Rotarod experiments.

<p>The average latencies to fall ± SEM (in sec) are shown for 3-month-old <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 8) and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 8) mice. The experiment was executed over a time range of 6 days. The day 6 experiment was performed in the dark to exclude compensation via visual cues. The difference between genotypes was not statistically significant at any given time point (p>0.05, one-way ANOVA, followed by Tukey's post test).</p

Targeting strategy for disruption of the <i>Trpml3</i> gene.

<p>A, Transmembrane-spanning domains are depicted as grey bars and numbered from 1–6. The orange frame indicates the part of the TRPML3 protein that is encoded by exon 11, which will be deleted (pore loop and TM6). Exons are shown as black and orange bars on the schematic genomic map below. B, Shown are the targeting vector and the targeted allele after homologous recombination. The blue bar represents the PGK promoter-driven <i>neo^R</i> expression cassette, which was used for positive selection. The DTA cassette, used for negative selection, is shown in green. The black and red arrowheads symbolize position and orientation of <i>FRT</i> and <i>loxP</i> sites. C, Targeted allele after Flp site-specific recombination in ES cells, resulting in excision of <i>neo^R</i> cassette, and leaving one FRT site behind. D, Excision of exon 11 using Cre site-specific recombinase, resulting in disruption of <i>Trpml3</i> gene.</p

Molecular and functional assessment of mutant TRPML3(Δ exon11) channels.

<p>A, Shown are representative cells expressing the respective murine (m) constructs of wild-type TRPML3 or mutant TRPML3(Δ exon11), C-terminally fused to yellow fluorescent protein, 24 h after transfection. Scale bar = 10 µm. B, Ca²⁺ imaging results showing relative [Ca²⁺]_i increases after application of TRPML3 activator SN-2 in HEK293 cells expressing wild-type TRPML3 or mutant TRPML3(Δ exon11). Shown are mean values ± SEM (numbers in parentheses are the numbers of independent experiments with 10–20 cells each). Statistical comparisons of means were made using Student's <i>t</i> test (unpaired); ***p<0.0001.</p

ABR measurements.

<p>A, Graph shows representative ABR waveforms of 3-week-old <i>Hprt^Cre/+; Trpml3^+/+</i> and <i>Hprt^Cre/+; Trpml3 ^Δ/Δ</i> mice in response to a click stimulus. ABRs were recorded at sound stimulation intensities of 25–70 dB. ABR waves I–V are indicated above the peaks. Red arrow highlights the hearing threshold, which is at 35 dB in this representative example pair. B, Shown are ABR thresholds (mean values ± SEM) to click, 8-, 16-, and 32 kHz stimuli of <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 6), <i>Hprt^Cre/+;Trpml3^Δ/+</i> (n = 6), and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 6) and C, of <i>Math1-CreER^Cre/+;Trpml3^+/+</i> (n = 3), <i>Math1-CreER^Cre/+;Trpml3^Δ/+</i> (n = 3), and <i>Math1-CreER^Cre/+;Trpml3 ^Δ/Δ</i> (n = 3), respectively. Statistical comparisons of means of different genotypes were made using one-way ANOVA followed by Tukey's post test; p>0.05. D, ABR threshold shifts of 3-month-old <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 7) and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 7) 1 week after the acoustic overexposure of 125 dB SPL at 4 kHz for 4 hr. Shown are mean values ± SEM. No significant differences were observed between genotypes (p>0.05, one-way ANOVA, followed by Tukey's post test).</p

Effects of dominant-negative and inactive isoforms of TRPML3 and TRPV5.

<p>(A) Representative whole cell currents shown were obtained from cells coexpressing TRPML3^D458K and TRPV5 (ML3^D458K/ V5), TRPV5^D535K and TRPV5 (V5^D535K/ V5), TRPML3 and TRPV5^D535K (ML3/V5^D535K), TRPV5^D550K and TRPV5 (V5^D550K/ V5), and TRPML3 and TRPV5^D550K (ML3/V5^D550K) under the four conditions indicated. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Average inward current densities at −150 mV of the various TRPML3 and TRPV5 channel pairs under the four different conditions as indicated. Bar diagrams represent mean±SD, numbers in parentheses are the number of cells analyzed. (<b>C</b>) Pharmacological properties of HEK293 cells expressing TRPV5 and TRPML3/TRPV5^D550K (ML3/V5^D550K). Representative traces were recorded during voltage steps from 0 mV to −150 mV under condition 3 before (blue) and after (cyan) application of 100 µM ruthenium red (RR). (<b>D</b>) Quantitative analysis of the percentage of inhibition at −150 mV (mean±SD, n = parenthesized).</p

Pharmacological properties of HEK293 cells expressing TRPV5, TRPML3, and both proteins.

<p>(A–C) Representative traces shown from transfected HEK293 cells expressing TRPV5, TRPML3, and TRPV5/TRPML3 in response to step polarization (from 0 mV to –150 mV) before (black lines) and after 1 mM dihydrostreptomycin (DHSM) (green lines), 100 µM Ruthenium Red (RR) (cyan lines) and 300 µM gadolinium chloride (Gd³⁺) (purple lines). (D) Quantitative analysis of the percentage of inhibition at −150 mV (mean±SD, n = 4–8).</p

Elovl4 5-bp deletion does not accelerate cone photoreceptor degeneration in an all-cone mouse

<div><p>Mutations in the elongation of very long chain fatty acid 4 (<i>ELOVL4</i>) gene cause Stargardt macular dystrophy 3 (STGD3), a rare, juvenile-onset, autosomal dominant form of macular degeneration. Although several mouse models have already been generated to investigate the link between the three identified disease-causing mutations in the <i>ELOVL4</i> gene, none of these models recapitulates the early-onset cone photoreceptor cell death observed in the macula of STGD3 patients. To address this specifically, we investigated the effect of mutant ELOVL4 in a mouse model with an all-cone retina. Hence, we bred mice carrying the heterozygously mutated <i>Elovl4</i> gene on the <i>R91W;Nrl</i>^<i>-/-</i> all-cone background and analyzed the retinal lipid composition, morphology, and function over the course of 1 year. We observed a reduction of total phosphatidylcholine-containing very long chain-polyunsaturated fatty acids (PC-VLC-PUFAs) by 39% in the <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i> mice already at 6 weeks of age with a pronounced decline of the longest forms of PC-VLC-PUFAs. Total levels of shorter-chain fatty acids (< C26) remained unaffected. However, this reduction in PC-VLC-PUFA content in the all-cone retina had no impact on morphology or function and did not accelerate retinal degeneration in the <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i> mice. Taken together, mutations in the <i>ELOVL4</i> gene lead to cone degeneration in humans, whereas mouse models expressing the mutant <i>Elovl4</i> show predominant rod degeneration. The lack of a phenotype in the all-cone retina expressing the mutant form of the protein supports the view that aberrant function of ELOVL4 is especially detrimental for rods in mice and suggests a more subtle role of VLC-PUFAs for cone maintenance and survival.</p></div

Stoichiometric analyses of TRPML3/TRPV5 currents.

<p>(A–D) Traces show currents at −150 mV recorded from HEK293 cells expressing TRPV5/TRPML3, in the presence of extracellular solutions containing 140 mM Na⁺ (condition 1, black), 150 mM K⁺ (condition 2, red), 140 mM Na⁺, 0.1 mM EGTA (condition 3, blue), and 150 mM K⁺, 0.1 mM EGTA (condition 4, pink). We hypothesized that the current recorded under condition 1 represents the novel conductance, the current under condition 2 consists of the novel conductance and TRPML3, the current under condition 3 is composed of the novel conductance and TRPV5, and finally, under condition 4, we expected that the recorded current consists of all three conductances. (E–F) Subtraction of A from B (in E) which represents pure TRPML3; subtraction of A from C (in F) which results in pure TRPV5. (G–I) HEK293 cells were transfected with TRPML3 and TRPV5 expression plasmids either pure (0% and 100%) and at molar ratios 1∶10 (10%), 1∶5 (20%), 2∶3 (40%), 1∶1 (50%), 3∶2 (60%), 5∶1 (80%) and 10∶1 (90%), respectively. Currents were elicited at −150 mV under conditions 1–3 and individual conductances for TRPML3 and TRPV5 were extracted as described in (E–F). Shown is the average fraction of novel current (G, black circles), TRPML3 (H, red triangles), and TRPV5 (I, blue triangles) is plotted against the fraction of TRPML3 over TRPV5 (G,H) and TRPV5 over TRPML3 (I) (n = 5–11).</p

Whole-cell currents of cells expressing TRPV5, TRPML3, and both proteins in different ionic conditions.

<p>(A) Traces show representative currents obtained from non-transfected HEK293 cells, and cells expressing TRPV5, TRPML3, and TRPV5/TRPML3, in the presence of extracellular solutions starting with 140 mM Na⁺ (condition 1, black), and successively switched to 150 mM K⁺ (condition 2, red), 140 mM Na⁺, 0.1 mM EGTA (condition 3, blue), followed by 150 mM K⁺, 0.1 mM EGTA (condition 4, pink), and a final set of measurements conducted in condition 1. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Mean values (±SD) of average inward current densities of TRPV5 (n = 10), TRPML3 (n = 12), and TRPV5/TRPML3 (n = 10) plotted against voltage in the presence of 140 Na⁺ (black circles), 150 K⁺ (red triangles), 140 Na⁺, 0.1 EGTA (blue triangles), and 150 K⁺, 0.1 EGTA (magenta diamonds), respectively. (C) Average inward current densities at −90 mV of HEK293 cells expressing the different channels in the four different conditions as indicated. Bar diagrams represent mean ± SD, numbers in parentheses are the number of cells analyzed. ** p<0.001 and * p<0.01, Student’s t-test, unpaired.</p

Interaction between TRPML channels and TRPV5/6.

<p>(A) Phylogenetic tree, based on full length sequence comparisons of human TRP channel proteins. (B) Phylogenetic tree, based on sequence comparisons of the pore forming domains of human TRP channel proteins. (C) Fluorescence energy resonance transfer (FRET) experiments showing representative FRET efficiencies among TRPML3, TRPV5, and TRPV6 channels. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. (D) Average FRET efficiencies reported as mean values± SEM (n = parenthesized). Shown are efficiencies for TRPML, TRPV5, and TRPV6 channel homo- and heteromers, as well as PKD2, TRPC6, TRPV2, and TRPA1 as controls. YFP and CFP indicate the fluorescent tag, which was carboxyl-terminal in all cases. Control indicates a pcDNA3.1-based expression vector for the corresponding fluorescent protein (YFP in the example shown). ***p<0.0001 and **p<0.001, Student’s t-test, unpaired, comparison with TRPC6/TRPML3 coexpression as negative control.</p