Free and bound intracellular calmodulin measurements in cardiac myocytes.
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.
Wu
Xu
X
Department of Physiology, Loyola University Chicago, 2160 S First Ave., Maywood, IL 60153, United States.
Bers
Donald M
DM
D000818:Animals; D015374:Biosensing Techniques; D002147:Calmodulin / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005464:Fluorobenzenes / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
C513915:AlexaFluor 488 carboxylic acid tetrafluorophenyl ester; D002147:Calmodulin; D005464:Fluorobenzenes