Ca2+-dependent interaction of the trpl cation channel and calmodulin.
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 (S710-I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) 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 Ki 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 Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 microM (calmodulin binding site 1) and 3.3 microM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+-calmodulin-dependent mechanism contributes to transient receptor potential-like cation channel modulation when expressed in CHO cells.
Trost
C
C
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany.
Marquart
A
A
Zimmer
S
S
Philipp
S
S
Cavalié
A
A
Flockerzi
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D050051:Transient Receptor Potential Channels
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D002118:Calcium