Activation of Na+,K+,Cl- cotransport in squid giant axon by extracellular ions: evidence for ordered binding.
Activation of the influx mode of the Na+,K+,Cl- cotransporter (NKCC) by extracellular Na+, K+ and Cl- was studied using the internally dialyzed squid giant axon. Cooperative interactions among the three transported ions were assessed using ion activation of NKCC-mediated 36Cl influx under two sets of experimental conditions. The first, or control condition, used high, non-limiting concentrations of two of the cotransported ions (the co-ions) while activating cotransport with the third ion. Under this non-limiting co-ion condition the calculated Vmax of the cotransporter was between 57 and 60 pmol/cm2/s. The apparent activation (KApp, or half-saturation) constants were: K+, 9 mM; Na+, 52 mM; and Cl-, 146 mM. The second condition used limiting co-ion concentration conditions. In this case, activation by each ion was determined when one of the other two co-ions was present at or near its apparent half-saturation concentration as determined above. Under these limiting conditions, the KApp values for all three co-ions were significantly increased regardless of which co-ion was present at a limiting concentration. The effects on the apparent Vmax were more complicated. When K+ was the limiting co-ion, there was little effect on the Vmax for Na+ or Cl- activation. In contrast, limiting concentrations of Na+ or Cl- both resulted in a large reduction of the apparent Vmax when activating with the other two co-ions. These results are consistent with an ordered binding mechanism for the NKCC in which K+ binds before Na+ or Cl-. Physiological implications for these results are discussed.
Altamirano
A A
AA
Department of Physiology, MCP Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129, USA.
Breitwieser
G E
GE
Russell
J M
JM
D000818:Animals; D001369:Axons / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology; D049832:Decapodiformes; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D002712:Chlorides; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D011188:Potassium