Rapid purification of an amiloride-sensitive Na+ channel from bovine kidney and its functional reconstitution.
We have used ion-exchange chromatography to isolate and partially purify an amiloride-sensitive Na+ channel protein from bovine renal papillae. The channel was purified by sequential chromatography on an anion-exchange and a cation-exchange cartridge. Five different approaches were applied to monitor the degree of purification of this Na+ channel. First, [3H]Br-benzamil binding activities to the purified proteins were determined. We found that no specific binding was measured in protein fractions eluted from a QUAT anion-exchange cartridge, whereas proteins eluted from an SP cation-exchange cartridge bound [3H]Br-benzamil specifically. Second, we examined the HPLC profiles of purified proteins through both ion-exchange chromatography steps and observed a major peak of very high molecular mass proteins (> 670 kDa) after final purification. Third, using anti-Na+ channel antibodies, Western and slot blot analyses were performed to assess qualitatively the enrichment of channel epitopes at each step of purification. Fourth, amiloride-sensitive 22Na+ influx could be demonstrated after the proteins eluted from the cation-exchange resin were reconstituted into liposomes. Fifth, the proteins eluted from the cation-exchange resin were reconstituted into planar lipid bilayers, and single amiloride-sensitive Na+ channel activity was measured. In summary, we have developed a new protocol applicable for speed- and scale-up purification of a functional amiloride-sensitive Na+ channel from bovine kidney papillae.
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Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Benos
D J
DJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004848:Epithelium / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism*; D008051:Lipid Bilayers; D008081:Liposomes; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D012995:Solubility
D008051:Lipid Bilayers; D008081:Liposomes; D015222:Sodium Channels; D000584:Amiloride; D012964:Sodium