Kinetic characterization of a stably expressed novel Na+/H+ exchanger (NHE-2).
We have recently reported the molecular cloning, sequencing and tissue distribution of a novel Na+/H+ exchanger (NHE-2). The cDNA for NHE-2 was cloned by screening a rat intestinal cDNA library. This clone was unique due to the fact that it lacks the first two transmembrane domains which are present in the other Na+/H+ exchanger isoforms (NHE-1, NHE-3, NHE-4). This structural change in the cDNA offered a unique opportunity to study in detail the properties of this stably expressed cDNA in chinese lung fibroblasts that lack the Na+/H+ exchanger (PS120) cells. Amiloride-sensitive Na+ uptake was linear up to 2 min in PS120 cells transfected with the cDNA. Kinetics of the amiloride-sensitive Na+ uptake showed a Vmax of 24.7 +/- 5 nmol/microliters ICW per min and a Km of 33.1 +/- 2.0 mM. The inhibitory constant (KI) for amiloride and its analogue 5-N-ethyl-N-isopropylamiloride (EIPA) was 0.15 microM and 0.66 microM, respectively. Epidermal growth factor, a known stimulator of NHE-1, also upregulated the expressed NHE-2. These results characterize the kinetic properties of this unique exchanger and suggests that the first two transmembrane domains of the Na+/H+ exchanger isoforms are not essential for the expression of amiloride-sensitive Na+ uptake.
Department of Pediatric Gastroenterology/Nutrition, Vanderbilt University Medical Center, Nashville, TN.
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Knobel
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Ghishan
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004815:Epidermal Growth Factor / Q000494:pharmacology; D007700:Kinetics; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D014162:Transfection
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D004815:Epidermal Growth Factor; D000584:Amiloride; D012964:Sodium