Simultaneous binding of basic peptides at intracellular sites on a large conductance Ca2+-activated K+ channel - Equilibrium and kinetic basis of negatively coupled ligand interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibit or (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-a ctivated K+ channels (maxi-K-Ca) by binding to an intracellular site outsid e of the pore to produce discrete substate events. In contrast, certain hom ologues of the Shaker ball peptide produce discrete blocking events by bind ing within the ion conduction pathway. In this study, we investigated ligan d interactions of these positively charged peptide molecules by analysis of single maxi-Kc, channels in planar bilayers recorded in the presence of DT X-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K-d, 16.5 nM) and BPTI (K-d, 1,490 nM) exhibit one-site binding kine tics when studied alone; however, records in the presence of DTX-I plus BPT I demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K-d, 17,500 nM) when DTX-I (n et charge, +10) is bound and, conversely, the affinity of DTX-I decreases b y 10.8-fold (K-d, 178 nM) when BPTI is bound. The ball peptide homologue (B P; net charge, +6) exhibits: high blocking affinity (K-d, 7.2 nM) at a sing le site when studied alone, but has 8.0-fold lower affinity (K-d, 57 nM) fo r blocking the DTX-occupied channel. The affinity of DTX-I likewise decreas es by 8.4-fold (K-d, 139 nM) when BP is bound. These results identify two t ypes of negatively coupled ligand-ligand interactions at distinct sites on the intracellular surface of maxi-K-Ca channels. Such antagonistic ligand i nteractions explain how the binding of BPTI or DTX-I to four potentially av ailable sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and as ymmetric changes in transition state energies for the interaction between D TX-I and BP originate from repulsive electrostatic interactions between pos itively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetrae thylammonium or charybdotoxin on the outside of the maxi-K-Ca channel.