Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; Graduate Programs of Neuroscience and Biochemistry and Cell Biology, The University of Texas MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, Texas, USA.
J Biol Chem. 2022 Mar;298(3):101664. doi: 10.1016/j.jbc.2022.101664. Epub 2022 Jan 30.
As high-conductance calcium- and voltage-dependent potassium channels, BK channels consist of pore-forming, voltage-, and Ca-sensing α and auxiliary subunits. The leucine-rich repeat (LRR) domain-containing auxiliary γ subunits potently modulate the voltage dependence of BK channel activation. Despite their dominant size in whole protein masses, the function of the LRR domain in BK channel γ subunits is unknown. We here investigated the function of these LRR domains in BK channel modulation by the auxiliary γ1-3 (LRRC26, LRRC52, and LRRC55) subunits. Using cell surface protein immunoprecipitation, we validated the predicted extracellular localization of the LRR domains. We then refined the structural models of mature proteins on the membrane via molecular dynamic simulations. By replacement of the LRR domain with extracellular regions or domains of non-LRR proteins, we found that the LRR domain is nonessential for the maximal channel-gating modulatory effect but is necessary for the all-or-none phenomenon of BK channel modulation by the γ1 subunit. Mutational and enzymatic blockade of N-glycosylation in the γ1-3 subunits resulted in a reduction or loss of BK channel modulation by γ subunits. Finally, by analyzing their expression in whole cells and on the plasma membrane, we found that blockade of N-glycosylation drastically reduced total expression of the γ2 subunit and the cell surface expression of the γ1 and γ3 subunits. We conclude that the LRR domains play key roles in the regulation of the expression, cell surface trafficking, and channel-modulation functions of the BK channel γ subunits.
作为高电导钙和电压依赖性钾通道，BK 通道由孔形成、电压和 Ca 感应的 α 和辅助亚基组成。富含亮氨酸重复（LRR）结构域的辅助 γ 亚基强烈调节 BK 通道激活的电压依赖性。尽管它们在整个蛋白质质量中占据主导地位，但 BK 通道 γ 亚基 LRR 结构域的功能尚不清楚。我们在这里研究了辅助 γ1-3（LRRC26、LRRC52 和 LRRC55）亚基的这些 LRR 结构域对 BK 通道调节的功能。通过细胞表面蛋白免疫沉淀，我们验证了 LRR 结构域的预测细胞外定位。然后，我们通过分子动力学模拟对膜上成熟蛋白的结构模型进行了细化。通过用细胞外区域或非 LRR 蛋白的结构域替换 LRR 结构域，我们发现 LRR 结构域对于最大的通道门控调节效应不是必需的，但对于 γ1 亚基对 BK 通道调节的全有或全无现象是必需的。γ1-3 亚基中 N-糖基化的突变和酶阻断导致 γ 亚基对 BK 通道的调节减少或丧失。最后，通过分析它们在整个细胞和质膜上的表达，我们发现 N-糖基化的阻断大大降低了 γ2 亚基的总表达和 γ1 和 γ3 亚基的质膜表达。我们得出结论，LRR 结构域在调节 BK 通道 γ 亚基的表达、质膜运输和通道调节功能方面发挥着关键作用。
