Functional Characterization of a Novel Intestinal NHE Isoform (NHE8)

Abstract

NHE8 is a newly identified NHE isoform from rat intestine. Our previous work has shown that NHE8 is located at the apical membrane of the intestinal epithelial cells, and it highly expressed in suckling rats. To date, the kinetic characteristics of this new NHE isoform has not been determined. This study was performed to determine the NHE8 transporter affinities to Na+, H+, and its sensitivity to HOE694. Rat NHE8 cDNA was subcloned into Mammalian Lumio Gateway Vectors (Invitrogen). PS120 cells were transfected with NHE8 cDNA constructs. Twenty-four hours after transfection, cells were treated with NH4Cl to produce an acid load. Non-transfected cells were killed by the acid load, while NHE8 cDNA construct transfected cells survived. NHE8 expressing cells were further used for intracellular pH (pHi) measurement to characterize NHE8 function using the pH indicator SNARF-4. Na+-dependent H+ transport was analyzed by measuring the rate of pHi recovery after an NH4Cl induced acid load. Our data indicate that: 1) pHi recovery in NHE8 expressing PS120 cells is Na+-dependent and mediated solely by NHE. 2) The extracelluar Na+ affinity of rat NHE8 transporter in PS120 cells is ~12 mM. 3) The estimated Km[pH] for NHE8 transporter in PS120 cells is ~pH6.65. 4) NHE8 is sensitive to HOE694, with a Ki of approximately 10 μM HOE694. In conclusion, our work suggests that NHE8 is a functional Na+/H+ exchanger with kinetic characteristics that differ from the other apical isoforms NHE2 and NHE3. This investigation was supported by NIH grant R37-DK33209.