Stereospecific Binding of an Intrinsically Disordered N-Terminal Inactivation Domain Distinguishes BK and Shaker Inactivation Mechanisms

Abstract

During fast inactivation of voltage-gated K+ channels (Kv) block of the conduction pathway occurs by the single-step insertion of mobile hydrophobic peptide segments into the channel inner cavity. In large-conductance, Ca2+ and voltage-dependent, K+ channels (BK) fast inactivation is mediated by cytosolic N-termini of accessory subunits, β2 or β3. Unlike Kv's, BK fast inactivation occurs in a 2-step mechanism: a binding step is postulated to precede the occurrence of the inactivated state. We hypothesized that, if the first step reflects binding, it may show stereospecificity. We therefore prepared two β3a(1-21) peptides corresponding, on one hand, to the naturally occurring peptide composed entirely of L-amino acids (L-β3a(1-21)), and then a peptide containing entirely the mirror-image D-amino acids (D-β3a(1-21)). By NMR, both peptides exhibit the hallmarks of intrinsically disordered proteins. The L-β3a(1-21) peptide fully mimics the 2-step inactivation produced by intact β3a subunits. In contrast, D-β3a(1-21) produces a 10-fold weaker block of BK current, with features characteristic of simple block. At the single channel level, we show that, whereas recovery from inactivation mediated by the β3a L-peptide passes through the 2-step sequence, recovery from block by the β3a D-peptide involves the classic single-step block behavior characteristic of the Shaker K+ channel. We also compared L- and D- forms of Shaker “ball domain” peptides. In both cases, recovery from inactivation occurs identically through a single-step mechanism. From these results two points emerge: first, binding of intrinsically disordered domains can occur either via stereospecific interactions (BK) or non-specifically (Shaker); second, the requirement for stereospecific binding in BK channels may relate to the large dimension of the BK inner pore such that stereospecific binding facilitates an unstructured domain to reach the blocking position.