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Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

ABSTRACT: Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.

SUBMITTER: Findeisen F

PROVIDER: S-EPMC3225732 | biostudies-literature | 2011 Jul-Aug

REPOSITORIES: biostudies-literature

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Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

Findeisen Felix F Tolia Alexandra A Arant Ryan R Kim Eun Young EY Isacoff Ehud E Minor Daniel L DL

Channels (Austin, Tex.) 20110701 4

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous pro ...[more]

PMID: 21712653

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Project description:Key pointsCav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant-derivatives, C-terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside-out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C-tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM-linked channels are useful tools for investigating Cav1.2 channels in the inside-out mode; the fast run-down is prevented by only ATP and the slow run-down is nearly absent.AbstractCalmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+ -dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C-terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co-expressed with β2a and α2δ subunits in HEK293 cells. In the inside-out mode, α1C and α1C∆ showed minimal open-probabilities in a basic internal solution (run-down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3-5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7-9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C-terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl-terminal tail is removed.

Dataset Information

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

Publications

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

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