TRPM3 proteins form steroid-activated, non-selective channel permeable to a wide variety of cations, including Ca²⁺, Mg²⁺ and Zn²⁺. Like many TRP channels, TRPM3 channels are not only permeable to, but are affected by divalent cations. For instance, Ca²⁺ and Mg²⁺ are permeant blockers inside the pore of TRPM3 channels.

However, intracellular Ca²⁺ also inhibits TRPM3 activity in a way independent from pore effects. We found that calmodulin binds to N-terminus of TRPM3 in a Ca²⁺-dependent manner and interferes with TRPM3 dependent Ca²⁺-signaling. This may indicate that calmodulin is involved in the regulation of the channel, namely in fast Ca²⁺-evoked inhibition. Six calmodulin binding sites were identified by protein pull-down assay and confirmed by dot-blot analysis. The second binding site displayed strongest calmodulin bindingin vitro. Point mutations introduced at this site allowed us to identify the amino acids essential for CaM binding. Mutations of both K203 and K208 to alanine abolished the binding of calmodulin at this site.

We investigated the effect of direct and rapid increase of Ca²⁺ on TRPM3 channel activity by flash-photolysis of caged Ca²⁺, which is dialyzed into the cells through a patch pipette in whole-cell patch-clamp experiments. We simultaneously monitored the current through wild-type and K203A / K208A double mutant TRPM3 channels and the free Ca²⁺ concentration by fluorescence. Our data show that both types of TRPM3 channels are inhibited by Ca²⁺ in a concentration-dependent way, with a Kd in the low micromolar range.