Abstract
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.
Highlights
32040 JOURNAL OF BIOLOGICAL CHEMISTRY proteins believed to encode ion channels of intracellular endosomes and lysosomes [1,2,3,4,5,6,7]
To identify additional Va-like mutations in TRPML1 that lead to plasma membrane (PM) expression and measurable whole cell currents, we constructed 20 Pro substitutions near the Va locus in the S4 –S5 linker and the bottom half of transmembrane 5 (TM5) (Fig. 1A)
Cells transfected with TRPML1Va or TRPML3Va exhibit elevated intracellular Ca2ϩ levels, i.e. Ca2ϩ overload [16, 18]
Summary
32040 JOURNAL OF BIOLOGICAL CHEMISTRY proteins believed to encode ion channels of intracellular endosomes and lysosomes [1,2,3,4,5,6,7]. A spontaneous mutation in mouse TRPML3 has shed some light on the physiological functions of TRPML channels. This alanine-to-proline mutation in TRPML3 at the cytoplasmic face of the predicted TM5 (TRPML3A419Por TRPML3Va) causes the vestibular and pigmentation defects of the Va phenotypes. ITRPML1 recorded in LEL (lysosomal ITRPML1) is reportedly inwardly rectifying [27], consistent with the I-V of whole cell ITRPML1-Va. Understanding the mechanisms of the Va-induced large whole cell current of TRPML1 may provide insight into the gating and/or trafficking mechanisms of TRPML1. We obtained several additional GOF Va-like mutations and related the channel activities of these mutants to their subcellular localizations and surface (PM) expression
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