Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+‐dependent inactivation

Abstract

The Ca²⁺-selective TRPV6 as well as the L-type Ca²⁺ channel are regulated by the Ca²⁺-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca²⁺, employing Ca²⁺-imaging and patch-clamp techniques. Additionally, confocal Förster resonance energy transfer (FRET) microscopy on living cells was utilized as a key method to visualize in vivo protein–protein interactions essential for CaM regulation of rTRPV6 activity. The effects of overexpressed CaM or its Ca²⁺-insensitive mutant (CaM_MUT) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca²⁺/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6–CaM interaction site. A significant reduction of rTRPV6 activity, as well as an increase in current inactivation, were observed when CaM was overexpressed in addition to endogenous CaM. The Ca²⁺-insensitive CaM_MUT, however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaM_MUT with rTRPV6, suggesting a strict Ca²⁺ dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca²⁺ levels, as observed by dynamic FRET measurements. An rTRPV6Δ_695–727 mutant with the very C-terminal end deleted, yielded Ca²⁺ currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results, in contrast with those for CaM-dependent L-type Ca²⁺ channel inactivation, demonstrate a dynamic association of CaM with the very C-terminal end of rTRPV6 (aa 695–727), and this enables acceleration of the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.