CLONING & EXPRESSION OF HUMAN EPITHELIAL NA/H EXCHANGERS

大多数腹泻疾病是通过绒毛细胞刷的抑制而发生的

Most diarrheal diseases occur through inhibition of villus cell brush border Na+/H+ exchange. We have cloned two 50% identical rabbit ileal villus cell Na+/H+ exchangers: NHE-1 and NHE-2. NHE-1 is the basolateral Na+/H+ exchanger by its localization to the ileal villus cell basolateral membrane in immunoblotting and immunocytochemical studies, its marked sensitivity to amiloride inhibition, and its activation by mitogens. NHE-2 is most likely the apical isoform of the rabbit ileal villus cell Na+/H+ exchangers; NHE-2 is greatly expressed in rabbit kidney and ileum and it is less sensitive to amiloride. The overall GOALS of this proposal is to clone the human apical epithelial Na+/H+ exchanger, examine its expression in the human gut and its regulation by second messengers known to inhibit brush border Na+/H+ exchange. We will first prove that rabbit NHE-2 is the apical epithelial isoform by demonstrating its localization to the apical membrane of rabbit ileum in immunoblotting and immunocytochemical studies, by showing that NHE-2 is solely expressed in neutral NaC1 absorbing epithelia and by determining NHE-2's K1 of amiloride and its internal pH set point. We will use NHE-2 to screen human kidney cortex and jejunum cDNA libraries in an attempt to clone the human apical exchanger. A full length clone will be expressed in Na+/H+ exchange deficient Chinese Hamster Lung fibroblast cell line PS120 to demonstrate that it codes for a functional Na+/H+ exchanger, allowing the fibroblasts to perform amiloride sensitive Na+/H+ exchange. Using a probe constructed from human NHE-2 in a quantitative method of Reverse Transcription-PCR analysis we will characterize the expression of human NHE-2 messenger RNA in tissue biopsies from the gastrointestinal tract of normal adults and from the jejunum of children with apical Na+/H+ exchange deficient congenital chronic secretory diarrhea. Our final goal will be to define second messenger regulation of human NHE-2 by determining if human NHE-2, as stably expressed in PS120 fibroblasts, can be regulated by known inhibitors of brush border Na+?H+ exchange, such as cAMP, protein kinase C, and Ca++/CaM kinase II. We will determine if such inhibition is accompanied by the phosphorylation of the exchanger. We will then determine which are the functional putative protein kinase consensus sequences located on NHE-2's C-terminus cytoplasmic tail, by examining the effects that unidirectional serial deletions of these kinase consensus sequences have on the ability of second messengers to regulate human NHE-2.