Transmembrane allosteric energetics characterization for strong coupling between proton and potassium ion binding in the KcsA channel

Abstract

The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K⁺ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 Å from the K⁺ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.

Keywords: allostery; inactivation; membrane protein; potassium channel; solid-state NMR.

Fig. 1.

(A) Structural transitions in KcsA during channel function. In each structure, the K⁺ binding selectivity filter (including conserved residues T74, T75, V76, G77, Y78, and G79) is highlighted in blue; the pH sensor (E118, E120, H25, mutated in 3FB5 and 3F5W) where protons bind during activation is highlighted in red; and the hinge of the inner helix, a site of significant conformational dynamics during activation and allosteric coupling, is highlighted in yellow. The resting state, designated as deactivated (PDB ID code 1K4C) has K⁺ ions bound in the selectivity filter and is conductive, but has a closed intracellular gate, with a deprotonated pH sensor and the TM2 bundle crossed and occluded. This stable species is observed in numerous X-rays structures of KcsA. Following a drop in pH, the activated state, the only conductive state in functional assays, is populated transiently. In this state both the selectivity filter and the intracellular gate are conductive (PDB ID code 3FB5). The activated state slowly and spontaneously decays to an inactivated state (PDB ID code 3F5W). Our working hypothesis for inactivation is that the inactivated state differs from the activated state by loss of the K⁺ ions in the selectivity filter and associated conformational and hydration changes. Electrophysiology studies show that protein can recover from the inactivated state to the deactivated state at high pH, here represented by the dashed lines through a putative state inactivated* or I* that is “closed at both gates,” meaning both deprotonated at the pH sensor and K⁺ deplete or possibly by way of the activated state. (B) A thermodynamic cycle for H⁺ and K⁺ ion binding in the coupling network of KcsA. The blue dashed and angled arrow represents the activation followed by C-type inactivation as observed in pH jump electrophysiology experiments; the red dashed and angled arrow represents our observations of the result of lowering [K⁺] at constant neutral or slightly elevated pH (35). The allosteric coupling factor defines the strength of the allosteric coupling and is calculated as: α = K_apparent(3.5)/K_apparent(7.5) or α′ = K_a(K⁺ bound)/K_a(K⁺ apo). (C) Similar changes in chemical shift implying similar structural transitions were seen for the selectivity filter of KcsA. Similar chemical shifts are seen in the selectivity filter for K⁺-bound state, regardless of pH; similar chemical shifts are seen in the selectivity filter for K⁺-apo state, regardless of pH marker peaks V76 (CB-CG1/CG2), T75 (CB-CA), T74 (CB-CA) obtained from ¹³C–¹³C 2D correlation spectra are shown for a variety of [K⁺] and pH values. (Left) Contrasts KcsA at pH 7.5 (the deactivated state, in yellow) to that at pH 3.5 (activated state, in blue); both experiments are at high ambient [K⁺] (bound states). (Right) Contrasts the channel at pH 7.5 (inactivated* state, in red) to pH 3.5 (inactivated state, in cyan); both experiments are at low ambient [K⁺] (apo states). There are significant chemical shift changes between high [K⁺] (yellow and blue) and low [K⁺](red and gray) conditions, due to ion release and associated structural changes in the selectivity filter. Excellent agreement in the overlay of this region of the spectra comparing neutral vs. acidic pH illustrates that the structure transition at the selectivity filter at neutral and acidic pH are very similar and the selectivity filter is clearly intact throughout this pH range, i.e., the structure of selectivity filter is K⁺ dependent, and pH does not directly perturb it. Superscripts refer to the K⁺ apo and bound states respectively.

Fig. S1.

NMR spectra of the glutamic acid residues in the intracellular pH sensor controlling the activation gate of KcsA show that the pH gate residues Glu118 and Glu120 are protonated at pH 3.5 across a broad range of [K⁺], whereas at pH 7.5, the behavior is more complex, and at high [K⁺], the pH sensor residues are deprotonated as expected, but at low [K⁺], due to coupling between K⁺ ion release and H⁺ binding, the sensor residues are protonated based on the NMR spectra. (A–D) Spectra for samples at (respectively) pH 7.5 and 50 mM [K⁺], pH 7.5 and 0.2 μM [K⁺], pH 3.5 and 10 mM [K⁺], and pH 3.5 and 500 μM [K⁺]. The signals associated with Glu118 and Glu120’s CG-CD correlation show comparable chemical shifts in B–D, indicating that they are protonated, whereas in A they show clear indication of deprotonation. We conclude that the local environment of these residues are similar at acidic pH across a range of [K⁺], and also at neutral pH if the [K⁺] is very low (35).

Fig. 2.

Differences in K⁺ binding at neutral vs. acidic pH values for WT-KcsA. (A) ¹³C–¹³C correlation spectra at pH 3.5 show changes in the populations of apo vs. bound state as the [K⁺] increases; the cross-peaks (V76 CB-CG1) were integrated to calculate the relative populations (using these and additional peaks shown in Fig. S2; Materials and Methods). (B) A significant change in potassium ion affinity (α = 3,500) is observed comparing channels with an open activation gate measured at pH 3.5 (magenta) vs. a closed gate measured at pH 7.5 (green). The K_d values calculated by fitting the data to a noncooperative binding equation (Hill coefficient n = 1) are 4 ± 1 μM for pH 7.5 and 14 ± 1 mM at pH 3.5.

Fig. S2.

Marker peaks in NMR spectra showing the structural transition at the selectivity filter at acidic pH 3.5 as the environmental [K⁺] changes from 500 μM to 80 mM. The volumes of cross-peaks indicated by arrows are used to calculate the bound state population ratio and K_apparent calculations in Fig. 2B.

Fig. 3.

F103 is a critical residue in the allosteric pathway. (A) The potassium ion titration curves of mutant F103A are compared at neutral and acidic pH with those for the wild-type channel to contrast the affinity and coupling changes. (B) The allosteric coupling strength is significantly weaker for mutant F103A compared with WT-KcsA. (C) Specific chemical shift perturbations are observed for the F103A mutant, compared with the WT-KcsA, underscoring the important interactions between F103 and I100 and with T74. Peaks in spectrum are I100 (CB-CG1, CB-CG1 CB-CD), respectively. In WT-KcsA, I100 shows a perturbation in the peaks associated with the bound, putatively activated state: the peaks are missing or broadened due to dynamics or heterogeneity. Similarly, the bound state peaks for I100 in F103A mutant, where allostery has been reduced, are also missing; but the apo state peaks shows significant chemical shift change, especially at CB. (D) Similarly T74 sidechain correlations show changes in the apo state (putative inactivated state) between the wild-type and the F103A mutant. (E–H) Crystal structure analysis reveals that the steric contact among T74, I100, and A103 is reduced in the F103A mutant compared with WT-KcsA. Steric contact between F103 (red) and I100 (magenta) occurs between monomers in the tetrameric structure. Contact between F103 (red) and T74/T75 (blue), particularly in the apo state, can be seen for the WT sample in both the bound state (E) (PDB ID code 3FB5) and in the apo state (F) (PDB ID code 3F5W). Our data are consistent with a model in which the clash is largest for the activated state. This steric clash is relieved when the bulky F103 sidechain is mutated to alanine (G and H) (made with PyMol).

Fig. S3.

Spectra of WT-KcsA (blue) are compared with those of the F103A mutant (orange), exhibiting marker peaks in the selectivity filter. Superscripts refer to the K⁺ apo and bound states respectively. The chemical shift values for WT and F103A are nearly identical in both the bound and apo states, which simplifies our analysis (no reassignment needed) and indicates that F103A minimally perturbs the structure (see Fig. S4 for the full overlay). In contrast, the relative populations of the states change significantly between F103A (pH 3.5 10 mM K⁺, 9% apo) and wild type (pH 3.5 10 mM K⁺, 57% apo).

Fig. S4.

Comparison of 2D homonuclear ¹³C spectra of wild-type KcsA vs. F103A shows that the chemical shift changes displayed in F103A are specific to the hinge residues. As illustrated by this overlay, the great majority of peaks in the wild-type channel are identical to the peaks in F103A. Specific exceptional changes have been seen in I100 and T74, however, as are discussed in the text and Fig. 3.