Thompson Jill L, Mignen Olivier, Shuttleworth Trevor J
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
J Biol Chem. 2009 Mar 13;284(11):6620-6. doi: 10.1074/jbc.M808346200. Epub 2008 Dec 15.
Homozygous expression of Orai1 bearing the R91W mutation results in the complete abrogation of currents through the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, resulting in a form of hereditary severe combined immune deficiency (SCID) syndrome (Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S. H., Tanasa, B., Hogan, P. G., Lewis, R. S., Daly, M., and Rao, A. (2006) Nature 441, 179-185). Although heterozygous carriers of the mutation show no clinical symptoms of immunodeficiency, store-operated Ca(2+) entry in their T cells is impaired, suggesting a gene-dosage effect of the mutation. We have recently demonstrated that the functional CRAC channel pore is composed of a tetrameric assembly of Orai1 subunits (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2008) J. Physiol. 586, 419-425). Therefore, to directly quantify the effect of the SCID mutant in the heterozygous situation, we generated a series of concatenated tetramers of Orai1 that included different numbers and arrangements of the R91W Orai1 subunits. The data obtained show that inclusion of increasing numbers of mutant subunits results in a graded reduction in CRAC channel currents and that this effect is independent of the spatial arrangement or order of the mutant subunits in the tetramer. Macroscopic biophysical properties of the channels were unchanged by inclusion of the mutant subunits, although the rate at which the current activates on store depletion was slowed. We conclude that incorporation of R91W mutant Orai1 subunits in the CRAC channel pore affects the overall magnitude of its conductance and that this effect is related solely to the number of mutant subunits incorporated. Predictions based on the tetrameric channel structure indicate that the graded effect of incorporation of SCID mutant subunits into such an assembly is quantitatively consistent with the previously demonstrated impaired effects on Ca(2+) entry recorded in the heterozygous carriers.
携带R91W突变的Orai1纯合表达导致通过储存操纵性Ca(2+)释放激活Ca(2+)（CRAC）通道的电流完全消失，从而引发一种遗传性严重联合免疫缺陷（SCID）综合征（费斯克，S.，格瓦克，Y.，普拉克里亚，M.，斯里坎特，S.，普佩尔，S.H.，塔纳萨，B.，霍根，P.G.，刘易斯，R.S.，戴利，M.，以及拉奥，A.（2006年）《自然》441卷，第179 - 185页）。尽管该突变的杂合携带者没有免疫缺陷的临床症状，但其T细胞中的储存操纵性Ca(2+)内流受损，这表明该突变存在基因剂量效应。我们最近证明功能性CRAC通道孔由Orai1亚基的四聚体组装而成（米涅恩，O.，汤普森，J.L.，以及沙特尔沃思，T.J.（2008年）《生理学杂志》586卷，第419 - 425页）。因此，为了直接量化SCID突变体在杂合情况下的效应，我们构建了一系列Orai1的串联四聚体，其中包含不同数量和排列的R91W Orai1亚基。所获得的数据表明，包含越来越多的突变亚基会导致CRAC通道电流逐渐降低，并且这种效应与突变亚基在四聚体中的空间排列或顺序无关。尽管储存耗尽时电流激活的速率减慢，但通道的宏观生物物理特性并未因包含突变亚基而改变。我们得出结论，在CRAC通道孔中掺入R91W突变Orai1亚基会影响其电导的总体大小，并且这种效应仅与掺入的突变亚基数量有关。基于四聚体通道结构的预测表明，将SCID突变亚基掺入这样一个组装体中的分级效应在数量上与先前在杂合携带者中记录的对Ca(2+)内流的受损效应一致。
