CA 2+-dependent interaction of the trpl cation channel and calmodulin

Abstract

The transient receptor potential-like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential-like fusion proteins to characterize calmodulin binding regions in the transient receptor potential-like channel through the use of [ 125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C-terminus of the transient receptor potential-like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S 710–I 725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K 859ETAKERFQRVAR 871) and binds both [ 125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent K i values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca 2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 μM (calmodulin binding site 1) and 3.3 μM Ca 2+ (calmodulin binding site 2) and give evidence that (iv) a Ca 2+-calmodulin-dependent mechanism contributes to transient receptor potential-like cation channel modulation when expressed in CHO cells.