Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel

Abstract

The activities of organellar ion channels are often regulated by Ca²⁺ and H⁺, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca²⁺/pH regulation of TRPML1, a Ca²⁺-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EM analyses confirmed that this architecture occurs in the full-length channel. Structure-function studies demonstrated that Ca²⁺ and H⁺ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.

Figure 1

Dual regulation of TRPML1 by Ca²⁺ and pH. (a) Families of TRPML1^vp currents at pH 7.4 and the indicated concentrations of extracellular Ca²⁺. (b) Current-voltage relationship of the currents in a at the indicated Ca²⁺ concentrations. NDF: nominal divalent cation free. (c) Dose-response relationships of Ca²⁺ inhibition of TRPML1^vp at pH 7.4 and 4.6, at a potential of -80 mV. Error bars represent SEM. Number of recordings is shown in parentheses. Solid curves are fits to the Hill equation. (d) Time course of TRPML1^vp currents at the indicated pH, with 1 mM Ca²⁺ and either Na⁺ or NMDG⁺ as the charge carrier.

Figure 2

Crystal structure of the TRPML1 I-II linker. (a, b) Ribbon representation a and electrostatic potential surface representation b of the I-II linker structure at pH 6.0. Upper panels, top down views from the extracellular/luminal side of the membrane. Lower panels, side views parallel to the membrane. (c) Stereo top down view of a protomer. (d) Stereo side view of the luminal pore-loop. The front subunit is removed for clarity.

Figure 3

Verification of the I-II linker structure in the full-length protein. (a) Location of three pairs of amino acids in the I-II linker structure, with distances between the C_β atoms indicated. Residue without or with parentheses correspond to human and C. elegans amino acids, respectively. (b) SDS-PAGE of full-length WT and mutant TRPML1 proteins under non-reducing (upper panel) and reducing (lower panel) conditions. The lanes are: 1 (WT), 2 (D104C/T293C), 3 (D104C/Y136C), 4 (D104C/D137C), 5 (H111C/T293C), 6 (H111C/Y136C), 7 (H111C/D137C), 8 (D158C/T293C), 9 (D158C/Y136C), 10 (D158C/D137C). (c,d) Cryo-EM structure of the I-II linker in full-length TRPML1, without c or with d, superposition of the I-II linker crystal structure. Upper panels, top down views from the extracellular/luminal side of the membrane. Lower panels, side views parallel to the membrane.

Figure 4

I-II linker intersubunit interactions contribute to TRPML1 assembly. (a) The I-II linker intersubunit interface. L144 and R146 are boxed in red, and their interacting partners are indicated. (b) Native gel electrophoresis of WT and mutant I-II linker proteins (upper panel) or full-length proteins (lower panel). (c) Left, families of currents of the indicated channels at pH 7.4 or pH 4.6. Right, average current amplitude at -80 mV of the indicated channels at pH 7.4 or pH 4.6. Number of recordings is shown in parentheses. Error bars represent SEM. (d) Confocal images of HeLa cells expressing the indicated GFP-tagged channels. Red indicates LysoTracker-labeled lysosomes.

Figure 5

Effect of MLIV-causing mutations. (a) Location of three MLIV-causing missense mutations in the I-II linker structure. (b) Circular dichroism spectra of WT and mutant I-II linker proteins. (c) Native gel electrophoresis of WT and mutant full-length proteins. (d) Fluorescence-detection size-exclusion chromatography profiles of the indicated GFP-tagged proteins. (e) Confocal images of HeLa cells expressing the indicated GFP-tagged channels. Red indicates LysoTracker-labeled lysosomes. The scale bar represents 5 μm. (f) Average current density at -80 mV of the indicated channels at pH 7.4. Number of recordings is indicated in above the bars. Error bars represent SEM.

Figure 6

Effect of luminal pore aspartate mutations. (a, b) Dose-response relationships of Ca²⁺ inhibition of the indicated channels at pH 7.4, a, and 4.6, b, at a potential of -80 mV. Error bars represent SEM. Number of recordings is shown in parentheses. Solid curves are fits to the Hill equation. (c-f) Time course of intracellular Ca²⁺ increase at pH 7.4 or pH 4.6, following a step increase of extracellular Ca²⁺ from 0 to 3 mM in cells expressing the indicated channels. Error bars represent SEM. Number of recordings is shown in parentheses.

Figure 7

Structures of the TRPML1 I-II linker at different pH and model of Ca²⁺/pH regulation. (a) Superposition of the I-II linker crystal structure obtained at pH 4.5, 6.0 and 7.5, viewed from the extracellular/luminal side of the membrane. (b) Side view of the superimposed luminal pore-loop structures obtained at pH 4.5, 6.0, and 7.5. (c) Model of Ca²⁺/pH dual regulation of TRPML1. Two of the four channels are schematized, with yellow representing the ED and green the TMD. The luminal pore aspartates are illustrated in red or pink, depending on luminal pH, and in yellow when mutated to glutamine. Light blue dots represent free Ca²⁺ ions, and the dark blue dot represents a bound Ca²⁺ ion. See text for details.