Accumulated experimental evidence suggests that annexin A6 (AnxA6) is involved in ion transport in various tissues. Such a biological function is related either to the modulation of ion transport systems by AnxA6 or to the ion channel activity of the protein. While AnxA6 channel activity at low pH seems to be associated with a large conformational transition in the protein, the mechanism of GTP-induced ion channel formation remains obscure. This activity is not accompanied by changes in protein structure. The existence of a domain binding the phosphate groups of GTP in AnxA6 [Bandorowicz-Pikula, J., Kirilenko, A., van Deursen, R., Golczak, M., Kuhnel, M., Lancelin, J. M., Pikula, S., and Buchet, R. (2003) Biochemistry 42, 9137-9146] may provide some clues about the molecular mechanisms of GTP-induced ion channel formation. In addition, we observed that one of the AnxA6 tryptophan residues, W192 or W343, may be involved in GTP binding. Therefore, we created several site-directed mutants of AnxA6 in which selected amino acid residues within a consensus sequence of a putative nucleotide-binding domain of AnxA6 were replaced with other amino acid residues without affecting the overall structure of protein as examined by circular dichroism and infrared spectroscopies. Their properties were analyzed and compared to those of the native protein. In contrast to mutant W192S and wild-type annexin, mutant W343S neither bound GTP nor exhibited GTP-induced ion channel activity. In addition, we detected the likely formation of AnxA6 trimers in the presence of GTP. The ability of mutant W343S to form trimers was significantly impaired. Our findings suggest that W343 participates in the formation of AnxA6 trimers. We hypothesize that such trimers could lead to a functional unit of the GTP-induced ion channels formed by the annexin molecules.