The human transient receptor potential vanilloid 3 channel is sensitized via the ERK pathway

Lenka Vyklicka,Stepana Boukalova,Lucie Macikova,Stepan Chvojka,Viktorie Vlachova

doi:10.1074/jbc.m117.801167

Abstract

The transient receptor potential vanilloid 3 (TRPV3) channel is a Ca2+-permeable thermosensitive ion channel widely expressed in keratinocytes, where together with epidermal growth factor receptor (EGFR) forms a signaling complex regulating epidermal homeostasis. Proper signaling through this complex is achieved and maintained via several pathways in which TRPV3 activation is absolutely required. Results of recent studies have suggested that low-level constitutive activity of TRPV3 induces EGFR-dependent signaling that, in turn, amplifies TRPV3 via activation of the mitogen-activated protein kinase ERK in a positive feedback loop. Here, we explored the molecular mechanism that increases TRPV3 activity through EGFR activation. We used mutagenesis and whole-cell patch clamp experiments on TRPV3 channels endogenously expressed in an immortalized human keratinocyte cell line (HaCaT) and in transiently transfected HEK293T cells and found that the sensitizing effect of EGFR on TRPV3 is mediated by ERK. We observed that ERK-mediated phosphorylation of TRPV3 alters its responsiveness to repeated chemical stimuli. Among several putative ERK phosphorylation sites, we identified threonine 264 in the N-terminal ankyrin repeat domain as the most critical site for the ERK-dependent modulation of TRPV3 channel activity. Of note, Thr264 is in close vicinity to a structurally and functionally important TRPV3 region comprising an atypical finger 3 and oxygen-dependent hydroxylation site. In summary, our findings indicate that Thr264 in TRPV3 is a key ERK phosphorylation site mediating EGFR-induced sensitization of the channel to stimulate signaling pathways involved in regulating skin homeostasis.

Highlights

Transient receptor potential vanilloid 3 (TRPV3)3 is a thermosensitive ion channel widely expressed in epithelial tissues of
A significant breakthrough in our understanding of how aberrant transient receptor potential vanilloid 3 (TRPV3) activity relates to skin pathophysiology has been made by Cheng et al [19], who elaborated an observation that the defects in hair morphogenesis in mice deficient in trpv3 strikingly resemble the mouse phenotype bearing mutations in the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor-␣ (TGF-␣)
We identify threonine 264 in the N-terminal ankyrin repeat domain (ARD) as a putative site for the ERK1-dependent modulation of TRPV3

Summary

Discussion

We identify threonine 264 in the N-terminal ARD as a putative site for the ERK1-dependent modulation of TRPV3. We cannot rule out the presence of some additional phosphorylation site(s) on TRPV3, because a mild increase in 2-APB current responses was observed in cells co-transfected with T264A and ERK1 Whereas Thr does not fulfill the consensus sequence for ERK, the phosphomimetic mutation at Thr in our experiments shown in Fig. 2B led to a significant decrease in initial response to 2-APB but, upon repeated stimulation, the sensitization was comparable with wild-type channels. These sites are probably not involved in the observed functional changes. The close vicinity of this residue from the structurally and functionally important region comprising an atypical finger 3 and oxygen-dependent hydroxylation site adds further complexity to the cellular signaling pathways involved in the regulation of skin homeostasis via growth factor downstream signaling

Cell culture and transfection of HaCaT cells

Data analysis

Patch clamp recording