Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.
Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.
复制标题
DOI：
10.1242/dmm.049704
复制
发表时间：
2023-06-01
影响因子：
4.3
通讯作者：
中科院分区：
文献类型：
--
作者：
低钾性周期性麻痹（HypoPP）是一种罕见的遗传性疾病，与编码电压门控Ca2+通道Cav1.1或电压门控Na+通道Nav1.4的CACNA1S或SCN4A突变相关。大多数hypopp相关的错义变化发生在这些通道的电压感应域（VSD）内的精氨酸残基上。已经确定，这种突变破坏了分离外部流体和内部细胞质缝隙的疏水密封，导致产生称为门控孔电流的异常泄漏电流。目前，门控孔电流被认为是HypoPP的基础。在这里，我们以HEK293T细胞为基础，利用睡美人转座子系统，生成了hypopp模型细胞系，它们共同表达小鼠内向流K+通道（mKir2.1）和hypopp2相关的Nav1.4通道。全细胞膜片钳测量证实，mKir2.1成功地将膜电位超极化到与肌纤维相当的水平，并且一些Nav1.4变体诱导了显著的基于质子的门控孔电流。重要的是，我们成功地通过使用比例pH指示剂在这些变体中荧光测量门控孔电流。我们的光学方法为高通量药物筛选提供了一个潜在的体外平台，不仅适用于HypoPP，也适用于由VSD突变引起的其他通道病变。摘要：低钾性周期性麻痹模型细胞，允许在多孔板格式的门控孔电流的荧光评估。
Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A encoding the voltage-gated Ca2+ channel Cav1.1 or the voltage-gated Na+ channel Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal that separates external fluid and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, based on HEK293T cells and by using the Sleeping Beauty transposon system, we generated HypoPP-model cell lines that co-express the mouse inward-rectifier K+ channel (mKir2.1) and HypoPP2-associated Nav1.4 channel. Whole-cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarizes the membrane potential to levels comparable to those of myofibers, and that some Nav1.4 variants induce notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants by using a ratiometric pH indicator. Our optical method provides a potential in vitro platform for high-throughput drug screening, not only for HypoPP but also for other channelopathies caused by VSD mutations. Summary: Hypokalemic periodic paralysis model cells that allow the fluorometric evaluation of gating pore currents in a multi-well-plate format.
登录
查看更多内容
影响因子：
16.8
作者：
Li Q;Wanderling S;Paduch M;Medovoy D;Singharoy A;McGreevy R;Villalba-Galea CA;Hulse RE;Roux B;Schulten K;Kossiakoff A;Perozo E
通讯作者：
Perozo E
影响因子：
5.6
作者：
Moreau A;Gosselin-Badaroudine P;Boutjdir M;Chahine M
通讯作者：
Chahine M
影响因子：
9.9
作者：
Matthews, E.;Labrum, R.;Hanna, M. G.
通讯作者：
Hanna, M. G.
影响因子：
9.9
作者：
Bulman, DE;Scoggan, KA;Ebers, GC
通讯作者：
Ebers, GC
DOI：
10.1073/pnas.1716461115
发表时间：
2017-12-26
影响因子：
11.1
作者：
Perni, Stefano;Lavorato, Manuela;Beam, Kurt G.
通讯作者：
Beam, Kurt G.