Rodrigues-Pinguet Nivalda, Jia Li, Li Maureen, Figl Antonio, Klaassen Alwin, Truong Anthony, Lester Henry A, Cohen Bruce N
Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, USA.
J Physiol. 2003 Jul 1;550(Pt 1):11-26. doi: 10.1113/jphysiol.2003.036681. Epub 2003 May 16.
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 (alpha4 or beta2), and studied the Ca2+ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca2+-induced increases in the 30 microM 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 microM. 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 microM ACh response, suggesting that the ADNFLE mutations reduce Ca2+ potentiation by enhancing Ca2+ block of the alpha4beta2 nAChR. Ca2+ modulation may prevent presynaptic alpha4beta2 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 alpha4beta2-mediated glutamate release during such bouts.
目前已知五个烟碱型乙酰胆碱受体（nAChR）突变与常染色体显性遗传性夜间额叶癫痫（ADNFLE）相关。它们临床症状的相似性表明，这些突变共同的功能异常是ADNFLE发作的基础。为了确定这种异常，我们构建了人类ADNFLE突变的大鼠同源物（S252F、+L264、S256L、V262L、V262M），并将它们与适当的野生型（WT）亚基（α4或β2）在非洲爪蟾卵母细胞中共同表达，研究其ACh反应对Ca2+的依赖性。所有突变均显著降低了2 mM Ca2+诱导的30 μM ACh反应的增加（P < 0.05）。与显性遗传模式一致，这种降低在注射了突变体和WT cRNA 1:1混合物的卵母细胞中持续存在。注射BAPTA表明，这种降低并非由于Ca2+激活的Cl-电流的二次激活减少所致。S256L突变也消除了2 mM Ba2+对ACh反应的增强作用。S256L、V262L和V262M突变对ACh浓度-反应关系有复杂影响，但在[ACh]≥30 μM时，这三个突变均使浓度-反应关系向左移动。V262M突变与消除Ca2+增强作用的突变（E180Q）共同表达，导致2 mM Ca2+对30 μM ACh反应产生阻断而非增强作用，这表明ADNFLE突变通过增强α4β2 nAChR的Ca2+阻断作用来降低Ca2+增强作用。Ca2+调节可能会防止突触前α4β2 nAChRs在同步、重复活动发作期间过度刺激中枢兴奋性突触处的谷氨酸释放。在这种发作期间，降低ACh反应对Ca2+的依赖性可能会通过增加α4β2介导的谷氨酸释放而引发癫痫发作。
