INHIBITION OF GLYCOSYLATION DECREASES NA+ H+ EXCHANGE ACTIVITY, BLOCKS NHE-3 TRANSPORT TO THE MEMBRANE, AND INCREASES NHE-3 MESSENGER-RNA EXPRESSION IN LLC-PK1 CELLS/

Recent studies have shown that NHE-3 is the luminal Na+/H+ exchanger i soform 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-PK 1 cells. Treatment of LLC-PK1 cells with 1.5 mu g/ml tunicamycin for 2 4 hours, which blocks glycosylation in the endoplasmic reticulum, sign ificantly 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 pH(i) recovery from an acid load (0.46 +/- 0. 05 pH/min in control cells vs 0.35 +/- 0.04 pH/min in tunicamycin-trea ted 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 illustrat ed that in tunicamycin-treated cells, NHE-3 mRNA expression increased threefold over control cells. Immunoblots of luminal membranes from co ntrol 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-tr eated cells showed only one strong band at 95 kd, NHE-3 immunoblots of whole cell extract from tunicamycin-treated cells showed that in addi tion to the 95 kd protein, an 87 kd band was also detected. These resu lts are consistent with the possibility that the two bands in the 94 a nd 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 L LC-PK1 cells. The results further suggest that glycosylation of NHE-3 mediates the translocation and insertion of this exchanger in the plas ma membrane.