Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells

Area of Science:

• Cell Biology
• Renal Physiology
• Membrane Transport

Background:

• The PKE20 mutant of LLC-PK1 renal epithelial cells exhibits elevated apical Na(+)-H+ antiporter activity.
• Previous studies indicated altered transporter function in whole cell experiments.

Purpose of the Study:

• To investigate the membrane-level properties of the Na(+)-H+ antiporter in the PKE20 mutant.
• To characterize the specific form of the Na(+)-H+ antiporter affected by the mutation.

Main Methods:

• Preparation of apical membrane vesicles from parent and PKE20 LLC-PK1 cells using magnesium-aggregation.
• Assay of Na(+)-H+ antiporter activity via 22Na influx under varying pH gradients.
• Inhibition studies using ethylisopropylamiloride (EIPA) to differentiate transporter forms.
• Analysis of other Na(+)-dependent transporters and polypeptide composition.

Main Results:

• PKE20 vesicles demonstrated a 4.2-fold increase in maximal velocity of Na(+)-H+ antiporter activity compared to parent cells.
• The enhanced activity was primarily attributed to the amiloride-resistant form of the antiporter.
• Minor increases were observed in other Na(+)-dependent transporters; no changes in polypeptide composition were detected.

Conclusions:

• The PKE20 mutation affects Na(+)-H+ antiporter activity at the membrane level in renal epithelial cells.
• The mutation specifically enhances the activity of the amiloride-resistant Na(+)-H+ antiporter.
• These findings provide insights into the molecular basis of altered renal transporter function.