Erler Isabell, Al-Ansary Dalia M M, Wissenbach Ulrich, Wagner Thomas F J, Flockerzi Veit, Niemeyer Barbara A
Department of Pharmacology and Toxicology, University of Saarland, Medical Campus, 66421 Homburg, Germany.
J Biol Chem. 2006 Dec 15;281(50):38396-404. doi: 10.1074/jbc.M607756200. Epub 2006 Oct 25.
TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.
瞬时受体电位褪黑素样（TRPM）通道在整体大小（>1000个氨基酸）、缺乏N端锚蛋白样重复序列以及疏水性预测方面与瞬时受体电位家族的许多其他成员不同，这些预测表明其可能有超过六个跨膜区域。每个TRPM成员的共同特征是有一个突出的C端卷曲螺旋区域。我们在此表明，TRPM8通道利用细胞内C端的假定卷曲螺旋区域组装成多聚体，并且该组装可被卷曲螺旋区域内的单点突变干扰。这种突变体既不会产生功能性通道，其亚基也不会相互作用或形成对应于多聚体的蛋白质复合物。然而，它们仍会被转运到质膜。此外，通过表达TRPM8的膜附着C端区域可抑制野生型电流。为了将组装与运输分开，我们通过鉴定和突变相关的N-连接糖基化位点来研究TRPM8蛋白的成熟过程，并表明糖基化对于多聚化本身以及转运到质膜并非必不可少，但似乎有助于高效的多聚化和运输。
