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Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca 2+ ‐dependent inactivation
Author(s) -
Derler Isabella,
Hofbauer Michael,
Kahr Heike,
Fritsch Reinhard,
Muik Martin,
Kepplinger Klaus,
Hack Marlene E.,
Moritz Sieglinde,
Schindl Rainer,
Groschner Klaus,
Romanin Christoph
Publication year - 2006
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2006.118661
Subject(s) - förster resonance energy transfer , biophysics , calmodulin , confocal microscopy , intracellular , mutant , chemistry , microbiology and biotechnology , patch clamp , confocal , calcium , biochemistry , biology , fluorescence , gene , receptor , physics , organic chemistry , quantum mechanics , geometry , mathematics
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 rTRPV6Δ 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.