Protonation state of E71 in KcsA and its role for channel collapse and inactivation

Abstract

The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K(+) titrations, we detect a local change at E71 that is coupled to channel collapse at low K(+). To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K(+) and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pK(a) (> 7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA-E71A does not inactivate and establish the ambient K(+) level as a means to populate the inactivated state of KcsA in a controlled way.

Fig. 1.

The network of hydrogen bonds that tether the selectivity filter to the pore helix and involve residues E71, D80, Y78, G77, T75, and W67 in KcsA are shown. (A) Shows the high K⁺, conductive state (PDB 1K4C). (B) Shows the collapsed, low K⁺ state (PDB 1K4D). Notice that although the E71–D80–W76 hydrogen bond network is generally maintained between the two states, the geometry of the network between E71 and D80 becomes more compact and the hydrogen bond Y78 N–E71 CO2 becomes much weaker at low potassium. A water molecule between E71 and D80 is included in the majority (approximately 80%) of all crystal structures of WT-KcsA with a resolution lower than 2.5 Å. Heavy-atom distances corresponding to the hydrogen bonds are listed in Table S1.

Fig. 2.

Sections of ¹³C–¹³C 2D spectra of both WT-KcsA and the inactivation-resistant mutant E71A are shown with key markers highlighted. Samples are in 9∶1 DOPE∶DOPS lipid bilayers and measured at 0–10 °C. (A) Spectra at pH 7.5 and two K⁺ concentrations, approximately 1 μM (red) and 25 mM (blue), show that the characteristic collapsed-state chemical shifts (V76* and T74*) are detected for the WT protein at low K⁺, but not detected for the mutant E71A at low ambient [K⁺], which instead remains conductive. The absence of the collapsed state for E71A and the presence for the WT channel is reproduced at several markers, including V76 Cβ-Cγ (30 ppm, 19.5 ppm) and T74 Cβ-Cγ (70.3 ppm, 20.5 ppm). (B) Spectra at high [K⁺] and two pH conditions, 3.5 (gray) and 7.5 (blue), are shown for both WT-KcsA and the mutant E71A. The WT low-pH spectra show some signature collapsed-state shifts suggesting that at low pH, a state similar to the collapsed state is sampled. These data show that, unlike the WT, the E71A filter does not collapse at low K⁺ or at low pH.

Fig. 3.

A summary of the linewidth and spectral quality of the KcsA mutant E71A at high and low potassium is shown. Samples are in 9∶1 DOPE∶DOPS lipid bilayers and measured at 0–10 °C. (A) The structural perturbation and heterogeneity observed at high potassium in a 2D correlation spectrum of the mutant (green) at sites W67, Y78, and D80 are shown and overlaid onto a WT-KcsA spectrum (blue) under the same conditions. The disordered and perturbed residues are mapped onto the structure of the KcsA filter with the conductive state (1K4C, blue) and the minor flipped conformation seen in some X-ray structures (like 2ATK, green). The narrow linewidth of marker peaks and other nearby residues indicates that at high K⁺, the mutant is correctly folded and the disorder likely results from two structurally distinct populations in a local region, and not a broad continuum of states. (B) An analysis of the Cα linewidths in the selectivity filter of wild-type KcsA and mutant E71A is shown. When [K⁺] is < 1 μM, residues in the wild-type filter shift but maintain a narrow NMR linewidth, whereas particular peaks in the mutant filter (Y78, V76, and T74) appear to be significantly broader. The data suggest that at low potassium, the selectivity filter in E71A exhibits significantly more disorder or dynamics than the wild-type filter.

Fig. 4.

The potassium-dependent isotropic chemical shifts of E71 and D80 are shown. (A) The ¹³C chemical shifts of E71 (Top) and D80 (Bottom) in WT-KcsA are plotted as a function of ambient [K⁺] at pH 7.5. The data show a large downfield movement in the side-chain carboxyl chemical shift of E71 (in yellow) as [K⁺] is increased, but no change in the backbone shifts. At [K⁺] = 10 μM, both states of E71 are observed, indicated by the filled and hollow markers. The D80 isotropic shifts remain unchanged by potassium. (B) Shows ¹³C–¹³C correlation spectra highlighting the E71 and D80 carboxyl chemical shifts at the endpoints of the titration. (C) The relative population of the deprotonated state of E71 based on its NMR signal intensity is overlaid with populations from another selectivity filter marker, V76. The V76 curve (fit to a two-state binding model) shows that the transition from the conductive state to the collapsed state occurs between [K⁺] = 1 μM and 10 μM. The data show that change in the E71 side chain occurs in the same potassium-concentration regime as the structural collapse of the filter based on K_d measurements of selectivity filter markers. Therefore these two processes are very likely coupled.

Fig. 5.

A summary of the chemical-shift anisotropy measurements at E71 and D80 is shown. Measurements were made at 25 mM K⁺, pH 7.5, and temperature of 0–10 °C. (A) The experimentally derived sideband intensities from slow magic-angle spinning spectra (black) are shown together with the best fit from SPINEVOLUTION (red). The error bars represent the average signal:noise of the spectrum. (A, Right) The simulated tensors based on the average values of a protonated (dark) and deprotonated (light) carboxyl are shown. The principle components of the chemical shift tensors of E71, D80, and two other control residues (A32 and E51) were derived from these fits and are listed in C. The errors reflect the standard deviation in the values obtained from the fit. (B) Plots the distribution of the δ11 component from a previously published database (22) of protonated (dark) and deprotonated (light) carboxyl tensors of known crystal structure. Our measured δ11 values for E71, D80, and E51 are overlaid in red onto this distribution. (The y-offset of E51 is for clarity.) From the data it is very clear that E71 is protonated and D80 and E51 are both deprotonated at neutral pH.