A computational structural hypothesis for a TRPV4–ANO1 interface, and a stapled peptide designed to test it

The calcium-permeable cation channel TRPV4 and the calcium-activated chloride channel ANO1 (TMEM16A) have been reported to colocalize and function together in several tissues — most clearly in eccrine sweat gland acini (Kashio et al., 2024), where TRPV4, ANO1, and AQP5 colocalize and TRPV4 is required for the heat-evoked component of sweat secretion. To our knowledge, a direct physical interaction between TRPV4 and ANO1 has not been demonstrated by co-immunoprecipitation, FRET, or proximity ligation in any published study; the UniProt annotation listing TRPV4 as interacting with ANO1 is flagged "by similarity," meaning predicted rather than measured. The structural basis of any such interaction is therefore unknown. Here we present a computational hypothesis. Using AlphaFold Server multimer prediction together with ClusPro rigid-body docking, we identify a candidate interface in which an arginine-rich amphipathic helix of TRPV4 (residues 817–830, sequence RLRRDRWSSV) docks into the first intracellular loop of ANO1 (residues 217–240 in the canonical isoform, 184–207 in the sweat gland-specific isoform 4). The two methods independently converge on the same central contact: tryptophan 823 of TRPV4 packs against tyrosine 222 and phenylalanine 224 of canonical ANO1, forming a tryptophan–aromatic hot spot in a buried surface area of approximately 1,600 Ų. We propose SP-TRPV4(817-827), an i,i+4 hydrocarbon-stapled peptide spanning this helix, together with a W823A staple-matched control, as tool reagents for testing whether the predicted interface is real. The single most informative experiment is W823A point mutagenesis combined with co-immunoprecipitation in a cell type co-expressing both proteins; a positive result establishes the interaction and motivates further work, a negative result falsifies the hypothesis. We discuss potential therapeutic implications across several indications conditionally, with explicit attention to where the published biology supports the mechanism and where it does not. This paper is a structural hypothesis paper; experimental validation is required before any therapeutic claim can be made.