Loss of calcineurin homologous protein-1 in chicken B lymphoma DT40 cells destabilizes Na⁺/H⁺ exchanger isoform-1 protein

Abstract

NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na(+)/H(+) exchangers (NHEs) functioning in maintaining intracellular concentrations of Na(+) and H(+) ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na(+)/H(+) activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca(2+) binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity.

Dual functional significance of calcineurin homologous protein 1 binding to Na+/H+ exchanger isoform 1

“…The sample preparation was performed without heat denaturing to minimize aggregation of NHE1, as described previously (5,16). SDS-PAGE and immunodetection was performed as described previously (16).…”

“…The sample preparation was performed without heat denaturing to minimize aggregation of NHE1, as described previously (5,16). SDS-PAGE and immunodetection was performed as described previously (16). Mouse monoclonal (clone 16B12) and rabbit polyclonal anti HA-tag antibody were purchased from Covance (Princeton, NJ) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.…”

“…Cells were fixed and stained with antibodies as described previously (16). For secondary antibodies, anti-rabbit IgG conjugated with Alexa Fluor 488 and anti-mouse IgG conjugated with Alexa Fluor 546 were obtained from Invitrogen.…”

“…Previously, knockout of CHP1 in a chicken B lymphoma DT40 cell line reduced NHE1 expression (16); however, the role of CHP1 in the localization of NHE1 to the plasma membrane remains controversial and further studies are required to clarify this. CHP1 expression is difficult to visualize microscopically because of the small size of the DT40 cells and the small amount of NHE1 in CHP1-deficient or CHP1-knockdown cells (16). Thus, to study the localization of NHE1, a monkey cell line (CV1) that overexpressed NHE1 was established, and the effect of the overexpression of CHP1 on the localization and stabilization of NHE1 was examined.…”

“…They concluded that CHP1 is an essential cofactor for the exchange activity of NHE1 but that it is not required for proper localization to plasma membranes. However, in CHP1 knockout chicken lymphoma cells (DT40), NHE1 protein levels were unexpectedly reduced to Ͻ10%, and very little NHE1 was localized to the plasma membrane (16). In CHP1 knockout cells, Na ϩ uptake activity by one NHE1 molecule was ϳ20% of wild-type cells.…”