Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein.
A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.
Laboratory of Physiology and Pathophysiology, School of Medicine, Université Libre de Bruxelles, Belgium.
P E
PE
Golstein
A
A
Sener
R
R
Beauwens
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D007082:Ileum / Q000378:metabolism; D007454:Iodides / Q000378:metabolism*; D007473:Ion Channels / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008871:Microvilli / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012995:Solubility; D013961:Thyroid Gland / Q000378:metabolism*
D003902:Detergents; D007454:Iodides; D007473:Ion Channels; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes