Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca2+-activated K+ channel

Large conductance Ca2+-activated K+ channels (BKCa) contain an intracellula r binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI m utants using single BKCa channels from rat skeletal muscle incorporated int o planar lipid bilayers. All of the mutants induced discrete substate event s at the single-channel level. The dwell time of the substate, which is inv ersely related to the dissociation rate constant of BPTI, exhibited relativ ely small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive co rrelation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding si te. The substate current level was unaffected by most of the mutations exce pt for substitutions of Lys15. Different residues at this position were fou nd to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state a t +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elasta se. The finding that Lys15 is a determinant of the conductance behavior of the BKCa channel when BPTI is bound implies that the same inhibitory loop t hat contacts SerP's is located close to the protein interface in the BKCa c hannel complex. This supports the hypothesis that the C-terminal region of the BKCa channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kun itz inhibitors based on current ideas for allosteric activation of BKCa cha nnels by voltage and Ca2+.