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Nature
2012 May 03;4857396:133-6. doi: 10.1038/nature10994.
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Stereospecific binding of a disordered peptide segment mediates BK channel inactivation.
Gonzalez-Perez V
,
Zeng XH
,
Henzler-Wildman K
,
Lingle CJ
.
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A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments, but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation. 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 permeation. Peptide segments that produce inactivation show little amino-acid identity and tolerate appreciable mutational substitutions without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures. 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 mechanism, inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary β-subunits involves two distinguishable kinetic steps. 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.
Figure 2. Intrinsically disordered D- and L- β3a peptides block BK channels, but only the L-peptide produces unique tail current behavior(a) BK current was activated with 10 μM cytosolic Ca2+ with the indicated voltage protocol; 0 (black), 4 (red), and 10 (blue) μM L-β3a(1-21) peptide. (b) Effects of 4 and 10 μM L-peptide shown on a faster time base. (c) Current integrals of tail currents from panel b. Same time base as in b. (d) Currents from another patch with 0, 10, and 40 μM D-β3a(1-21) peptide. (e) Tail currents from (d) on a faster time base. (f) Current integrals of tail currents from panel (e). Same time base as (e). (g) Single BK channel (10 μM Ca2+) showing control trace, two traces with 10 μM L-peptide and two traces with 10 μM D-peptide. (h) Faster time base examples of traces in (g) highlighting differences in tail current openings, consistent with indicated models. Red bar: time of repolarization.
Figure 3. L- and D- Shaker peptides block Shaker-IR channels in a similar fashion(a) Currents in inside-patches activated with the indicated voltage protocol with (blue) and without (black) 10 μM L-Shaker peptide. (b) Currents with (red) and without (black) 10 μM D-Shaker peptide. (c) Tail currents from (a) at expanded time base. (d) Tail currents from (b) at expanded time base. (a) and (b) are from the same patch. (e) A single Shaker-IR channel was activated and tail currents monitored during application of 100 μM L- or D-Shaker peptides. Bottom panel shows averages of 95 sweeps for no peptide (black), and 105 sweeps for D-(red) and L-peptides (blue). (f) Traces from (e) at higher time resolution. Red bar: time of repolarization. Tail openings after block by D- or L-peptide occur with an average delay of 0.51 ms (97 openings) and 0.57 ms (95 openings), respectively.
Figure 4. BK pore blockers compete with inactivation, but not N-terminus binding(a) Tail currents simulated from Model 3 (Fig. S8a3) as [blocker] increases. (b) Model predictions for normalized tail current amplitude (IBlocker/ICntrl) and tail current decay (τBlocker/τCntrl) as a function of [Blocker]/Kd(0) (binding constant at 0 mV). (c) BK α subunits coexpressed with construct D20A (β3a N-terminus appended to β2 subunit, see Methods). Currents were activated with 10 μM Ca2+, with the indicated voltage steps, along with 2 (red) or 10 (blue) mM TBA. (d) Effect of [TBA] on tail current amplitude and time constant plotted assuming Kd(0) for TBA is 0.9 mM. (e) BK α+D20A traces for control solution and with 200 and 400 μM bbTBA. (f) Effect of bbTBA on tail current amplitude and time constant with a Kd(0) for bbTBA block of 6 μM.
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