Free and bound intracellular calmodulin measurements in cardiac myocytes

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Calmodulin (CaM) is a ubiquitous Ca2+ binding protein and Ca2+-CaM activates many cellular targets and functions. While much of CaM is thought to be protein bound, quantitative data in cardiac myocytes is lacking regarding CaM location, [CaM]free and CaM redistribution during changes in [Ca2+]i. Here, we demonstrated that in adult rabbit cardiac myocytes, CaM is highly concentrated at Z-lines (confirmed by Di-8-ANEPPS staining of transverse tubules) using three different approaches: immunocytochemistry (endogenous CaM), Alexa Fluor 488 conjugate CaM (F-CaM) in both permeabilized cells (exogenous CaM) and in patch clamped intact cells (via pipette dialysis). Using 100 nM [CaM]free we washed F-CaM into permeabilized myocytes and saw a two-phase (fast and slow) CaM binding curve with a plateau after 40 min of F-CaM wash-in. We also measured myocyte [CaM]free using two modified null-point titration methods, finding [CaM]free to be 50-75 nM (which is only 1% of total [CaM]). Higher [Ca2+]i increased CaM binding especially in the nucleus and at Z-lines and significantly slowed F-CaM dissociation rate when F-CaM was washed out of permeabilized myocytes. Additionally, in both permeabilized and intact myocytes, CaM moved into the nucleus when [Ca2+]i was elevated, and this was reversible. We conclude that [CaM]free is very low in myocytes even at resting [Ca2+]i, indicating intense competition of CaM targets for free CaM. Bound CaM is relatively concentrated at Z-lines at rest but translocates significantly to the nucleus upon elevation of [Ca2+]i, which may influence activation of different targets and cellular functions.

Original languageEnglish (US)
Pages (from-to)353-364
Number of pages12
JournalCell Calcium
Volume41
Issue number4
DOIs
StatePublished - Apr 2007
Externally publishedYes

Fingerprint

Calmodulin
Cardiac Myocytes
Muscle Cells
Calmodulin-Binding Proteins

Keywords

  • [CaM]
  • CaM
  • CaM binding
  • CaM redistribution

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

Cite this

Free and bound intracellular calmodulin measurements in cardiac myocytes. / Wu, Xu; Bers, Donald M.

In: Cell Calcium, Vol. 41, No. 4, 04.2007, p. 353-364.

Research output: Contribution to journalArticle

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abstract = "Calmodulin (CaM) is a ubiquitous Ca2+ binding protein and Ca2+-CaM activates many cellular targets and functions. While much of CaM is thought to be protein bound, quantitative data in cardiac myocytes is lacking regarding CaM location, [CaM]free and CaM redistribution during changes in [Ca2+]i. Here, we demonstrated that in adult rabbit cardiac myocytes, CaM is highly concentrated at Z-lines (confirmed by Di-8-ANEPPS staining of transverse tubules) using three different approaches: immunocytochemistry (endogenous CaM), Alexa Fluor 488 conjugate CaM (F-CaM) in both permeabilized cells (exogenous CaM) and in patch clamped intact cells (via pipette dialysis). Using 100 nM [CaM]free we washed F-CaM into permeabilized myocytes and saw a two-phase (fast and slow) CaM binding curve with a plateau after 40 min of F-CaM wash-in. We also measured myocyte [CaM]free using two modified null-point titration methods, finding [CaM]free to be 50-75 nM (which is only 1{\%} of total [CaM]). Higher [Ca2+]i increased CaM binding especially in the nucleus and at Z-lines and significantly slowed F-CaM dissociation rate when F-CaM was washed out of permeabilized myocytes. Additionally, in both permeabilized and intact myocytes, CaM moved into the nucleus when [Ca2+]i was elevated, and this was reversible. We conclude that [CaM]free is very low in myocytes even at resting [Ca2+]i, indicating intense competition of CaM targets for free CaM. Bound CaM is relatively concentrated at Z-lines at rest but translocates significantly to the nucleus upon elevation of [Ca2+]i, which may influence activation of different targets and cellular functions.",
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