Abstract

Calmodulin (CaM) is a ubiquitous calcium (Ca) binding protein which bind myosin light chain kinase (MLCK), caldesmon (CaD), calponin (Calp) and a sarcolemmal Ca-ATPase to regulate the contraction of vascular smooth muscle. Ca binding to CaM stimulates it binding to each of these proteins but little is known concerning the rates at which Ca-CaM binds and dissociates from these important target proteins during a Ca transient. We will use fluorescence stopped-flow with our fluorescent CaM's to determine the Kd's and the rates of association and dissociation of each of these CaM-target protein complexes and determine the rate at which each complex is disrupted by Ca removal. We will determine if these target proteins have differential effects on Ca exchange rates with CaM's N- and C-terminal Ca binding sites and relate Ca dissociation from these sites to the rate of EGTA induced complex disruption. We will study naturally occurring mutant CaM's to determine if altered Ca binding to their N- and C-terminal Ca binding sites is responsible for their inability to activate Na and K ion channels, respectively. This should yield important new information on the mechanism by which Ca-CaM binds and activates more than 20 different cellular target proteins. Hormonal activation of the Ca phospholipid dependent protein kinase C (PKC) results in its activation at resting Ca levels and its phosphorylation o MLCK, CaD, Calp, and Ca-ATPase to modulate and maintain smooth muscle contraction. We will determine if PKC phosphorylation of these target proteins alters CaM banding and activation. We have, for the first time directly measured Ca off-rates from purified PKC and the Ca-ATPase. This will allow us to determine the mechanism and extent that physiological activators of these two enzymes modulate their Ca affinity and their activity. These studies should increase our understanding of how hormonal activation modulates these Ca binding proteins and CaM's interaction with its target proteins in smooth muscle.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK033727-09
Application #
3232127
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
1985-07-01
Project End
1998-06-30
Budget Start
1993-07-15
Budget End
1994-06-30
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Ohio State University
Department
Type
Schools of Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210

  Publications

Black, D J; Halling, D Brent; Mandich, David V et al. (2005) Calmodulin interactions with IQ peptides from voltage-dependent calcium channels. Am J Physiol Cell Physiol 288:C669-76
Tang, Wei; Halling, D Brent; Black, D J et al. (2003) Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel. Biophys J 85:1538-47
Van Lierop, Jacquelyn E; Wilson, David P; Davis, Jonathan P et al. (2002) Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40). J Biol Chem 277:6550-8
Davis, Jonathan P; Rall, Jack A; Reiser, Peter J et al. (2002) Engineering competitive magnesium binding into the first EF-hand of skeletal troponin C. J Biol Chem 277:49716-26
Tikunova, Svetlana B; Rall, Jack A; Davis, Jonathan P (2002) Effect of hydrophobic residue substitutions with glutamine on Ca(2+) binding and exchange with the N-domain of troponin C. Biochemistry 41:6697-705
Johnson, J David; Tikunova, Svetlana B (2002) Fluorescence methods for measuring calcium affinity and calcium exchange with proteins. Methods Mol Biol 173:89-102
Tikunova, S B; Black, D J; Johnson, J D et al. (2001) Modifying Mg2+ binding and exchange with the N-terminal of calmodulin. Biochemistry 40:3348-53
Adak, S; Santolini, J; Tikunova, S et al. (2001) Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis. J Biol Chem 276:1244-52
Black, D J; Tikunova, S B; Johnson, J D et al. (2000) Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association. Biochemistry 39:13831-7
Lee, S H; Johnson, J D; Walsh, M P et al. (2000) Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration. Biochem J 350 Pt 1:299-306

Showing the most recent 10 out of 39 publications