Mechanism of swelling activation of K-Cl cotransport in inside-out vesicles of LK sheep erythrocyte membranes.
Stimulation by swelling of K-Cl cotransport was studied in inside-out vesicles (IOVs) made from membranes of LK sheep erythrocytes. The purpose was to understand this stimulation in terms of the three-state process proposed for regulation of the cotransporter (P.B. Dunham, J. Klimczak, and P.J. Logue. J. Gen. Physiol. 101: 733-765, 1993). The first step in this process, A --> B, is rate limiting and controlled by transphosphorylation reactions. The second step, B --> C, is fast; its control is unknown. Predictions were that maximum velocity (Jmax) of cotransport increases with A --> B and concentration at one-half Jmax (K1/2) of K+ as a substrate decreases with B --> C. We tested the hypothesis that most transporters in IOVs are in the B state and that swelling activates cotransport in vesicles by the B --> C conversion. In accordance with this hypothesis, swelling should activate K+ influx with no discernable delay. It did. K1/2 for K+ should decrease with swelling and Jmax should not change. K1/2 decreased 10-fold, and Jmax did not change. Inhibitors of transphosphorylation, reactions of A --> B, should not affect K+ flux into IOVs, and they did not. The results support the hypothesis: swelling activation of K+ flux into IOVs corresponds to B --> C. A mechanical change in the membrane causes a specific change in the cotransporter: an increase in apparent affinity for K+.
Kelley
S J
SJ
Department of Biology, Syracuse University, New York 13244, USA.
Dunham
P B
PB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006706:Homeostasis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D012756:Sheep; D019311:Staurosporine / Q000494:pharmacology; D027981:Symporters; D013997:Time Factors; D000096922:K Cl- Cotransporters
D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D002712:Chlorides; D005033:Ethylmaleimide; D008274:Magnesium; D008387:Marine Toxins; D010080:Oxazoles; D011188:Potassium; D019311:Staurosporine; D027981:Symporters; D000096922:K Cl- Cotransporters; C059041:calyculin A