Dong Xian-ping, Wang Xiang, Shen Dongbiao, Chen Su, Liu Meiling, Wang Yanbin, Mills Eric, Cheng Xiping, Delling Markus, Xu Haoxing
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2009 Nov 13;284(46):32040-52. doi: 10.1074/jbc.M109.037184. Epub 2009 Jul 28.
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 whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.
黏脂蛋白瞬时受体电位（TRP）（TRPML）蛋白是一类定位于内溶酶体的阳离子通道，在人类和啮齿动物中具有重要的遗传学意义。人类TRPML1基因突变会导致IV型黏脂贮积症，这是一种严重的儿童神经退行性疾病。我们最近的电生理研究表明，虽然使用溶酶体膜片钳技术只能在晚期内体和溶酶体（LEL）中记录到TRPML1介导的电流，但TRPML1中的脯氨酸替代（TRPML1(V432P)）会导致产生一个大的全细胞电流。因此，目前尚不清楚TRPML1(V432P)介导的大电流是由表面表达增加（转运）、通道活性升高（门控）还是两者共同作用导致的。在此，我们在先前与多种6跨膜阳离子通道门控相关的区域进行了系统性的脯氨酸替代。我们发现，几个脯氨酸替代在质膜（PM）和内溶酶体膜上均表现出功能获得性（GOF）组成型活性。虽然野生型TRPML1和非GOF脯氨酸替代仅定位于LEL，在PM中几乎检测不到，但具有高组成型活性的GOF突变并不局限于LEL区室，最重要的是，表现出显著的表面表达。由于溶酶体胞吐作用依赖于Ca(2+)，脯氨酸替代导致的组成型Ca(2+)通透性可能导致了不依赖刺激的溶酶体内Ca(2+)释放，从而导致了TRPML1的表面表达和全细胞电流。事实上，在表达GOF TRPML1通道的细胞中，溶酶体相关膜蛋白-1（Lamp-1）的表面染色显著增加。我们得出结论，TRPML1是一种内向整流、质子不可渗透、Ca(2+)和Fe(2+)/Mn(2+)双重通透的阳离子通道，可能通过跨膜5（TM5）胞质面的构象变化，由未知的细胞机制进行门控。此外，LEL中TRPML1的激活可能导致TRPML1蛋白出现在PM上。
