Potassium-chloride cotransport in resealed human red cell ghosts

Area of Science:

• Cell Physiology
• Membrane Transport
• Erythrocyte Biology

Background:

• Potassium (K) influx in human erythrocytes is crucial for maintaining cell volume and function.
• Furosemide-sensitive K influx and K-Cl cotransport are known mechanisms in intact red blood cells.
• N-ethylmaleimide (NEM) is known to stimulate K influx in intact erythrocytes via sulfhydryl group alkylation.

Purpose of the Study:

• To investigate the differences in furosemide-inhibitable K influx between resealed erythrocyte ghosts and intact cells.
• To elucidate the mechanisms underlying the enhanced K influx observed in resealed erythrocyte ghosts.
• To compare the effects of N-ethylmaleimide (NEM) on K influx in intact cells versus resealed ghosts.

Main Methods:

• Measurement of furosemide-inhibitable potassium (K) and sodium (Na) influx in resealed human erythrocyte ghosts and intact cells.
• Assessment of the role of chloride (Cl) and sodium (Na) in the enhanced K influx in ghosts.
• Investigation of the effect of N-ethylmaleimide (NEM) and dithiothreitol (DTT) on K influx in ghosts and intact cells.
• Evaluation of K influx sensitivity to changes in cell volume in resealed ghosts.

Main Results:

• Furosemide-inhibitable K influx was threefold higher in resealed erythrocyte ghosts compared to intact cells.
• This enhanced K influx was specific for K, required chloride (Cl), and was independent of sodium (Na).
• N-ethylmaleimide (NEM) did not enhance, and slightly inhibited, K influx in resealed ghosts, unlike in intact cells.
• Dithiothreitol (DTT) prevented the increase in K influx in ghosts, indicating a role for sulfhydryl group oxidation.
• K influx in resealed ghosts demonstrated sensitivity to cell volume changes, similar to NEM-treated intact cells.

Conclusions:

• The enhanced furosemide-inhibitable K influx in resealed erythrocyte ghosts is attributed to oxidation of membrane protein sulfhydryl groups during the lysis process.
• This oxidation mechanism differs from the alkylation mechanism targeted by N-ethylmaleimide (NEM) in intact cells.
• The findings suggest that membrane protein sulfhydryl group status significantly influences K transport in erythrocytes, particularly under conditions of cell lysis and volume changes.