title: Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation.
abstract: The Ca(2+)-selective TRPV6 as well as the L-type Ca(2+) channel are regulated by the Ca(2+)-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca(2+), employing Ca(2+)-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 Ca(2+)-insensitive mutant (CaM(MUT)) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca(2+)/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 Ca(2+)-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 Ca(2+) dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca(2+) levels, as observed by dynamic FRET measurements. An rTRPV6Delta(695-727) mutant with the very C-terminal end deleted, yielded Ca(2+) 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 Ca(2+) 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.
authors.affiliation: Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria.
authors.forename: Isabella
authors.initials: I
authors.lastname: Derler
authors.forename: Michael
authors.initials: M
authors.lastname: Hofbauer
authors.forename: Heike
authors.initials: H
authors.lastname: Kahr
authors.forename: Reinhard
authors.initials: R
authors.lastname: Fritsch
authors.forename: Martin
authors.initials: M
authors.lastname: Muik
authors.forename: Klaus
authors.initials: K
authors.lastname: Kepplinger
authors.forename: Marlene E
authors.initials: ME
authors.lastname: Hack
authors.forename: Sieglinde
authors.initials: S
authors.lastname: Moritz
authors.forename: Rainer
authors.initials: R
authors.lastname: Schindl
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
authors.forename: Christoph
authors.initials: C
authors.lastname: Romanin
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D051381:Rats; D050916:TRPV Cation Channels / Q000502:physiology*
chemical_list: D002147:Calmodulin; D050916:TRPV Cation Channels; C120776:TRPV6 channel; D002118:Calcium