PMC

Subcellular localization of TRPML3 loop mutants. A, the table summarizes the laser scanning microscopy and calcium imaging results. PM, plasma membrane localization; +, plasma membrane localization comparable with the wild type; +/−, mostly intracellular localization with some potential distribution in the plasma membrane; −, obvious intracellular localization of the channel protein; =, unchanged basal calcium levels compared with the wild-type control; <, >, and ≫, decreased, increased, and strongly increased basal calcium levels in HEK293 cells transfected with the respective mutant isoforms. B, subcellular localization of YFP-tagged wild-type TRPML3 and selected mutant isoforms overexpressed in HEK293 cells and analyzed by laser scanning microscopy. Scale bar = 10 μm.

Effect of different chemical stimuli on TRPML2. A, average inward current densities in the presence and absence of different chemical stimuli in SBS from experiments as shown in B at −80 and −200 mV and normalized by cell capacitance (picofarads (pF)). Shown are means ± S.E. ***, p < 0.0001; **, p < 0.001 (unpaired Student's t test). + #, with compound; − #, without compound. Compounds were used at a final concentration of 10 μm each. B, steady-state current-voltage plots of whole-cell currents as summarized for A in the presence of different chemical stimuli in SBS. The major cation in the pipette solution was 150 mm Cs⁺ (pH 7.2). C, chemical formulae of compounds SF-21, SF-41, and SF-81.

Currents recorded from HEK293 cells expressing TRPML3 loop mutants E361A and D371A. A and B, steady-state current-voltage plots of whole-cell currents (A) and average inward current densities (B) measured in HEK293 cells transfected with the extracellular loop mutants or wild-type TRPML3 in response to 10-ms voltage steps from a holding potential of +10 mV between −200 and +100 mV in 20-mV incremental steps (normalized by cell capacitance (picofarads (pF)). Constitutively active inwardly rectifying currents were present in TRPML3(E361A)-expressing cells and were not detectable in TRPML3(D371A)-expressing cells. Shown are means ± S.E. Numbers in parentheses indicate the number of experiments. ***, p < 0.0001 (unpaired Student's t test) compared with the wild type. C and D, average inward current densities (C) and steady-state current-voltage plots (D) of whole-cell currents at −80 and −200 mV from experiments similar to the ones described for A and B and normalized to cell capacitance. Shown are means ± S.E. Numbers in parentheses indicate the number of experiments.

Effect of LSS on HEK293 cells expressing TRPML1, TRPML2, or TRPML3. A, steady-state current-voltage plots of whole-cell currents elicited in HEK293 cells transfected with wild-type TRPML1, TRPML2, or TRPML3 in response to 10-ms voltage steps from a holding potential of +10 mV between −200 and +100 mV in 20-mV incremental steps (normalized by cell capacitance (picofarads (pF)). Shown are the currents before and after perfusion with extracellular solution containing 2 mm NaCl, 150 mm KCl, 0.25 mm CaCl₂, 10 mm HEPES, and 10 mm d-glucose at pH 7.4 (LSS). The major cation in the pipette solution was 150 mm Cs⁺ (pH 7.2). B, average inward current densities at −80 and −200 mV from the experiments in A and normalized by cell capacitance. Shown are means ± S.E. Numbers in parentheses indicate the number of experiments. ***, p < 0.0001; *, p < 0.01 (unpaired Student's t test). C, expression of the three TRPML channels in HEK293 cells. Shown are representative confocal micrographs of HEK293 cells overexpressing C-terminally YFP-fused constructs of TRPML1, TRPML2, and TRPML3 (yellow). Significant amounts of TRPML2 and TRPML3 were associated with the plasma membrane, which is visualized with Cy5-conjugated anti-pan-cadherin antibody (pan-Cad-Cy5; red; Abcam). Scale bar = 10 μm.

Extracellular loop mutation increases TRPML3 open probability in high extracellular sodium. A, schematic displaying the estimated positions of all negatively charged amino acids in the extracellular loops, as well as the varitint-waddler mutations A419P and I362T and the H283A mutation previously shown to result in constitutive TRPML3 activity. Color-coded scissors represent the positions where TRPML3 amino acid sequences were excised to generate TRPML3 mutants with smaller first extracellular loops. B, shown are base lines of E361A mutant and wild-type TRPML3-expressing HEK293 cells as well as non-transfected control cells (NT) of a representative fura-2 calcium imaging experiment. C, effect of the substitution of negatively charged amino acids in the first and second extracellular loops with alanine on [Ca²⁺]_i. Shown are means ± S.E. Numbers in parentheses indicate the number of experiments. All experiments are means of at least 10–15 cells each. For background subtraction, non-transfected control cells on the same coverslip were used. ***, p < 0.0001; **, p < 0.001; *, p < 0.01 (unpaired Student's t test). D, TRPML1, TRPML2, and TRPML3 species protein sequence alignment of the region surrounding Glu-361, which is indicated with a blue arrow. Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Pt, Pan troglodytes; Pa, Pongo abelii; Nl, Nomascus leucogenys; Mmu, Macaca mulatta; Cj, Callithrix jacchus; Bt, Bos taurus; Ec, Equus caballus; La, Loxodonta africana; Cf, Canis familiaris; Cg, Cricetulus griseus; Am, Ailuropoda melanoleuca; Xl, Xenopus laevis.