Recent studies have shown that NHE-3 is the luminal Na+/H+ exchanger isoform in cultured renal proximal tubule cells LLC-PK1 and OK (J Biol Chem 1994;269:15613-8). The purpose of the current experiments was to study the role of NHE-3 glycosylation on antiporter activity in LLC-PK1 cells. Treatment of LLC-PK1 cells with 1.5 μg/ml tunicamycin for 24 hours, which blocks glycosylation in the endoplasmic reticulum, significantly decreased antiporter activity as assessed by acid-stimulated sodium 22 uptake (9.52 ± 1.0 nmol/mg protein in control cells vs 5.85 ± 0.7 nmol/mg protein in tunicamycin-treated cells, p < 0.01, n = 4) and sodium-dependent pH1 recovery from an acid load (0.46 ± 0.05 pH/min in control cells vs 0.35 ± 0.04 pH/min in tunicamycin-treated cells, p < 0.02, n = 6). Lactate dehydrogenase (LDH) concentration in the medium was the same in both groups (p > 0.05), indicating that the inhibitory effect of tunicamycin was not caused by cell toxicity. Northern hybridization of poly(A)+ RNA from LLC- PK1 cells illustrated that in tunicamycin-treated cells, NHE-3 mRNA expression increased threefold over control cells. Immunoblots of luminal membranes from control LLC-PK1 cells with specific NHE-3 antiserum showed a doublet at 94 to 95 kd and a band at 90 kd. Luminal membranes from tunicamycin-treated cells showed only one strong band at 95 kd. NHE-3 immunoblots of whole cell extract from tunicamycin-treated cells showed that in addition to the 95 kd protein, an 87 kd band was also detected. These results are consistent with the possibility that the two bands in the 94 and 90 kd areas became deglycosylated and did not reach the membrane in the presence of tunicamycin. We conclude that glycosylation of the Na+/H+ exchanger isoform NHE-3 is essential for antiporter activity in LLC-PK1 cells. The results further suggest that glycosylation of NHE-3 mediates the translocation and insertion of this exchanger in the plasma membrane.
ASJC Scopus subject areas
- Pathology and Forensic Medicine