激活改变或钙离子阻断增强是否导致了这种降低，我们将大鼠ADNFLE突变与一个α4 N端突变α4（E180Q）共表达，该突变消除了α4β2的变构钙离子激活。在每种情况下，双突变体的钙离子抑制作用都小于钙离子阻断机制所预期的。实际上，钙离子对M2区域细胞内末端附近的ADNFLE突变——α4（S252F）和α4（S256L）——的作用符合一种直接的变构机制。相反，钙离子对M2区域细胞外末端附近的ADNFLE突变——α4（+L264）β2、β2（V262L）和β2（V262M）——的作用符合一种涉及变构激活改变和阻断增强的混合机制。然而，2 mM钙离子对α4β2、α4（+L264）β2和α4β2（V262L）单通道电导的作用、膜电位对β2（V262L）介导的钙离子增强作用降低的影响以及消除细胞外环中的负电荷对这种降低的影响，均未能提供突变增强钙离子阻断的任何直接证据。此外，对α4β2、α4（S256L）和α4（+L264）钙离子浓度-增强关系的分析表明，ADNFLE突变通过改变变构激活而非增强阻断来降低α4β2对乙酰胆碱反应的钙离子增强作用。
