The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration

Abstract

Varitint-waddler (Va and Va(J)) mice are deaf and have vestibular impairment, with inner ear defects that include the degeneration and loss of sensory hair cells. The semidominant Va mutation results in an alanine-to-proline substitution at residue 419 (A419P) of the presumed ion channel TRPML3. Another allele, Va(J), has the A419P mutation in addition to an I362T mutation. We found that hair cells, marginal cells of stria vascularis, and other cells lining the cochlear and vestibular endolymphatic compartments express TRPML3. When heterologously expressed in LLC-PK1-CL4 epithelial cells, a culture model for hair cells, TRPML3 accumulated in lysosomes and in espin-enlarged microvilli that resemble stereocilia. We also demonstrated that wild-type TRPML3 forms channels that are blocked by Gd(3+), have a conductance of 50-70 pS and, like many other TRP channels, open at very positive potentials and thus rectify outwardly. In addition to this outward current, TRPML3(419P) and (I362T+A419P) generated a constitutive inwardly rectifying current that suggests a sensitivity to hyperpolarizing negative potentials and that depolarized the cells. Cells expressing TRPML3(A419P) or (I362T+A419P), but not wild-type TRPML3, died and were extruded from the epithelium in a manner reminiscent of degenerating hair cells in Va mice. The increased open probability of TRPML3(A419P) and (I362T+A419P) at physiological potentials likely underlies hair cell degeneration and deafness in Va and Va(J) mice.

Fig. 1.

Expression of TRPML3 mRNA in hair cells, stria vascularis, Reissner's membrane, and other cells lining the scala media of the cochlea and the endolymphatic spaces of the vestibule. In situ hybridization with antisense probes to the 3′ half of TRPML3 mRNA (a, e, i, l, n, and p), control with sense probes (c, g, and k) and nuclear DAPI label (b, d, f, h, j, m, o, q). (a–d) Crista ampullaris: TRPML3 in vestibular hair cells (CA HC) and adjacent transient epithelium and dark cells (TE+DC). (e–h) Sacculus: TRPML3 in vestibular hair cells (HC) and in cells of the vestibular membrane. (i–k) Cochlear scala media: TRPML3 in stria vascularis (SV), Reissner's membrane (RM), spiral limbus (SL), hair cells of the organ of Corti (OC), and support cells of the marginally located lesser epithelial ridge (H & C, Hensen's and Claudius' cells). (l and m) Marginal cells of the stria vascularis express TRPML3 mRNA. (n–q) Cochlear inner and outer hair cells (IHC and OHC) express TRPML3 mRNA. All sections are from P5 mice.

Fig. 2.

Localization of TRPML3::GFP in LLC-PK1-CL4 cells. (a–d) CL4 cells coexpressing TRPML3::GFP (green) and espin, which results in enlarged microvilli that resemble hair cell stereocilia. Phalloidin (red) labels F-actin. (a–c) Optical section through apical end of cell demonstrating localization of (a) TRPML3 to (b) enlarged microvilli (merged in c). (d) Optical section through the cell body demonstrating localization of TRPML3 to intracellular vesicles.

Fig. 3.

Extrusion and death of LLC-PK1-CL4 cells expressing TRPML3(A419P). (a and c) Cells expressing wild-type TRPML3 remain in the epithelium. (b and d) Cells expressing TRPML3(A419P) round up and become extruded from the epithelium. (a and b) Top views. (c and d) Orthogonal (x and z) sections. (f and h) Cells expressing TRPML3(A419P) take up the cell death marker propidium iodide (PI), whereas (e and g) cells expressing wild-type TRPML3 do not. The optical section in f cuts through the transfected cells, above the monolayer of untransfected cells. (e and f) Top views. (g and h) Lateral 3D projections. There was a 1:1 correlation between epithelial extrusion and PI uptake. DAPI labels nuclei and phalloidin F-actin. (i and j) TRPML3 currents in CL4 cells demonstrate channel expression in their plasma membrane. (i) Current–voltage (IV) plot of CL4 cells expressing TRPML3::GFP. (j) IV plot of CL4 cells expressing TRPML3(A419P)::GFP or TRPML3(I362T+A419P)::GFP, both of which add an inwardly rectifying current. Identical currents were obtained in HEK 293 cells expressing these constructs or untagged ones (Fig. 4). The currents found in CL4 cells expressing TRPML3 (n = 8), TRPML3(A419P) (n = 8), or TRPML3(I362T+A419P) (n = 4) were not detected in untransfected CL4 cells (n = 6).

Fig. 4.

The degeneration causing A419P mutation results in depolarization-sensitive opening of TRPML3 channels at negative voltages. (a) IV plot of cells expressing TRPML3, TRPML3(A419P), or TRPML3(I362T+A419P) shows outward currents at positive potentials in wild type and mutants and an additional inward current at negative potentials in mutants. (b) Apparent open probabilities (G/Gmax) at various voltages. The A419P mutation (with or without I362T) does not affect the increase of channel opening with membrane potential increases in the positive voltage range, but induces opening at hyperpolarized negative voltages. Gmax is defined as the steady-state conductance of TRPML3(A419P) or (I362T+A419P) at −120 mV. G is the steady-state conductance at the plotted membrane potentials. (c) Whole-cell currents elicited by voltage steps from −120 mV to up to +120 mV. A use-dependent increase in current at −120 mV was detected in cells expressing mutant or wild-type TRPML3. (d) IV plots cells expressing TRPML3(A419P) demonstrating that inward rectification is not affected by the presence or absence of external Ca²⁺. External solution with 0 mM Ca²⁺ contained 4 mM EGTA. Both external solutions lacked Mg²⁺. (e) IV plots of cells expressing TRPML3(A419P) showing block by Gd³⁺. (Inset) Dose–responses indicate that block is independent of voltage and unaffected by the A419P mutation [IC₅₀ of TRPML3(A419P) at −80 mV is 20.6 ± 2.0 μM; IC₅₀ of TRPML3(A419P) at +80 mV is 24.6 ± 6.7 μM; IC₅₀ of wild-type TRPML3 at +80 mV is 14.8 ± 2.5 μM; n = 3 or 4]. (f) IV plots of cells expressing TRPML3(A419P) in the presence of external solutions containing either 140 mM NaCl, 140 mM KCl, or 20 mM CaCl₂ plus 98 mM NMDGCl. The reversal potential was near 0 mV with Na⁺ or K⁺, but more positive with Ca²⁺ (there were no inward currents with 140 mM NMDGCl externally, from which we deduce that neither Cl⁻ nor NMDG⁺ permeate). Cells were HEK 293T.

Fig. 5.

Single-channel currents of TRPML3 and TRPML3(A419P). (a) Voltage ramps of outside-out membrane patches from cells expressing TRPML3 reveal preferential channel opening at positive potentials and block by Gd³⁺. (b) Voltage steps of an outside-out membrane patch from a cell expressing TRPML3(A419P) reveal increased channel opening at negative potentials. (c) IV plots of single-channel current amplitudes show the same single-channel conductance for TRPML3 and TRPML3(A419P).