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

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 gainof-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, protonimpermeable, 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.

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

“…Likewise, C430P and C431P mutants were found to be constitutively active. In contrast, V436P rendered the channel completely inactive 13 . Similarly, V436C may render the channel inactive by a mechanism which cannot be overcome by compounds of the SF-22 type.…”

“…TMD5 mutations in TRPML channels have recently been shown to have a critical impact on TRPML channel opening (varitint-waddler mutant isoforms and analogues). Thus, mutation of V432 to proline renders TRPML1 constitutively active 10,13 . Likewise, C430P and C431P mutants were found to be constitutively active.…”

“…In recent years, TRPML1 has been characterized using electrophysiological tools and its basic cation channel properties have been investigated [9][10][11][12][13][14][15][16][17][18][19] , its protein-protein interaction network has been explored [20][21][22][23][24] , and knockout mouse models have been generated and investigated [25][26][27][28] . However, effective treatment options for MLIV patients are still missing.…”

Phosphoinositide isoforms determine compartment-specific ion channel activity

“…Using whole-endolysosome recordings, we recently found that TRPML1 conducts both Ca 2+ and Fe 2+ , and can be activated specifically by PI(3,5)P 2 but not other phosphoinositides (12,16,17). Interestingly, TRPML1 can also be detected in nonlysosome cellular compartments, in particular, the PM, upon exocytosis from the Golgi apparatus (the biosynthetic pathway) (13,14) or the lysosome (lysosomal exocytosis) (18,19). Indeed, when lysosomal exocytosis was dramatically increased by a constitutively active TRPML1 mutant (TRPML1 Va ), large whole-cell TRPML1 currents were detected (18).…”

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs

“…Four linker helices surround the S6 bundle crossing and stabilize the closed channel pore. It is interesting to note that several gain-of-function mutations similar to the V432P varitint-waddler phenotype 15,27-29 have been identified on S5 in a proline-scanning mutagenesis study 30 , including R427P, C430P and C431P, which are all clustered at the interaction interface between the S5 and S4-S5 linker (Fig. 4c).…”