From the Département de Physiologie, Membrane Protein Research Group, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
J Biol Chem. 2013 Oct 11;288(41):29281-93. doi: 10.1074/jbc.M113.500975. Epub 2013 Aug 22.
T-type CaV3 channels are important mediators of Ca(2+) entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial NaV channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG(interact) ≥ 1.5 kcal mol(-1)) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type CaV3 channel.
T 型钙通道在静息膜电位附近是钙离子内流的重要调节剂。目前对于负责通道激活的分子机制知之甚少。基于细菌 NaV 通道高分辨率结构的同源模型预测 II 域（IIS4-S5）的 S4-S5 螺旋与 II 域（IIS6）和 III 域（IIIS6）的远端 S6 孔区之间存在相互作用。通过双突变体循环分析研究了功能内和跨域相互作用。针对 47 个单突变体和 20 对突变体测量了激活门控和通道动力学。在 IIS4-S5 和 IIS6 之间以及 IIS4-S5 和 IIIS6 之间引入的 4 对特定突变体之间测量到显著的偶联能量（ΔΔG（相互作用）≥1.5 kcal/mol）。与计算机模型一致，IIS4-S5 中的 Thr-911 在通道激活过程中与 IIS6 中的 Ile-1013 具有功能偶联。然而，发现 IIS4-S5 中的 Val-907 与 IIS6 中的 Ile-1013 之间的相互作用能更强。此外，Val-907 与 IIIIS6 中的 Asn-1548 显著偶联，但与 IVS6 中的 Asn-1853 不偶联。总之，我们的结果表明，在低电压门控 T 型钙通道的激活过程中，相邻结构域的 S4-S5 和 S6 螺旋在能量上是偶联的。
