Yang Huanghe, Hu Lei, Shi Jingyi, Cui Jianmin
Department of Biomedical Engineering and Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, Missouri 63130, USA.
Biophys J. 2006 Oct 15;91(8):2892-900. doi: 10.1529/biophysj.106.090159. Epub 2006 Jul 28.
Intracellular Mg(2+) at physiological concentrations activates mSlo1 BK channels by binding to a metal-binding site in the cytosolic domain. Previous studies suggest that residues E374, Q397, and E399 are important in Mg(2+) binding. In the present study, we show that mutations of E374 or E399 to other amino acids, except for Asp, abolish Mg(2+) sensitivity. These results further support that the side chains of E374 and E399 are essential for Mg(2+) coordination. To the contrary, none of the Q397 mutations abolishes Mg(2+) sensitivity, suggesting that its side chain may not coordinate to Mg(2+). However, because Q397 is spatially close to E374 and E399, its mutations affect the Mg(2+) sensitivity of channel gating by either reducing or increasing the Mg(2+) binding affinity. The pattern of mutational effects and the effect of chemical modification of Q397C indicate that Q397 is involved in the Mg(2+)-dependent activation of BK channels and that mutations of Q397 alter Mg(2+) sensitivity by affecting the conformation of the Mg(2+) binding site as well as by electrostatic interactions with the bound Mg(2+) ion.
生理浓度下的细胞内镁离子(Mg²⁺)通过与胞质结构域中的金属结合位点结合来激活mSlo1 BK通道。先前的研究表明,E374、Q397和E399残基在Mg²⁺结合中起重要作用。在本研究中,我们发现将E374或E399突变为除天冬氨酸以外的其他氨基酸会消除Mg²⁺敏感性。这些结果进一步支持E374和E399的侧链对于Mg²⁺配位至关重要。相反,Q397的突变均未消除Mg²⁺敏感性,这表明其侧链可能不与Mg²⁺配位。然而,由于Q397在空间上靠近E374和E399,其突变通过降低或增加Mg²⁺结合亲和力来影响通道门控的Mg²⁺敏感性。突变效应模式以及Q397C的化学修饰效应表明,Q397参与BK通道的Mg²⁺依赖性激活,并且Q397的突变通过影响Mg²⁺结合位点的构象以及与结合的Mg²⁺离子的静电相互作用来改变Mg²⁺敏感性。