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 Cl-36 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 V-max of the cotransporter was between 57 and 60 pmol/cm(2)/s. The apparent activation (K-App. 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 K-App 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 V-max were more complicated. When K+ was the limiting co-ion, there was little effect on the V-max for Na+ or Cl- activation. In contrast, limiting concentrations of Na+ or Cl- both resulted in a large reduction of the apparent V-max 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. (C) 1999 Elsevier Science B.V. All rights reserved.