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. 2015 Jul 7;54(26):4033-41.
doi: 10.1021/acs.biochem.5b00235. Epub 2015 Jun 26.

Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse

Sonya M Hanson  1   2 , Mark S P Sansom  2 , Esther B E Becker  3
Affiliations

Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse

Sonya M Hanson et al. Biochemistry. .

Abstract

A gain-of-function mutation (T635A) in the transient receptor potential (TRP) channel TRPC3 results in abnormal channel gating and causes cerebellar ataxia in the dominant Moonwalker (Mwk) mouse mutant. However, the underlying molecular and structural mechanisms are unclear. Here, we used a combined approach of computational modeling and functional characterization of proposed TRPC3 mutants. Our findings support a mechanism by which the hydrogen bonding capability of threonine 635 plays a significant role in maintaining a stable, closed state channel. This capability is lost in the Mwk mutant, suggesting a structural basis for the disease-causing phenotype in the Mwk mouse.

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Figures

Figure 1
Figure 1
Structural overview and sequence alignments of TRPC3 and the S4–S5 linker region. (A) Schematic of the six-transmembrane TRPC3 channel protein, in which the six transmembrane helices, S1–S6 (numbered cylinders), are preceded by a coiled coil domain (gray box) and an ankyrin repeat domain (black diamonds) and followed by the TRP domain (checkered box), another predicted coiled coil domain (gray box), and a predicted CIRB domain (light gray box). The Mwk mutation (T635A) resides within the S4–S5 linker region and is indicated by a yellow circle. The sequence alignment of the wild-type and mutant mouse TRPC3 S4–S5 linker region is shown below. Figure adapted from ref (6). (B) Sequence alignment of the S4–S5 linker regions of several TRP channels and Kv1.2. The position of the Mwk mutation is highlighted in yellow. Other residues found to have significant effects on channel function when mutated are colored red (see the text for details). (C) Structure of apo TRPV1 (PDB entry 3J5P) with mutants in the S4–S5 linker region found to have significant effects on TRP channel function highlighted with red dots. (D) Sequence conservation diagram of the S4 transmembrane helix and the S4–S5 linker region of Kv and TRP channels showing that the threonine mutated in the Mwk mouse is surprisingly more conserved in Kv channels than in TRP channels. The analysis includes Kv’s 1.x–10.x for a total of 40 sequences analyzed for Kv channels, and all representatives of the TRPC, TRPM, TRPV, TRPA, TRPP, and TRPML subfamilies for a total of 28 sequences analyzed for TRP channels. Figures made using WebLogo and MAFFT sequence alignments of all Kv and TRP channels identified in ref (38).
Figure 2
Figure 2
The gain-of-function Mwk mutation in TRPC3 causes increased cell death and calcium signaling in neuronal cells. (A) Equal amounts of TRPC3 are expressed at the cell surface in the mouse cerebellum. Biotinylated cerebellar slice cultures from wild-type (WT) and Mwk mice were lysed and subjected to pull-down experiments using streptavidin beads, followed by immunoblotting for TRPC3 and actin. Abbreviations: I, input; S, supernatant; B, pellet (biotinylated fraction). (B) Overexpression of Mwk (T635A) but not wild-type (WT) TRPC3 significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test; p < 0.005). V denotes vector control. (C and D) Overexpression of Mwk (T635A) but not wild-type (WT) TRPC3 causes significant nuclear localization of co-expressed GFP-tagged NFAT (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test; p < 0.001). Cells were fixed 24 h after transfection and subjected to indirect immunofluorescence using antibodies against FLAG, GFP, and the DNA dye DAPI. Cells transfected with mutant TRPC3 have a more rounded appearance because of their impending cell death. (E) The in vitro gain-of-function phenotype of Mwk TRPC3 is not likely to be mediated by phosphorylation. Overexpression of the phospho-mimic T635D mutation but also of the control T635N mutation did not induce cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test). (F) Overexpression of the phospho-mimic T635D mutation but also of the control T635N mutation did not cause significant nuclear translocation of GFP-NFAT (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test).
Figure 3
Figure 3
Homology modeling reveals potential for hydrogen bonding. (A) Sequence alignment of mouse TRPC3 to rat TRPV1 and to the S4–S5 linker of the Kv 1.2/2.1 chimera used to create the homology models. A cut of 21 amino acids (indicated by double lines) was made at the end of S3 to facilitate modeling. Transmembrane helices are indicated by black boxes with the corresponding label for each S1–S6 transmembrane helix above. Coloring is according to the Clustal coloring scheme. The TRPC3 Mwk mutation in the S4–S5 linker region is indicated by a double-lined box. Arrows indicate residues highlighted in other panels of the figure. Image made with JalView. (B) Top-ranked (according to MODELLER score) mouse TRPC3 homology model on the TRPV1 apo structure (PDB entry 3J5P) with the Kv 1.2/2.1 channel chimera structure for the S4–S5 linker shown from a top view and a side view. Coloring indicates separate chains of the tetrameric channel. The S1–S4 helices of the purple chain have been removed for the sake of clarity in the side view figure. (C) Homology models of TRPC3 indicate that the threonine 635 (in green) mutated in the Mwk mouse has the potential for hydrogen bonding with the end of helix S6. The first quadrant shows an overlap of four models and the resulting T635 orientation. The remaining quadrants show three possible hydrogen bonding partners: S735 (cyan), Y736 (magenta), and the adjacent R634 (yellow). (D) Overexpression of the TRPC3 mutants Mwk (T635A) and T635V significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test; p < 0.001 and p < 0.05). (E) Overexpression of the TRPC3 mutants Mwk (T635A) and T635V caused significant nuclear translocation of GFP-NFAT (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test; p < 0.0001). (F) Overexpression of the TRPC3 mutants S735A and Y736F did not induce cell death upon overexpression in Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test). See also Figure S2 of the Supporting Information. (G) Other possible hydrogen bonding partners on the S6 helix (indicated as wheat mesh) were not seen as being viable for experimental validation as they pointed away from the site in question. Building homology models using the capsaicin-bound open state TRPV1 structure, however, indicated a further possible hydrogen bonding interaction with N726 (red) on helix S6. This interaction is with an adjacent subunit, and not the same subunit as with residues predicted with models based on the apo TRPV1. N726 is 10 residues toward the center of the membrane along S6 compared to other residues suspected to interact with T635.
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References

    1. Venkatachalam K.; Montell C. (2007) TRP channels. Annu. Rev. Biochem. 76, 387–417. - PMC - PubMed
    1. Ramsey I. S.; Delling M.; Clapham D. E. (2006) An introduction to TRP channels. Annu. Rev. Physiol. 68, 619–647. - PubMed
    1. Sekerková G.; Kim J.-A.; Nigro M. J.; Becker E. B. E.; Hartmann J.; Birnbaumer L.; Mugnaini E.; Martina M. (2013) Early onset of ataxia in moonwalker mice is accompanied by complete ablation of type II unipolar brush cells and Purkinje cell dysfunction. J. Neurosci. 33, 19689–19694. - PMC - PubMed
    1. Hartmann J.; Dragicevic E.; Adelsberger H.; Henning H. A.; Sumser M.; Abramowitz J.; Blum R.; Dietrich A.; Freichel M.; Flockerzi V.; Birnbaumer L.; Konnerth A. (2008) TRPC3 channels are required for synaptic transmission and motor coordination. Neuron 59, 392–398. - PMC - PubMed
    1. Becker E. B. E.; Oliver P. L.; Glitsch M. D.; Banks G. T.; Achilli F.; Hardy A.; Nolan P. M.; Fisher E. M. C.; Davies K. E. (2009) A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc. Natl. Acad. Sci. U.S.A. 106, 6706–6711. - PMC - PubMed

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