Mutations in calmodulin-binding domains of TRPV4/6 channels confer invasive properties to colon adenocarcinoma cells

Abstract

Transient receptor potential (TRP) channels form a family of polymodal cation channels gated by thermal, mechanical, or chemical stimuli, with many of them involved in the control of proliferation, apoptosis, or cell cycle. From an evolutionary point of view, TRP family is characterized by high conservation of duplicated genes originating from whole-genome duplication at the onset of vertebrates. The conservation of such "ohnolog" genes is theoretically linked to an increased probability of generating phenotypes deleterious for the organism upon gene mutation. We aimed to test experimentally the hypothesis that TRP mutations, in particular gain-of-function, could be involved in the generation of deleterious phenotypes involved in cancer, such as gain of invasiveness. Indeed, a number of TRP channels have been linked to cancer progression, and exhibit changes in expression levels in various types of cancers. However, TRP mutations in cancer have been poorly documented. We focused on 2 TRPV family members, TRPV4 and TRPV6, and studied the effect of putative gain-of-function mutations on invasiveness properties. TRPV channels have a C-terminal calmodulin-binding domain (CaMBD) that has important functions for regulating protein function, through different mechanisms depending on the channel (channel inactivation/potentiation, cytoskeleton regulation). We studied the effect of mutations mimicking constitutive phosphorylation in TRPV4 and TRPV6 CaMBDs: TRPV4 S823D, S824D and T813D, TRPV6 S691D, S692D and T702. We found that most of these mutants induced a strong gain of invasiveness of colon adenocarcinoma SW480 cells, both for TRPV4 and TRPV6. While increased invasion with TRPV6 S692D and T702D mutants was correlated to increased mutant channel activity, it was not the case for TRPV4 mutants, suggesting different mechanisms with the same global effect of gain in deleterious phenotype. This highlights the potential importance to search for TRP mutations involved in cancer.

Keywords: Transient receptor potential (TRP) channels; cancer; evolution theory; gain of function; invasion.

Figure 1.

Ohnologs in TRP family. Phylogenetic tree was obtained from Nilius et al. [38], and asterisks correspond to the two whole-genome duplications on each branch (shown for ohnolog genes) [37].

Figure 2.

Mutations in TRPV4 and TRPV6 CaMBDs confer increased invasive properties to SW480 cells. (a) TRPV4 and TRPV6 CaMBD domains, with serine/threonine residues as potential phosphorylation sites (arrows). (b–c) Invasion assay on TRPV6 (b) and TRPV4 (c) CaMBD mutants mimicking constitutive phosphorylation. The number of cells passing through Matrigel after 22 h invasion (out of 20,000 seeded cells) is indicated. For TRPV4, bicistronic vectors allowing selection of transfected cells were used. A common reference (non-transfected cells) is indicated in (b) and (c) (black). (d–e) Typical images on cells harvested in invasion assays, on Corning BioCoat Matrigel invasion chambers with 8 μm pores, for TRPV6 (d) and TRPV4 (e). Images shown correspond to experiments with non-bicistronic vectors, both for TRPV6 (b) and TRPV4 (see SI).

Figure 3.

Electrophysiology of TRPV4/6 CaMBD mutants. Whole-cell patch-clamp experiments on HEK cells were performed 24 h after transfection with TRPV6 (a) or TRPV4 (b). Current–voltage relations obtained from the TRPV6 wild type and the three mutants (a) and for the TRPV4 wild type and mutant channels before (open symbol) and after GSK treatment (closed symbols). Each datapoint represents the mean ± SEM.