We have investigated the interactions of synthetic charybdotoxin (ChTX), synthetic iberiotoxin (IbTX), and recombinant mutant ChTX peptides with the Ca(2+)-activated K+ channel (Gardos pathway) in human and rabbit erythrocytes. We measured the binding of 125I-ChTX to erythrocytes, the displacement of bound 125I-ChTX by unlabeled toxin and analogs, and then compared these data with isotopic and electrical indices of channel activity measured under the same conditions. We found that a major portion of 125I-ChTX bound to red cells was displaceable by excess unlabeled ChTX. This specific 125I-ChTX binding to human red cells was markedly increased in low ionic strength conditions as compared with that measured at physiological ionic strength and at alkaline pH as compared with normal pH. At pH 8 and low ionic strength, specific binding could be described most simply as a single class of sites of Kd = 94 +/- 49 pM and Bmax = 120 +/- 36 sites/cell (n = 3). Ca(2+)-activated 86Rb 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 Kd for 125I-ChTX = 37 +/- 17 pM, with 126 +/- 24 binding sites/cell and an ID50 for inhibition of 86Rb influx by ChTX = 25 pM. Whereas IbTX neither inhibited Ca(2+)-activated 86Rb influx nor displaced 125I-ChTX in human red cells, it partially displaced 125I-ChTX and partially inhibited 86Rb influx in rabbit red cells. Studies with recombinant mutant ChTX peptides showed that the mutant toxin K27Q was inactive as a transport inhibitor and displayed a large reduction in ability to displace 125I-ChTX. The mutation K31Q resulted in abolition of ionic strength dependence of the inhibitory effect on the Ca(2+)-activated K+ permeability. In view of the similarity between the 125I-ChTX binding constant and the transport inhibition constant of ChTX, we examined the potency of 125I-ChTX as a transport inhibitor. 125I-ChTX inhibited Ca(2+)-activated K+ transport 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 physiologic ionic strength 125I-ChTX and ChTX are indistinguishable as inhibitors of erythroid Ca(2+)-activated K+ transport. However, iodination of Y36 is associated with abolition of the 200-fold increase in inhibitory potency shown by ChTX at low ionic strength.
Ca(2+)-activated K+ transport in erythrocytes. Comparison of binding and transport inhibition by scorpion toxins
DE FRANCESCHI, Lucia;
1993-01-01
Abstract
We have investigated the interactions of synthetic charybdotoxin (ChTX), synthetic iberiotoxin (IbTX), and recombinant mutant ChTX peptides with the Ca(2+)-activated K+ channel (Gardos pathway) in human and rabbit erythrocytes. We measured the binding of 125I-ChTX to erythrocytes, the displacement of bound 125I-ChTX by unlabeled toxin and analogs, and then compared these data with isotopic and electrical indices of channel activity measured under the same conditions. We found that a major portion of 125I-ChTX bound to red cells was displaceable by excess unlabeled ChTX. This specific 125I-ChTX binding to human red cells was markedly increased in low ionic strength conditions as compared with that measured at physiological ionic strength and at alkaline pH as compared with normal pH. At pH 8 and low ionic strength, specific binding could be described most simply as a single class of sites of Kd = 94 +/- 49 pM and Bmax = 120 +/- 36 sites/cell (n = 3). Ca(2+)-activated 86Rb 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 Kd for 125I-ChTX = 37 +/- 17 pM, with 126 +/- 24 binding sites/cell and an ID50 for inhibition of 86Rb influx by ChTX = 25 pM. Whereas IbTX neither inhibited Ca(2+)-activated 86Rb influx nor displaced 125I-ChTX in human red cells, it partially displaced 125I-ChTX and partially inhibited 86Rb influx in rabbit red cells. Studies with recombinant mutant ChTX peptides showed that the mutant toxin K27Q was inactive as a transport inhibitor and displayed a large reduction in ability to displace 125I-ChTX. The mutation K31Q resulted in abolition of ionic strength dependence of the inhibitory effect on the Ca(2+)-activated K+ permeability. In view of the similarity between the 125I-ChTX binding constant and the transport inhibition constant of ChTX, we examined the potency of 125I-ChTX as a transport inhibitor. 125I-ChTX inhibited Ca(2+)-activated K+ transport 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 physiologic ionic strength 125I-ChTX and ChTX are indistinguishable as inhibitors of erythroid Ca(2+)-activated K+ transport. However, iodination of Y36 is associated with abolition of the 200-fold increase in inhibitory potency shown by ChTX at low ionic strength.
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