Kurejová Martina, Lacinová L'ubica, Pavlovicová Michaela, Eschbach Martin, Klugbauer Norbert
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, Bratislava, Slovakia.
Pflugers Arch. 2007 Dec;455(3):527-39. doi: 10.1007/s00424-007-0302-7. Epub 2007 Jul 19.
The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca(V)3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca(V)3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it.
通过用中性半胱氨酸取代T型Ca(V)3.1钙通道结构域I至IV中电压感应S4片段的最上端带电荷精氨酸残基，研究了这些片段对通道门控的贡献。在每个构建体中，引入了单个（R180C、R834C、R1379C或R1717C）或双重（两个相邻结构域）突变。我们发现，IS4、IIS4和IIIS4片段中最上端精氨酸的中和使通道激活的电压依赖性向超极化方向移动，其中IS4突变体的影响最为显著。相反，在所有四个单突变通道中，通道失活的电压依赖性向更负的膜电位移动，并且这些影响比通道激活的影响更明显。失活恢复受IS4和IIIS4突变的影响。在双重突变体中，对通道失活和失活恢复的影响是相加的，但对通道激活没有影响。将突变通道暴露于还原剂二硫苏糖醇不会改变通道特性。总之，我们的数据表明，Ca(V)3.1钙通道所有四个结构域中的S4片段在通道失活期间对电压感应有贡献，而只有结构域I、II和III中的S4片段在通道激活中起这种作用。此外，从IVS4和IIIS4以及在较小程度上从IS4片段中去除最外层碱性氨基酸稳定了通道的开放状态，而从IIS4中去除则使其不稳定。
