Calneuron I (CalnI) is a calmodulin-like protein that contains two functional EF-hand motifs at the N-terminal and a hydrophobic segment at the C-terminal. CalnI was cloned from the adult rat cortex and fused with GFP at its N-terminal. When expressed in bovine chromaffin cells, wild-type CalnI was localized at the plasma membrane. However, a mutant that lacked the hydrophobic segment was localized in the cytosol and nucleus, while a Ca2+-binding-deficient mutant was found in the cytosol and at the plasma membrane. Evaluation using the whole-cell patch-clamp technique revealed that Ca2+ currents were inhibited by both wild-type CalnI and the Ca2+-binding-deficient mutant. When the bovine N-type Ca2+ channel was expressed in 293T cells, Ca2+ currents were mostly inhibited by co-expression of CalnI, but not by the mutant without the hydrophobic tail. These results suggest that CalnI attenuates Ca2+ channel activity and that its subcellular localization is important for this effect. (C) 2009 Elsevier Inc. All rights reserved.