When it binds Ca2+, calmodulin (CaM) activates an enormous array of different enzymes with critical functions in the cell. Our preliminary results indicate the maximum global free concentration of Ca2+-CaM in cells is approximately 30 nM, and the total concentration of CaM is well below the concentration of CaM-binding proteins. While many CaM-dependent enzymes bind Ca2+-CaM with dissociation constants of approximately 1 nM, some have affinities hundreds of times lower. Among this group are the CaM-dependent adenylate cyclases, which are robustly activated by Ca2+ in cells. This is not consistent with the low global free concentrations of Ca2+-CaM that we have measured. However, conditions that produce local increases in the free Ca2+ concentration at the plasma membrane appear to be required to significantly activate the cyclases in several cell types. And they co-localize in the brain with neuromodulin or neurogranin, membrane-associated proteins thought to function as CaM """"""""sinks"""""""". We have therefore hypothesized the existence of two mechanisms that enhance the free Ca2+-CaM concentrations at the plasma membrane to produce the observed activation of the cyclases: (1) Diffusional recruitment of CaM due to local elevations in Ca2+, and (2) Concentration of CaM at the plasma membrane by CaM sink proteins. We propose to test this hypothesis by determining how the free concentrations of Ca2+-CaM produced at the plasma membrane are affected by local elevations in the free Ca2+ concentration and expression of neuromodulin, and how this relates to changes in the activity an expressed CaM-dependent cyclase activity (AC1). We also,,will investigate whether PKC- catalyzed phosphorylation of neuromodulin, which abolishes its CaM sink function, can dynamically control free Ca2+-CaM concentrations and cyclase activity. Given the limiting amount of CaM in the cell, it is clearly of general importance to understand how it is distributed among CaM-dependent enzymes, and how this impacts cellular function. This proposal emphasizes factors that enhance the free Ca2+-CaM concentrations at the plasma membrane and control the activities of CaM-dependent cyclases, as well as other low affinity CaM-dependent enzymes. The cyclases are a particularly important class of low affinity CaM targets, having been specifically been implicated in secretion and steroidogenesis in parotid acinar and adrenal glomerulosa cells, and in some forms of learning.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Physical Biochemistry Study Section (PB)
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Sato, Sheryl M
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University of Missouri Kansas City
Schools of Medicine
Kansas City
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Black, D J; Persechini, Anthony (2011) In calmodulin-IQ domain complexes, the Ca(2+)-free and Ca(2+)-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites. Biochemistry 50:10061-8
Black, D J; Persechini, Anthony (2010) Variations at the semiconserved glycine in the IQ domain consensus sequence have a major impact on Ca2+-dependent switching in calmodulin-IQ domain complexes. Biochemistry 49:78-83
Black, D J; LaMartina, David; Persechini, Anthony (2009) The IQ domains in neuromodulin and PEP19 represent two major functional classes. Biochemistry 48:11766-72
Tran, Quang-Kim; Black, D J; Persechini, Anthony (2005) Dominant affectors in the calmodulin network shape the time courses of target responses in the cell. Cell Calcium 37:541-53
Isotani, Eiji; Zhi, Gang; Lau, Kim S et al. (2004) Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse. Proc Natl Acad Sci U S A 101:6279-84
Black, D J; Tran, Quang-Kim; Persechini, Anthony (2004) Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+. Cell Calcium 35:415-25
Geguchadze, Ramaz; Zhi, Gang; Lau, Kim S et al. (2004) Quantitative measurements of Ca(2+)/calmodulin binding and activation of myosin light chain kinase in cells. FEBS Lett 557:121-4
Tran, Quang-Kim; Black, D J; Persechini, Anthony (2003) Intracellular coupling via limiting calmodulin. J Biol Chem 278:24247-50
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