Activating mutations of the TRPML1 channel revealed by proline-scanning mutagenesis.
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.
Dong
Xian-ping
XP
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Wang
Xiang
X
Shen
Dongbiao
D
Chen
Su
S
Liu
Meiling
M
Wang
Yanbin
Y
Mills
Eric
E
Cheng
Xiping
X
Delling
Markus
M
Xu
Haoxing
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004594:Electrophysiology; D005089:Exocytosis; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008247:Lysosomes; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D050053:TRPM Cation Channels / Q000235:genetics* / Q000378:metabolism*; D050051:Transient Receptor Potential Channels
C416674:MCOLN1 protein, human; D050053:TRPM Cation Channels; D050051:Transient Receptor Potential Channels; D008345:Manganese; D011392:Proline; D002118:Calcium