Toxin pharmacology of the large-conductance Ca2+-activated K+ channel in the apical membrane of rabbit proximal convoluted tubule in primary culture

The patch-clamp technique was used to study the toxin pharmacology of the large-conductance Ca²⁺-activated K⁺ channel (BKCa) present in the apical membrane of rabbit proximal convoluted tubules (PCT) in primary culture. Experiments were performed with the inside-out configuration. This channel was very selective for K⁺ against Na⁺ and had a conductance of 180 pS with 140 mmol/l in the pipette and the bath. The action of toxins was studied on the extracellular side of the channel by using the pipette perfusion technique. Experimental conditions were 140 mmol/l KCl in the pipette and 140 mmol/l Nad in the bath. Pipette potential was maintained at 0 mV. Perfusion of crude venom from Leiurus quinquestriatus hebraeus inhibited reversibly the open probability (P_o) in a concentration-dependent fashion (IC₅₀=0.8 mg/l; n=3). The following synthetic or purified toxins were tested: synthetic charybdotoxin (ChTX) IC₅₀=7.3×10^–9 M (n=5); iberiotoxin (IbTX) IC₅₀=5.5×10^–7 mol/l (n=3); and kaliotoxin (KTX) IC₅₀=4.8×10^–7 mol/l (n=3). The suppression of the six first N-terminal amino-acids slightly reduced the affinity of ChTX (IC₅₀=1.2×10^–8 mol/l, n=4). Neither Dendroaspis polylepis venom nor purified dendrotoxin modified P_o even at high concentrations (20 mg/l and 10^–6 mol/l respectively). Apamin, which blocked the small-conductance K⁺ channel in cultured PCT, did not act on BKCa. These results indicate that ChTX is the most efficient known toxin against the epithelial BKCa in primary cultures of PCT. In spite of there being considerable homology of sequence between ChTX, IbTX and KTX, ChTX was about 100 times more effective than the others. Truncated ChTX kept a high affinity for this channel and could be used to obtain a labelled probe.