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Endolysosomal Cation Channels of the Transient Receptor Potential Superfamily

Physiology, Pharmacology, and Mouse Models

Professorial Dissertation 2014 44 Pages

Medicine - Pharmacology

Excerpt

Table of contents

1 List of original publications selected for this „Habilitationsschrift“

2 Introduction

3 Analysis of the TRPML3 varitint-waddler mouse mutants

4 Development of small molecule agonists for TRPML channels

5 Functional investigation of MLIV causing TRPML1 mutants

6 Novel TRPML channel interaction partners

7 Functional analysis of the TPC2-/- knockout mouse

8 Novel TPC channel interaction partners

9 Summary

10 References

11 Curiculum vitae

12 Complete list of original publications

13 Personal contributions to the selected original publications

14 Acknowledgement

1 List of original publications selected for this „Habilitationsschrift“

I. Grimm, C., Cuajungco, M.P., van Aken, A.F.J., Schnee, M., Jörs, S., Kros, C.J., Ricci, A.J., and Heller, S. (2007) A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse. PNAS, 104:19583-19588

II. Grimm, C.*, Jörs, S.*, Heller, S. (2009) Life and death of sensory hair cells expressing constitutively active TRPML3. J. Biol. Chem., 284:13823-13831

III. Grimm, C.*, Jörs, S.*, Saldanha, S.A.*, Obukhov, A.G., Pan, B., Oshima, K., Cuajungco, M.P., Chase, P., Hodder, P., Heller, S. (2010) Small molecule activators of TRPML3, Chem. & Biol. (Cell Press), 17:135-148

IV. Aneiros, E., Cao, L., Papakosta, M., Stevens, E.B., Phillips, S.C., Grimm, C.# (2011) Biophysical and molecular basis of TRPV1 proton gating. EMBO J., 30:994-1002

V. Grimm, C.#, Jörs, S., Gao, Z., Obukhov, A.G., Heller, S. (2012) Constitutive activity of TRPML2 and TRPML3 channels versus activation by low extracellular sodium and small molecules, J. Biol. Chem., 287:22701-22708

VI. Chen, C.-C., Keller, M., Hess, M., Schiffmann, R., Urban, N., Wolfgardt, A., Schaefer, M., Bracher, F., Biel, M., Wahl-Schott, C., Grimm, C.# (2014) A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV. Nature Commun., 5:4681. doi: 10.1038/ncomms5681

VII. Cuajungco, M.P., Basilio, L.C., Silva, J., Hart, T., Tringali, J., Chen, C.-C., Biel, M., Grimm, C.# (2014) Cellular zinc levels are modulated by TRPML1-TMEM163 interaction. Traffic, doi: 10.1111/tra.12205. [Epub ahead of print]

VIII. Grimm, C., Holdt, L.M., Chen, C.-C., Hassan, S., Müller, C., Jörs, S., Cuny, H., Kissing, S., Schröder, B., Butz, E., Northoff, B., Castonguay, J., Luber, C.A., Moser, M., Spahn, S., Lüllmann-Rauch, R., Fendel, C., Klugbauer, N., Griesbeck, O., Haas, A., Mann, M., Bracher, F., Teupser, D., Saftig, P., Biel, M., Wahl-Schott, C. (2014) High susceptibility to fatty liver disease in two-pore channel 2-deficient mice. Nature Commun., 5:4699. doi: 10.1038/ncomms5699

* authors contributed equally; # corresponding author

2 Introduction

TRP (transient receptor potential) channels are mostly non-selective cation channels involved in almost all kinds of our sensory modalities, from vision and hearing, mechano-, osmo-, and taste to pain sensation (Gees et al., 2010; Nilius and Owsianik, 2011). There are 28 mammalian TRP channels known today (Figure 1) and at least 13 different hereditary diseases are associated with TRP channel dysfunction in humans. The TRP channels TRPML1, TRPML2 and TRPML3, also called mucolipins or MCOLN1-3 are, like most TRP channels, non-selective cation channels permeable for sodium, potassium, and calcium. TRPML1 has also been shown to be permeable for heavy metals such as iron or zinc (Dong et al., 2008).

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Figure 1. Shown is a phylogenic analysis of human TRPs and two-pore channels (TPCs) based on the amino acid sequences in the pore region (TMD5 – TMD6). The corresponding alignment was generated using DNAMAN software, the phylogenic tree was generated using NJPlot software.

A special feature of TRPML channels is their intracellular expression (Figure 2). They mostly reside in membranes of organelles of the endolysosomal system such as early and late endosomes, recycling endosomes, lysosomes, or lysosome-related organelles (Abe and Puertollano, 2011).

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Figure 2. Cartoon illustrating putative subcellular localizations of endolysosomal cation channels of the TRP superfamily and endolysosomal degradation and recycling pathways. In the degradation pathway (endo/lysosomal axis) EGF/EGFR and LDL are degraded. In the recycling pathway LDLR and Tf/TfR are recycled. RE = recycling endosome.

Mutations in TRPML1 (MCOLN1) are causative for mucolipidosis type IV (MLIV), an autosomal recessive lysosomal storage disorder characterized by severe psychomotor retardation and ophthalmologic abnormalities, including corneal opacity, retinal degeneration, and strabismus. Most patients are unable to speak or walk independently. ML IV patients also have abnormalities in white matter, indicative of a developmental brain disorder (Slaugenhaupt, 1999; Bargal et al., 2000; Altarescu et al., 2002; Bach et al., 2005). Mutations in TRPML3 (varitint-waddler mutations) cause deafness and circling behavior in mice (DiPalma et al., 2002; Cuajungco and Samie, 2008). No disease causing mutations are known for the Trpml2 gene. While the human TRPML1 mutations that are associated with ML IV (Figure 3) generally cause a loss-of-function, the TRPML3 varitint-waddler mutations (Figure 3) have been found to be gain-of-function mutations. Correspondingly, the knockout mouse phenotype of TRPML1 resembles very much the human clinical ML IV phenotype (Chandra et al., 2011; Venugopal et al., 2007). The varitint-waddler gain-of-function mutations, rendering TRPML3 constitutively active were found to cause massive calcium overload in inner ear hair cells and skin melanocytes and consequently cell death (Kim et al., 2007; Grimm et al., 2007; Xu et al., 2007; Nagata et al., 2008; Dong et al., 2009; Grimm et al., 2009; Samie et al., 2009). Quite in contrast to the varitint-waddler mice, TRPML3 knockout mice do not show a hearing or pigmentation phenotype (Jörs et al., 2010; Castiglioni et al., 2011).

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Figure 3. Cartoon illustrating the putative topologies of TRPML and TPC channels. LTS = lysosomal targeting sequence, LLBCS = legume lectin β-chain signature, PRD = proline rich domain. Shown in red are mutations in TRPML1 causing MLIV in humans, mutations in TRPML3 causing the varitint-waddler phenotype in mice, and polymorphisms in TPC2 responsible for a blond versus brown phenotype in humans.

Further investigations will be necessary to assess the physiological role of TRPML3 in the inner ear. This role may well be a more global function affecting various other TRPML3 expressing cells and tissues ranging from kidney and liver to skin melanocytes (Martina et al., 2009; Lelouvier and Puertollano, 2011).

A second group of recently emerged endolysosomal cation channels are the two-pore channels (TPCs) which are distantly related to TRP and in particular TRPML channels (Figure 1). They and comprise two members in mammals: TPC1 and TPC2. TPC1 is mainly present in the proximal endosomal system, while TPC2 is predominantly found on late endosomes and lysosomes (Figure 2). While no disease causing mutations have been identified for TPCs, polymorphisms in TPC2 (M484L and G734E; Figure 3) have been postulated to play a role in pigmentation (blond versus brown phenotype; Sulem et al., 2008). The activation mechanism of TPCs is complex and has been suggested to involve the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and the endolysosomal membrane lipidphosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) (Calcraft et al., 2009; Brailoiu et al., 2009; Zong et al., 2009; Brailoiu et al., 2010, Ruas et al., 2010; Schieder et al., 2010a, 2010b; Wang et al., 2012; Cang et al., 2013)

Endosomes and lysosomes are important for the breakdown of proteins, lipids, and other molecules and have been found to be implicated not only in endolysosomal storage disorders (LSDs) such as mucolipidoses or sphingolipidoses but also in several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Morgan et al., 2011; Saftig and Klumperman, 2009), pigmentation disorders (Dell’Angelica et al., 2000), infectious diseases (McGourty et al., 2012; Nakamura et al., 2014; Jae et al., 2014), and cancer (Saftig and Sandhoff, 2013).

Endolysosomal organelles contain a plethora of different receptors, ion channels and transporters that are crucial for their normal functions (Schröder et al., 2010). Ion homeostasis, regulation of distinct proton concentrations in different organelles and regulation of heavy metal concentrations such as copper, zinc or iron are essential not only for lysosomes and endosomes but also for lysosome-related organelles (LROs) such as melanosomes, lytic granules, or platelet-dense granules. It has been known for a while that acidification of lysosomes and LROs is primarily regulated by ATP-driven proton pumps (vacuolar H+-ATPases). However, little is known about the fine regulation of luminal pH in endosomes, lysosomes, or lysosome related organelles, as well as the regulation of intraluminal cation concentrations such as calcium, sodium and potassium, or heavy metals (Luzio et al., 2007; Dong et al., 2008; Lloyd-Evans and Platt, 2011; Scott and Gruenberg, 2011; Mindell, 2012). Further unclear is how the different endolysosomal fusion processes are regulated and how calcium that appears to be necessary for such fusion processes is provided (Luzio et al., 2010). TRPML channels and TPCs are likely to play decisive roles in the regulation of the above mentioned processes (Shen et al., 2012; Wang et al., 2012). By analyzing knockout mouse models, activation mechanisms using small molecule agonists, or novel protein-protein interaction partners the physiological role and function of TRPML channels and TPCs in such processes have recently been further elucidated as will be outlined in this thesis. The identification of small molecule chemical activators for TRPML channels has opened up the possibility to study TRPML mutant and wild-type isoforms both in vitro and in vivo in more detail. Ultimately, these drugs or their derivatives may be used to rescue disease symptoms in mucolipidosis type IV patients or to treat other disorders and diseases which would benefit from activation or blockage of either TRPML or TPC channels.

3 Analysis of the TRPML3 varitint-waddler mouse mutants

Original publications I and II

Varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3, are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. In publication I we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current and we hypothesized that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels are not affected. We concluded that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. Our study provided the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.

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Figure 4. (a) Images of WT and Va mice. The Va mouse shows almost complete loss of the WT fur color. (b-c) Representative ABR measurements of a WT and a heterozygous Va mouse. (d-e) Elevated intracellular calcium and sodium levels. Shown are results from imaging experiments with HEK293 cells overexpressing human (Hs) and murine (Mm) TRPML3 and the respective varitint-waddler mutant isoforms, A419P (Va) or I362T/A419P (VaJ). All experiments were performed 10-15 h after transfection (mean ± SEM; n = number of experiments. Transfected cells were identified by YFP fluorescence of the expressed proteins. In e, after incubation in 2 mM EGTA for 5 min, the external buffer solution was switched to 2 mM calcium. (f-g) Patch-clamp analysis of TRPML3 WT and mutant variants in HEK293 cells. Shown in f are currents elicited from WT, A419P, and I362/A419P mutants in response to 5-ms voltage steps from a holding potential of -50 mV between -200 mV and +100 mV in 20-mV incremental steps. Black bars indicate zero current line. Shown in g are steady-state current–voltage plots from f normalized to cell capacitance. Lower panel in g: Same plot, but normalized to maximal current elicited at -200 mV to demonstrate similarity in responses. (h) Quantification of the number of transfected HEK293 cells that bound Cy5-conjugated annexin V, an indication of early signs of apoptosis. Shown are time points after transfection with expression vectors for the mutants indicated. We found no annexin V-positive cells expressing WT TRPML3 or the I362T mutant 25 h after transfection. (i) Relative calcium levels of HEK293 cells overexpressing TRPML3(A419P) and analogous mutations in other related TRP channels such as TRPML1, TRPML2, TRPV5, and TRPV6 as well as only distantly related TRP channels such as TRPV2, TRPM2, and TRPC6. The relative calcium levels of the respective WT isoforms are shown in black. (j) Cartoon of the murine TRPML3 channel protein showing the relative locations of the varitint-waddler muations.

Since the varitint-waddler mutation A419P rendered TRPML3 constitutively active, resulting in massive cationic overload, particularly in sustained influx of Na+ and Ca2+, we were intrigued by the fact that hair cells expressing the TRPML3(A419P) isoform are able to cope with the high intracellular Ca2+ for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal well with high Ca2+ loads due to the exceptionally high abundance of plasma membrane calcium ATPases (PMCAs). Thus, in publication II we show that PMCA2 significantly reduces the intracellular Ca2+ increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca2+ loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice completely lacked auditory brainstem responses, while residual responses were detectable in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca2+ loading, which implies that Ca2+ loading is an unlikely primary cause of hair cell death in ototoxic stress situations.

4 Development of small molecule agonists for TRPML channels

Original publications III and V

With the aim to identify and further develop small molecule activators for TRPML channels, we initially conducted a high-throughput screen (publication III) for activators of the TRPML3 channel. Stable TRPML3 HEK293 cell lines were generated and screened using Fluo-based calcium imaging methods. Briefly, a library of 217,969 compounds was tested and candidate compounds were subsequently validated using Fura-2 based single cell ratiometric calcium imaging and whole-cell patch-clamp methods (publication III). Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. The nine chemotypes identified were (pyrazol-5-yl)isoxazole-benzenesulfonamides (SF-1), secondary arylsulfonamides (SF-2), tertiary arylsulfonamides (SF-3), sulfonylarylpiperazines (SF-4), 1-(2,2,4-trimethylquinolinyl)-alkylones (SF-5), spirobenzoannulene-arylpyrazolones (SF-6), t-butyl-3-methyl-4-(arylsulfonyl)-pyrazol-5-ols (SF-7), arylsulfonylpyridin-2-ones (SF-8), and t-butyl-3methylfuran-2-carboxamides (SF-9). For further evaluation at least one representative of each chemical scaffold was selected as well as two singletons, SN-1 and SN-2 (publication III). Criteria for this selection were low EC50 values for TRPML3 activation and no or low activation of the negative control (publication III). The candidate compounds were found to be inactive against other TRP channels tested, including members of the TRPC, TRPV, TRPM, TRPA, and TRPN subfamilies (publication III). Candidate compounds were also found to be inactive against a plethora of other targets (generally more than 500, each; for further information see http://pubchem.ncbi.nlm.nih.gov; AID: 1448, 1525, 1526, 1562, 2719, 1809, and 2694).

We further showed that agonists strongly potentiated TRPML3 activation when exposed to low extracytosolic [Na+]. However, testing compounds on sensory hair cells endogenously expressing TRPML3 revealed absence of activator-responsive channels in the plasma membrane. Likewise, epidermal melanocytes showed only weak or no responses to the compounds when applied to the extracellular medium. These results suggested that in native cells TRPML3 is likely absent or largely absent from plasma membranes and that expression may be limited to endolysosomal compartments in vivo. In publication III and V we further demonstrated that the TRPML3-related cation channels TRPML1 and TRPML2 can be activated by subsets of the small chemical compounds that were previously identified as activators of TRPML3, suggesting similar gating mechanisms for all TRPML channels.

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Details

Pages
44
Year
2014
ISBN (eBook)
9783656849759
ISBN (Book)
9783656849766
File size
4.7 MB
Language
English
Catalog Number
v284634
Institution / College
LMU Munich – Pharmacy/Pharmacology
Grade
Passed
Tags
endolysosomal cation channels transient receptor potential superfamily physiology pharmacology mouse models

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Title: Endolysosomal Cation Channels of the Transient Receptor Potential Superfamily