TOXIN PHARMACOLOGY OF THE LARGE-CONDUCTANCE CA2-ACTIVATED K+ CHANNEL IN THE APICAL MEMBRANE OF RABBIT PROXIMAL CONVOLUTED TUBULE IN PRIMARYCULTURE()

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 conduct ance 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 usi ng the pipette perfusion technique. Experimental conditions were 140 m mol/l KCl in the pipette and 140 mmol/l NaCl in the bath. Pipette pote ntial was maintained at 0 mV. Perfusion of crude venom from Leizurus q uinquestriatus hebraeus inhibited reversibly the open probability (P-0 ) in a concentration-dependent fashion (IC50 = 0.8 mg/l; n = 3). The f ollowing synthetic or purified toxins were tested: synthetic charybdot oxin (ChTX) IC50 = 7.3 X 10(-9) M (n = 5); iberiotoxin (IbTX) IC50 = 5 .5 X 10(-7) mol/l (n = 3); and kaliotoxin (KTX) IC50 = 4.8 X 10(-7) mo l/l (n = 3). The suppression of the six first N-terminal amino-acids s lightly reduced the affinity of ChTX (IC50 = 1.2 X 10(-8) mol/l, n = 4 ). Neither Dendroaspis polylepis venom nor purified alpha dendrotoxin modified P-0 even at high concentrations (20 mg/l and 10(-6) mol/l res pectively). 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. Ln spite of there being considerable homology of seq uence between ChTX, IbTX and KTX, ChTX was about 100 times more effect ive than the others. Truncated ChTX kept a high affinity for this chan nel and could be used to obtain a labelled probe.