Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca 2 1 -activated K 1 channels (maxi-K Ca ) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K Ca channels in planar bilayers recorded in the presence of DTX-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 kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, 1 6) decreases by 11.7-fold ( K d , 17,500 nM) when DTX-I (net charge, 1 10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold ( K d , 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, 1 6) exhibits high blocking affinity ( K d , 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity ( K d , 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold ( K d , 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K Ca channel. key words: aprotinin • bovine pancreatic trypsin inhibitor • charybdotoxin • dendrotoxin • Kunitz inhibitor i n t r o d u c t i o n Large conductance ( z 250 pS) Ca 2 1 -activated K 1 channels (BK or maxi-K Ca channels) 1 are an intriguing class of ion channels that are activated in a synergistic fashion by intracellular Ca 2 1 and depolarizing voltage (Latorre, 1994). In Drosophila , maxi-K Ca channels are encoded by the Slowpoke gene, part of which is homologous to the family of voltage-gated K 1 channels (K V channels), including Shaker (Atkinson et al., 1991; Adelman et al., 1992). Both K V and Slowpoke channels contain a characteristic motif of six membrane-spanning segments called S1–S6. This motif includes the S4 segment involved in voltage sensing and the extracellular P-region between S5 and S6 that determines ionic selectivity. Besides these common structural elements, Slowpoke maxi-K Ca channels have an additional transmembrane segment at the NH 2 terminus, called S0, plus a unique sequence of z 800 residues at the COOH terminus (Meera et al., 1997). Substantial evidence supports the notion that the COOH-terminal portion of the maxi-K Ca channel protein corresponds to a large cytoplasmic domain that contains Ca 2 1 -binding site(s) involved in channel activation (Wei et al., 1994; Schreiber and Salkoff, 1997). To understand the mechanism of Ca 2 1 activation and biochemical regulation of maxi-K Ca channels, it is necessary to define the structure and function of this cytoplasmic domain. An underlying objective of the work described in this paper is to characterize intracellular binding sites for peptide Address correspondence to Dr. Edward Moczydlowski, Department of Pharmacology, Yale University School of Medicine, Sterling Hall of Medicine, P.O. Box 208066, New Haven, CT 06520-8066. Fax: 203785-7670; E-mail: [email protected] 1 Abbreviations used in this paper: BP, Shaker ball peptide homolog; BPTI, bovine pancreatic trypsin inhibitor; ChTX, charybdotoxin; DTX-I, Toxin I of Dendroaspis polylepis ; maxi-K Ca , large conductance Ca-activated K 1 channel; TEA, tetraethylammonium. on M ay 8, 2017 D ow nladed fom Published February 1, 1999