Exploring functional conservation of the cooling agent binding site in the transient receptor potential melastatin subfamily.

Abstract

Transient Receptor Potential (TRP) cation channels act as cellular sensors of a wide variety of stimuli and play a role in many biological processes, making them attractive pharmacological targets. However, the lack of specific agonists or antagonists for many subfamily members has impeded studies of both their biophysical properties and their broader biological functions. The architecture of the TRP channel transmembrane domain, which contains both the ion conducting pore and several ligand binding sites, is conserved across the family, and previous studies transferring ligand sensitivity among TRP channels revealed conserved channel gating mechanisms within the Vanilloid subfamily. Sequence-based and structure-based alignments have shown conservation within other TRPM family members of key residues mediating TRPM8 sensitivity to cooling agents and antagonists, even though no other TRPM channels have been previously described to be sensitive to cooling agents. We have been particularly interested in TRPM4 because few pharmacological agents are known to modulate the activity of this channel and it is expressed in many tissues, including cardiac, immune, and cancer cells, and mutations have been causally linked to heritable Familial Heart Block. We have explored the sensitivity of TRPM4 to TRPM8 ligands using inside-out patch clamp recordings and found that cooling agents can modulate TRPM4 activity in response to intracellular Ca2+ and that this sensitivity likely arises from the equivalent binding pocket to TRPM8. This structural and functional conservation may facilitate the use of the rich pharmacology of TRPM8 to guide identification or development of TRPM4-specific ligands.