MECHANISMS AND REGULATION OF EPITHELIAL TRANSPORT

肾近端小管和肠刷边界膜包含

Renal proximal tubule and intestinal brush border membranes contain transporters which catalyze active cotransport of Na+ and glucose. Using immunological and functional criteria, we have identified a 75 kD glycoprotein in renal brush border membranes as a subunit of the Na+/glucose symporter, purified the 75 kD protein to homogeneity and obtained functional reconstitution in proteoliposomes. Our monoclonal and polyclonal antibodies also recognize the 75 kD subunit in LLC-PK1 cells, a long term line from renal proximal tubule. The overall goals of the continuation of this project are to further characterize the structure and function of the renal Na+/D- glucose symporter and to investigate the regulation of its differentiated expression in cell culture. The cDNA encoding the 75 kD subunit of the renal Na+/glucose symporter will be cloned and sequenced. An LLC-PK1 lambda gt11 expression library enriched in symporter cDNA will be screened using our polyclonal antibody to the symporter; alternately, a LLC- PK1 lambda gtlO library will be screened using synthetic oligonucleotides corresponding to the sequence of a peptide derived from our pure symporter preparation. A predicted amino acid sequence and secondary structure will be calculated based on the cDNA sequence. The oligomeric structure of the symporter will be investigated by gel filtration and comparative sedimentation in sucrose gradients made from D2O and H2O and by cross-linking studies. The possibility that Na+, glucose or phlorizin affect oligomeric structure will also be tested. Epitopes of the symporter on the inner and outer surface of the membrane and transmembrane segment will be mapped using monoclonal antibodies to the purified protein and site-directed polyclonal antibodies. Site-directed mutagenesis studies will be initiated to investigate functional domains of the symporter. The regulation of Na+/glucose symporter expression in LLC-PK1 cells will be investigated using specific antibodies to monitor biosynthesis and turnover of the protein and cDNA probes to measure specific mRNA levels, rates of transcription and half-life. These studies will focus on the dramatic (20-fold) induction of symporter activity achieved after treatment with the differentiation inducer hexamethylene bisacetamide (HMBA).