Structure−Activity Relationships for the Interaction of Bovine Pancreatic Trypsin Inhibitor with an Intracellular Site on a Large Conductance Ca2+-Activated K+ Channel

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Abstract
Large conductance Ca2+-activated K+ channels (BKCa) contain an intracellular 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 mutants using single BKCa channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (Ca channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BKCa channel 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 Kunitz inhibitors based on current ideas for allosteric activation of BKCa channels by voltage and Ca2+.