Functional reconstitution of ICln in lipid bilayers

Reconstitution of purified ICln in lipid bilayer leads to functional ion ch annels showing varying rectification. The reconstituted single channels hav e a conductance of congruent to 3 pS and their open probability is sensitiv e to nucleoside analogues. Mutation of a putative nucleotide binding site i dentified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstitute d ICln channels can be permeated both by cations and anions. The relative p ermeability of cations over anions depends on the presence of calcium. In t he presence of calcium reconstituted ICln channels are more permeable to br omide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Sit e-directed mutagenesis revealed the calcium-binding site responsible for th e shift of the selectivity from cations towards anions of reconstituted ICl n channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experime nts show that ICln can act as an ionic channel, which does not exclude addi tional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads t o an impaired regulatory volume decrease (RVD) after cytoplasmic swelling a nd reconstituted ICln channels show several biophysical features of ion cha nnels activated after swelling, ICln is a molecular candidate for these cha nnels.