Stereospecific binding of a disordered peptide segment mediates BK channel inactivation

Two-step BK channel inactivation mediated by an intrinsically disordered BK β-subunit peptide involves a stereospecific binding interaction that precedes blockade. After K+ ion channels open, they frequently 'inactivate' when a cytosolic segment of the channel moves into a position that can physically occlude ion permeation through the channel pore. This study shows that a specific intrinsically disordered peptide domain of an ion-channel subunit of the Ca2+-activated BK channel produces its functional effects through stereospecific binding. The BK β3a auxiliary subunit peptide inactivates the associated BK channel only when it is composed of L-amino acids, and not D-amino acids. This means that the two-step inactivation process requires that a stereospecific-binding event — not just simple steric occlusion of the pore — must occur if the auxiliary subunit is to block the channel. A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments1, but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation2,3,4,5. Many K+ channel proteins, after initial channel opening, show a time-dependent reduction in current flux, termed ‘inactivation’, which involves movement of mobile cytosolic peptide segments (approximately 20–30 residues) into a position that physically occludes ion permeation6,7,8. Peptide segments that produce inactivation show little amino-acid identity6,9,10,11,12,13 and tolerate appreciable mutational substitutions13 without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures14,15,16,17. Channel inactivation mechanisms may therefore help us to decipher how intrinsically disordered regions mediate functional effects. Whereas many aspects of inactivation of voltage-dependent K+ channels (Kv) can be described by a simple one-step occlusion mechanism6,7,18,19, inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary β-subunits involves two distinguishable kinetic steps20,21. Here we show that two-step inactivation mediated by an intrinsically disordered BK β-subunit peptide involves a stereospecific binding interaction that precedes blockade. In contrast, blocking mediated by a Shaker Kv inactivation peptide is consistent with direct, simple occlusion by a hydrophobic segment without substantial steric requirement. The results indicate that two distinct types of molecular interaction between disordered peptide segments and their binding sites produce qualitatively similar functions.