PMU-Autor/inn/en
Jakab MartinAbstract
Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide 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. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln 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 experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.
Useful keywords (using NLM MeSH Indexing)
3T3 Cells
Animals
Bromides/metabolism
Calcium/metabolism
Calcium/pharmacology
Cell Line
Cell Size/drug effects
Chelating Agents/pharmacology
Chlorides/metabolism
Dogs
Ion Channels/antagonists*
inhibitors
Ion Channels/metabolism*
Ion Transport/drug effects
Lipid Bilayers/metabolism*
Mice
Mutagenesis, Site-Directed
Nickel/pharmacology
Nucleosides/metabolism
Nucleosides/pharmacology
Oocytes/cytology
Oocytes/metabolism
Patch-Clamp Techniques
Potassium/metabolism
Protein Structure, Tertiary
Proteins/antagonists*
inhibitors
Proteins/genetics
Proteins/metabolism*
Substrate Specificity/drug effects
Transfection
Xenopus Proteins
Xenopus laevis
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ICln