cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein

Abstract

NHE3 is the Na+/H+ exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na+ absorption. The brush border Na+ absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing approximately 50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK1 cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25-30% inhibition in these cells.

Figure 1

(A) Alignment of the amino acid sequences of E3KARP and NHERF. Identical amino acid residues are indicated by |. The conserved PDZ domains are boxed. C42 and C54 were identical in size, encoding amino acids 130–268, and C16 extends from amino acid 9 to the 3′ poly(A) tail. The amino-terminal sequence of E3KARP (underlined) was obtained by PCR based on the sequence in GenBank. (B) In vitro and in vivo interaction of C16 and C42 with NHE3. (a) MBP-16, MBP-42, and MBP control proteins were immobilized on amylose–agarose beads and were incubated with detergent-solubilized lysates from PS120/NHE3V cells. Shown are immunoblots of NHE3V bound to MBP, MBP-16, and MBP-42. (b) PS120/NHE3V fibroblasts expressing GST, GST-42, or GST-16 were lysed and GST fusion proteins were purified on glutathione–Sepharose beads. Copurified NHE3V proteins were detected by Western immunoblot. (Upper) Immunoblots of copurified NHE3V. (Lower) Immunoblots of GST fusion proteins expressed in PS120/NHE3V cells. (Left) GST and GST-42 transiently transfected in PS120/NHE3V cells. (Right) GST-16 and GST stably transfected in PS120/NHE3V cells.

Figure 2

(A) Northern blot analysis of E3KARP and NHERF expression in rabbit ileal villus cells, PS120, and Caco-2 cells. Two micrograms of poly(A)⁺ enriched RNA from either PS120 fibroblasts or Caco-2 cells was electrophoresed per lane. For the rabbit ileal villus cells, 30 μg of total RNA was used. Hybridization was done under high-stringency conditions to avoid crosshybridization between E3KARP and NHERF. (B) Western blot analysis of expression of NHERF. Lanes: 1, brush border membrane from rabbit kidney and crude membranes from 2, CHO; 3, OK; 4, PS120; and 5, LLC-PK₁ cells. All lanes were loaded with 15 μg of membranes and were probed with polyclonal antibody against NHERF.

Figure 3

cAMP-induced inhibition of NHE3. Stably transfected PS120 fibroblasts acidified with NH₄Cl were either recovered in Na⁺ medium (○) or treated with 0.5 mM 8-Br-cAMP for 5–10 min before the recovery in Na⁺ medium (▴). Na⁺/H⁺ efflux rates were calculated at various pH_i, and lines were fitted to the data using an allosteric model. Treatment of (A) PS120/NHE3V/NHERF with 0.5 mM 8-Br-cAMP (▴) inhibited the Na⁺/H⁺ exchange activity with a decrease in V_max by ≈30% with no effect on K′(H_i⁺) or n_app. (C) Similarly, PS120/NHE3V/GST-E3KARP was inhibited by 8-Br-cAMP by a decrease in V_max by ≈28%. In contrast, PS120/NHE3 (B) and PS120/NHE3/GST (D) were not affected by 8-Br-cAMP. Shown here are data from four or more experiments for each condition.

Figure 4

Expression of E3KARP and NHERF mRNAs in human tissues. E3KARP and NHERF cDNAs that do not crosshybridize were used as probes. Both blots (Left, MTN blot II; Right, MTN blot) were commercially prepared and purchased from CLONTECH. Northern blot analyses were performed at four different times under the same stringency conditions and therefore cannot be used to compare the relative intensity between the blots.