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

J Physiol. 2006 Nov 15;577(Pt 1):31-44. doi: 10.1113/jphysiol.2006.118661. Epub 2006 Sep 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Calcium Signaling / physiology*
  • Calmodulin / metabolism*
  • Cell Line
  • Humans
  • Ion Channel Gating / physiology*
  • Kidney / physiology*
  • Kinetics
  • Membrane Potentials / physiology*
  • Rats
  • TRPV Cation Channels / physiology*

Substances

  • Calmodulin
  • TRPV Cation Channels
  • TRPV6 channel
  • Calcium