CA2-ACTIVATED K+ TRANSPORT IN ERYTHROCYTES - COMPARISON OF BINDING AND TRANSPORT INHIBITION BY SCORPION TOXINS()

We have investigated the interactions of synthetic charybdotoxin (ChTX ), synthetic iberiotoxin (IbTX), and recombinant mutant ChTX peptides with the Ca2+-activated K+ channel (Gardos pathway) in human and rabbi t erythrocytes. We measured the binding of I-125-ChTX to erythrocytes, the displacement of bound I-125-ChTX by unlabeled toxin and analogs, and then compared these data with isotopic and electrical indices of c hannel activity measured under the same conditions. We found that a ma jor portion of I-125-ChTX bound to red cells was displaceable by exces s unlabeled ChTX. This specific I-125-ChTX binding to human red cells was markedly increased in low ionic strength conditions as compared wi th that measured at physiological ionic strength and at alkaline pH as compared with normal pH. At pH 8 and low ionic strength, specific bin ding could be described most simply as a single class of sites of K(d) = 94 +/- 49 pM and B(max) = 120 +/- 36 sites/cell (n = 3). Ca2+-activ ated Rb-86 influx measured under identical conditions revealed an ID50 for ChTX of 21 +/- 15 pM (n = 6) at low ionic strength and 4 +/- 2.4 nM (n = 4) at physiological ionic strength. Similar studies in rabbit erythrocytes at low ionic strength revealed a K(d) for I-125-ChTX = 37 +/- 17 pM, with 126 +/- 24 binding sites/cell and an ID50 for inhibit ion of Rb-86 influx by ChTX = 25 pM. Whereas IbTX neither inhibited Ca 2+-activated Rb-86 influx nor displaced I-125-ChTX in human red cells, it partially displaced I-125-ChTX and partially inhibited Rb-86 influ x in rabbit red cells. Studies with recombinant mutant ChTX peptides s howed that the mutant toxin K27Q was inactive as a transport inhibitor and displayed a large reduction in ability to displace I-125-ChTX. Th e mutation K31Q resulted in abolition of ionic strength dependence of the inhibitory effect on the Ca2+-activated K+ permeability. In view o f the similarity between the I-125-ChTX binding constant and the trans port inhibition constant of ChTX, we examined the potency of I-125-ChT X as a transport inhibitor. I-125-ChTX inhibited Ca2+-activated K+ tra nsport with ID50 values of 3.3 +/- 1 nM (n = 7) at low ionic strength and 4.1 +/- 3 nM (n = 6) at physiologic ionic strength. Thus, at physi ologic ionic strength I-125-ChTX and ChTX are indistinguishable as inh ibitors of erythroid Ca2+-activated K+ transport. However, iodination of Y36 is associated with abolition of the 200-fold increase in inhibi tory potency shown by ChTX at low ionic strength.