Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR

Abstract

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.

Keywords: KcsA; ion channels; solid-state NMR; transmembrane allostery.

Fig. 1.

[K⁺] dependence of apo and bound state cross-peaks (marker peaks) near the selectivity filter in 2D ¹³C-¹³C correlation spectra of H25R/E118A KcsA at pH 7.5. (A) T75 CB-CA, (B) T74 CB-CA, (C) V76 CB-CG2, and (D) T75 CA-CG show that the marker peaks shift from apo state in low [K⁺] to bound state in high [K⁺]. The contour levels of the spectrum were set at 5 times noise level. Marker peaks could be integrated to calculate the population ratio of apo and bound states.

Fig. 2.

Potassium affinity of the selectivity filter of KcsA based on NMR is presented as a titration plot, presenting the ratio of bound K⁺ to bound-plus-apo as a function of ambient [K⁺]. The H25R/E118A mutant (blue) is compared with previously reported data for wild-type pH 7.5 (green) and wild-type pH 3.5 (magenta) (13). K_app of the open pH gate mutant was calculated to be 81 ± 1 mM by fitting the data to a Hill binding model, compared with 4 ± 1 μM for wild type at pH 7.5 and 14 ± 1 mM for wild type at pH 3.5 (13). These data show that the H25R/E118A mutant with a constitutively open pH gate exhibits a K⁺ affinity that is somewhat looser still as compared to the open wild-type KcsA at pH 3.5.

Fig. 3.

The effect of [K⁺] on the E120 residue in the pH gate of the H25R/E118A KcsA mutant at pH 7.5. (A) The 2D ¹³C-¹³C correlation spectrum of open pH gate KcsA at 75 mM [K⁺]. The peaks in the red and blue rectangles are the E120 CG−CD peaks in deprotonated and protonated state, respectively. (B) The protonated and deprotonated state change of the E120 peak across various [K⁺]. Protonated and deprotonated E120 CG−CD peaks at 50 μM and 10, 50, 75, and 80 mM are shown. At pH 7.5, E120 is protonated at low [K⁺]; while, at 80 mM [K⁺] and above, E120 is completely deprotonated. In samples with [K⁺] larger than 80 mM, the E120 CG−CD peaks are all in the deprotonated state. This protonation state change provides clear evidence for pK_a change of the glutamic acid residue.

Scheme 1.

The potassium channel KcsA changes are depicted undergoing a conversion from a conductive state (with selectivity filter loaded with K⁺ ions) to an inactivated K⁺-depleted state. We hypothesize that such a change provides the basis for spontaneous inactivation and is caused by the allosteric coupling between the pH gate and the selectivity filter. In this work, we characterize the H25R/E118A KcsA mutant where the pH gate is open, to study the K⁺ binding and inactivation. In our previous work, channel opening was achieved by lowering the pH, raising the possibility of nonspecific effects of pH (13). The conductive state and the inactivated state shown in the scheme are based on Protein Data Bank entries 5VK6 and 5VKE, respectively (44). The selectivity filter pH gate and potassium ions are rendered in blue yellow and magenta, respectively.