Metal-driven operation of the human large-conductance voltage- and Ca2+-dependent potassium channel (BK) gating ring apparatus

Abstract

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

FIGURE 1.

Amino acid sequence, purification, and secondary structure analysis of the recombinant BK CTD. A, BK channel model based on the transmembrane domain of the Kv1.2–2.1 chimera (Protein Data Bank code 2R9R) (18) and the BK gating ring (code 3NAF) (23). The gating ring, formed by four RCK1 (green) and four RCK2 (blue) domains, assembles below the transmembrane domain. Residues known to be involved in high-affinity Ca²⁺ sensitivity in the BK gating ring are depicted in red (Asp³⁶²/Asp³⁶⁷ in RCK1 and the calcium bowl in RCK2) or orange (Met⁵¹³ in RCK1). B, SDS-PAGE of the purified CTD shows a single band of estimated molecular mass (lane M) of ∼72 kDa. C, the experimental far-UV CD spectrum of the purified BK CTD is shown as a black curve, and the theoretical CONTINLL-calculated curve is shown superimposed in red. A change in molar absorption is defined as Δϵ m⁻¹ cm⁻¹. See supplemental Fig. S2A for secondary structure parameters.

FIGURE 2.

BK C termini assemble into tetramers in solution. A, representative elution profile of BK CTD from a Superdex 200 10/300 column. The peak (at 10.3 ml) corresponds to M_r = 326,000, as estimated from the calibration curve constructed with proteins of known molecular mass (thyroglobulin, β-amylase, bovine serum albumin, and carbonic anhydrase (Carb. Anhyd.)), shown in B. The mean molecular weight estimated from 15 column elutions was 324,000 ± 4,000. As a theoretical CTD tetramer would have M_r = 78,000 × 4 = 312,000, these data support the view that in solution, the purified CTD forms tetrameric structures, likely corresponding to the BK gating ring.

FIGURE 3.

Ca²⁺ and Mg²⁺ induce conformational changes in the BK gating ring. A and B show superimposed time-resolved fluorescence intensity decays of the gating ring native Trp residues recorded in increasing [Ca²⁺] and [Mg²⁺], respectively. The intrinsic Trp fluorescence emission was measured at 340 nm (λ_ex = 296 nm). The fluorescence lifetime is progressively reduced by the addition of micromolar free Ca²⁺ or millimolar Mg²⁺, suggesting cation-induced conformational change(s). The instrument response time (IRF) is shown in gray. Fitting parameters for the Ca²⁺ and Mg²⁺ experiments are in supplemental Tables S1 and S2, respectively.

FIGURE 4.

The divalent cation selectivity of the purified BK gating ring. A, normalized representative steady-state emission spectra of the gating ring in solution when excited at 295 nm, acquired under increasing [Ca²⁺]. Note the dose-dependent quenching effect. B, as in A, except for increasing [Mg²⁺] in a nominally Ca²⁺-free buffer. C, as in B, except for increasing [Ba²⁺] up to 13 mm. Note that the lack of Ba²⁺ effects on the gating ring intrinsic Trp fluorescence. After adding Ba²⁺, the [Ca²⁺] was increased to 1.2 μm to ascertain gating ring functionality (red trace). D, the fluorescence intensity (at 340 nm) from experiments as in A–C is plotted versus the concentration of divalent cations (●, Ca²⁺; ■, Mg²⁺; ▴, Ba²⁺). The experimental points were fit to double (Ca²⁺) or single (Mg²⁺) Hill functions. The apparent affinity of the gating ring for Ca²⁺ was: K1½ = 0.29 ± 0.0043 μm (n₁ = 3.4 ± 0.16) and K2½ = 3.5 ± 0.51 μm (n₂ = 2.1 ± 0.35), and for Mg²⁺: K½ = 154 ± 20.7 μm (n = 2.02 ± 0.44) (n = 3).

FIGURE 5.

Neutralization of the calcium bowl region reduces the Ca²⁺ sensitivity of the gating ring and abolishes Mg²⁺ sensitivity. A, the intrinsic Trp fluorescence intensity decay of a gating ring carrying the D894A,D895A,D896A,D897A,D898A neutralization within the RCK2 domain, recorded in nominal zero (blue) or 35 μm (red) free Ca²⁺. Increasing the [Ca²⁺] to 35 μm Ca²⁺ produced a modest change in the τ_avg, as compared with the wild-type gating ring (Fig. 3A). IRF, instrument response time. B and C are fluorescence emission spectra of the calcium bowl mutant gating ring in solution excited at 295 nm. B, the Trp fluorescence intensity decreased as the free [Ca²⁺] of a solution containing the calcium bowl mutant gating ring was increased from nominal zero to 35 μm. C, unlike the wild-type gating ring, the calcium bowl mutant did not exhibit Mg²⁺ sensitivity, up to 12 mm. D, the fluorescence intensity at 350 nm from experiments in panels B and C is plotted versus the concentration of divalent cations (●, Ca²⁺; , ■, Mg²⁺). The experimental points are fit to a Hill function with K½ = 2.0 ± 0.19 μm and n = 2.1 ± 0.35.

FIGURE 6.

Ca²⁺ reversibly reduces the hydrodynamic radius of the gating ring in solution. A, a top view of the BK gating ring (23), which has a toroidal structure with a diameter ∼9–12 nm, a height ∼5 nm, and a central aperture of ∼2 nm. Residues known to be involved in high-affinity Ca²⁺ sensitivity in the BK gating ring are depicted in red (Asp³⁶²/Asp³⁶⁷ in RCK1 and the calcium bowl in RCK2) or orange (Met⁵¹³ in RCK1). B, gating ring particle distributions obtained from a characteristic dynamic light scattering experiment. Only the dominant (70%) distributions below 60 nm are shown. The mean R_H of the particle distribution in nominally 0 Ca²⁺ (blue) was 10.1 nm. Increasing the free [Ca²⁺] to 35 μm shifted the distribution toward smaller R_H values, with a mean of 7.8 nm (red), consistent with a transition to a more compact conformation. Reducing free Ca²⁺ from 35 to 0.07 μm by the addition of EGTA (green) practically restored the original distribution (9.5 nm), demonstrating that the Ca²⁺-induced rearrangements of the gating ring are reversible. C, a characteristic DLS experiment on the gating ring before (R_H = 9.70 nm; blue) and after (R_H = 8.65 nm; orange) the addition of 12 mm Mg²⁺. D, overall R_H reduction by the addition of saturating ligand concentration to gating ring particles in solution, expressed as the percentile change from the mean ligand-free R_H (R_{H_Apo}). Error bars indicate S.E.