Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis

作者：Xian‐Ping Dong, Xiang Wang, Dongbiao Shen, Su Chen, Meiling Liu, Yanbin Wang, Eric Mills, Xiping Cheng, Markus Delling, Haoxing Xu · 发表于：Journal of Biological Chemistry · 年份：2009 · DOI：10.1074/jbc.m109.037184 · 被引用次数：130 · 研究领域：Calcium signaling and nucleotide metabolism、Ion Channels and Receptors、Piperaceae Chemical and Biological Studies

The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and who...