Stac3 enhances expression of human Ca(V)1.1 in Xenopus oocytes and reveals gating pore currents in HypoPP mutant channels.
Stac3 enhances expression of human Ca(V)1.1 in Xenopus oocytes and reveals gating pore currents in HypoPP mutant channels.
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DOI：
10.1085/jgp.201711962
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发表时间：
2018-03-05
期刊：
影响因子：
--
通讯作者：
Cannon SC
中科院分区：
文献类型：
--
作者：
Wu F;Quinonez M;DiFranco M;Cannon SC
低钾型周期性麻痹（HypoPP）被认为是由通过突变的Na+或Ca 2+通道的电压传感器的异常内向电流引起的。Wu等人使用Stac 3增强卵母细胞中两种HypoPP CaV1.1突变体的膜表达，并发现两者都支持门控孔电流。CaV1.1（骨骼肌中L型Ca 2+通道的成孔亚基）的突变是低钾型周期性麻痹（HypoPP）的既定原因。然而，HypoPP突变体通道的功能评估一直受到阻碍，在实现足够的质膜表达的细胞，不是肌肉来源的困难。在这项研究中，我们发现Stac 3的共表达显著增加了非洲爪蟾卵母细胞质膜上人CaV1.1（加上α2-δ1b和β1a亚基）的表达。在使用切开卵母细胞钳的电压钳研究中，我们观察到1 μA数量级的离子电流和0.5-1 nC的门控电荷位移。重要的是，这种高表达水平足以确定HypoPP突变体通道是否由于电压传感器结构域的S4区段中精氨酸残基的错义突变而泄漏。我们发现，R528 H和R528 G结构域II的S4都支持门控孔电流，但与其他R/H HypoPP突变，R528 H不进行质子。Stac 3增强的CaV1.1在卵母细胞中的膜表达增加了疾病相关突变的功能研究的通量，并且是研究CaV1.1的电压依赖性的新平台，而没有骨骼肌中横小管网络的复杂性。
Hypokalemic periodic paralysis (HypoPP) is thought to be caused by an aberrant inward current through the voltage sensors of mutant Na+ or Ca2+ channels. Wu et al. use Stac3 to enhance the membrane expression of two HypoPP CaV1.1 mutants in oocytes and find that both support gating pore currents. Mutations of CaV1.1, the pore-forming subunit of the L-type Ca2+ channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). However, functional assessment of HypoPP mutant channels has been hampered by difficulties in achieving sufficient plasma membrane expression in cells that are not of muscle origin. In this study, we show that coexpression of Stac3 dramatically increases the expression of human CaV1.1 (plus α2-δ1b and β1a subunits) at the plasma membrane of Xenopus laevis oocytes. In voltage-clamp studies with the cut-open oocyte clamp, we observe ionic currents on the order of 1 μA and gating charge displacements of ∼0.5–1 nC. Importantly, this high expression level is sufficient to ascertain whether HypoPP mutant channels are leaky because of missense mutations at arginine residues in S4 segments of the voltage sensor domains. We show that R528H and R528G in S4 of domain II both support gating pore currents, but unlike other R/H HypoPP mutations, R528H does not conduct protons. Stac3-enhanced membrane expression of CaV1.1 in oocytes increases the throughput for functional studies of disease-associated mutations and is a new platform for investigating the voltage-dependent properties of CaV1.1 without the complexity of the transverse tubule network in skeletal muscle.
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影响因子：
16.6
作者：
通讯作者：
--
DOI：
10.1073/pnas.1612441113
发表时间：
2016-09-27
影响因子：
11.1
作者：
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通讯作者：
Beam, Kurt G.
影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
5.6
作者：
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通讯作者：
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