NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.

Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (approximately 100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone.