Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: individual differences in the number of channels

Abstract

We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K+ (Ca-K) channel of human red cells (RBC). Cytosolic Ca2+ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na+ media containing CaCl2. The Ca-K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8 +/- 2.4 mmol/liter cell.hr, to 3.7 +/- 1.0 mmol/liter cell.hr by 6 nM CTX (n = 3). The kinetic of Ca-K efflux was studied by increasing cell ionized Ca2+ using A23187 (60 mumol/liter cell), and buffering with EGTA or citrate; initial rates of net K+ efflux (90 mmol/liter cell K+) into Na+ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca-K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca2+ was raised, with a Km of 0.37 microM and saturating between 2 and 10 microM Ca2+. Ca-K efflux was partially blocked (71 +/- 7.8%, mean +/- SD, n = 17) by CTX with high affinity (IC50 0.8 nM), a finding suggesting that is a high affinity ligand of Ca-K channels. CTX also blocked 72% of the Ca-activated K+ efflux into 75 mM K+ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K+ channel in Na+ media. CTX did not block Valinomycin-activated K+ efflux into Na+ or K+ medium and therefore it does not inhibit K+ movement coupled to anion conductive permeability. The Vmax, but not the Km-Ca of Ca-K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell.min (FU). In red cells with Hb A, Vmax was 9.36 +/- 3.0 FU (mean +/- SD, n = 17). The Vmax of the CTX-sensitive, Ca-K efflux was 6.27 +/- 2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75 +/- 3.2 FU, n = 8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of the Vmax of Ca-K+ efflux. Estimation of the number of CTX-sensitive Ca-activated K+ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K+ efflux (2.7 +/- 0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.