State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

Abstract

Large-conductance voltage- and calcium-dependent potassium channels (BK, "Big K+") are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a "gating-ring" structure has been proposed to transduce Ca(2+) binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca(2+) binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.

Fig. 1.

Localization of the fluorescent reporters in the BK gating-ring structure. (A) Topological representation of the BK α-subunit. The transmembrane region includes the voltage sensing domain (S1–S4), the pore domain (S5–S6) and an additional transmembrane helix (S0) that leads the N terminus to the extracellular side. The large cytoplasmic region consists of two RCK domains (RCK1 and RCK2). The orange asterisk indicates high-affinity Ca²⁺ binding site at RCK2 (calcium bowl). Amino acid positions where the fluorescent protein sequence was inserted are shown in blue (BK-860) or in magenta (BK-667). The green asterisks indicate amino acids flanking the disordered loop containing the 667 site. The native human BK numbering (23) is given for the constructs used in this study. (B) Structures of fluorescent proteins CFP and YFP (cyan and yellow, respectively) are represented as linked at position 667 in each subunit of the Ca²⁺-free gating ring structure (3NAF) (10). In this model, the neighboring fluorophores are 95 Å apart. Quantified RNA was injected in a 3:1 ratio into Xenopus oocytes to express most frequently the fluorescent heterotetramer 3CFP:1YCFP BK (Fig. S1) that is illustrated. (C) Ca²⁺-free (PDB ID code 3NAF; Left) and Ca²⁺-bound “open” (PDB ID code 3U6N; Right) structures of the gating ring, viewed toward the membrane plane. Colors follow the same code as the scheme shown in A. The 667 sites were resolved only in the Ca²⁺-free structure (Left; highlighted in magenta). For comparison, boundaries of regions comprising the 667 sites (F609 and K684) are labeled in both structures (green). The 860 site resides in an unresolved loop region between positions D833 and T872 (blue). (D) Side view of the gating ring with the same sites highlighted. The transmembrane region would lie to the right in this view. (Scale bars in C and D: 100 Å.)

Fig. 2.

Ca²⁺ and voltage induce structural rearrangements of the gating ring of intact BK channels in membrane macropatches. (A) Depolarizing pulses (upper traces) were applied to a Xenopus oocyte inside-out patch in 12 µM Ca²⁺, evoking the current traces shown beneath. Depolarizations were followed by a −70 mV repolarizing pulse and the holding potential was −100 mV. (B) Fluorescence spectra collected from the patch in A during steady-state activation (gray bar in A; Fig. S2). Excitation with 458-nm light produced mixed CFP and YFP emission (Upper), whereas 488-nm light drove only YFP emission (Lower). Traces are drawn with the same color coding according to voltage (color scale bar). (C) FRET efficiency E calculated from spectra at all studied voltages. The solid line is a Boltzmann fit. (D) Representative BK-860CY currents recorded from a Xenopus oocyte inside-out patch in various Ca²⁺ concentrations but with a fixed depolarization to +80 mV. Current traces are color-coded according to the free Ca²⁺ concentration (legend in E). (E) Synchronous fluorescence recordings for the same patch as D. (F) The dependence of FRET efficiency (E) on [Ca²⁺] (circles) was fitted to a Hill function (solid line) with k_d = 16 µM and n = 1.8.

Fig. 3.

Relative movement of the 860 sites shows Ca²⁺ dependence. G–V curves (upper panels) and FRET efficiency (E–V) curves (lower panels) were determined synchronously from patches at various free Ca²⁺ concentrations, for BK-860CY heterotetramers (A), and (B) mutants with disrupted Ca²⁺ sensitivity at the Ca²⁺ bowl (BK-860/5D5A/CY), or (C) at both the calcium bowl and the RCK1 Ca²⁺ binding site (BK-860/5D5A/M513I/CY). Data are color-coded in all graphs according to free Ca²⁺ concentration. The solid curves in A (Upper) represent fits to the Sweet and Cox (16) model with parameters shown in Table S1. Data points and error bars represent average ± SEM (n = 3–13).

Fig. 4.

Relative movement of the 667 sites is Ca²⁺ and voltage dependent. G–V curves (Upper) and the corresponding E–V curves (Lower) were determined synchronously at various free Ca²⁺ concentrations, for (A) BK-667CY heterotetramers, (B) mutants with reduced Ca²⁺ sensitivity at the Ca²⁺ bowl (BK-667/5D5A/CY), or (C) mutations at both the Ca²⁺ bowl and the RCK1 Ca²⁺ binding site (BK-667/5D5A/M513I/CY). Data are color-coded in all graphs according to free Ca²⁺ concentrations. The solid lines in A–C (Upper) represent fits to the Sweet and Cox (16) model with parameters shown in Table S1. Data points and error bars represent average ± SEM (n = 3–13).

Fig. 5.

Model of the gating ring movement. (A) Side view of the Ca²⁺-free gating ring (3NAF structure) before (Left) and after (Right) applying a rotation of 30° (indicated with arrows) of each subunit around the individual rotation axes shown. The transmembrane region would lie above the gating ring in this view. The Ca²⁺ bowl is shown in orange, and the S6 linkers in red. (B) Corresponding top view. Note the predicted separation of the S6 segments (black square). (C) Bottom view of the fluorescent fusion BK-667CY gating ring model. In addition to separating the S6 linker segments, the 30° rotation of each RCK1–RCK2 module moves the fused fluorescent proteins at positions 667 closer together by 40 Å (Right).