Expression of amiloride-sensitive sodium channel: a strategy for the coexpression of multimeric membrane protein in Sf9 insect cells.
The amiloride-sensitive epithelial sodium channel (ENaC) mediates Na(+) reabsorption in many epithelial tissues including the distal nephron, colon, lung, and secretory glands and plays an important role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric integral membrane protein formed by the association of highly homologous,alpha-, beta-, and gamma-ENaC subunits. Here we explored the Sf9 insect cell-baculovirus expression system as a source to obtain high yields of recombinant ENaC for functional and structural studies. Although this expression system is widely used, coexpression of ENaC subunits could not be accomplished by the conventional procedures. We thus developed a protocol in which the alpha- and gamma-ENaC cDNA's were first fused individually with polyhedrin promoters at their 5'-ends and then inserted in the multiple cloning sites of pVL1393 transfer vector carrying the beta-ENaC cDNA. Utilizing this transfer vector, a recombinant baculovirus carrying all of the three ENaC cDNA's was prepared. Infection of Sf9 insect cells with this recombinant baculovirus resulted in the expression all of the three ENaC subunits in high yield. Planar lipid bilayer reconstitution procedure revealed the presence of approximately 6 pS sodium channels that are amiloride-sensitive. The results presented point out certain underlying rules for the expression of multiple genes in Sf9 cells, which may be useful in the expression other multimeric proteins and in the studies of protein-protein interactions as well.
Rao
U S
US
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA. usrao@unmc.edu
Mehdi
A
A
Steimle
R E
RE
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D053503:Epithelial Sodium Channels; D015870:Gene Expression; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics*; D018411:Spodoptera
D053503:Epithelial Sodium Channels; D008565:Membrane Proteins; D011994:Recombinant Proteins; D015222:Sodium Channels; D000584:Amiloride