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.

Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR

“…Since NOE signals between the methyl group of Val-76 in SF and water were detected only in the impermeable state, we think that a water-binding instead of K + ion leads to the blockage of the ion permeabilization at the SF. It should be noted that the reduction of K + ion binding to the open state was also confirmed by recent solid-state NMR analysis of the constitutively open H25R/E118A mutant of KcsA embedded in a lipid bilayer at neutral pH [ 19 ].…”

Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface

“…Small perturbations therefore will be able to influence ionization states and membrane protein function. For example, a key glutamic acid residue E120 in the pH “gate” of the KcsA potassium channel is near a membrane interface and has a p K a that varies with the K + concentration . When measured in 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl- sn -glycero-3-phosphoserine (DOPS) at pH 7.5, this residue, E120, is protonated at 50 μM [K + ], yet deprotonated at 80 mM [K + ] .…”

Unraveling of a Strongly Correlated Dynamical Network of Residues Controlling the Permeation of Potassium in KcsA Ion Channel

“…This dynamical network ( Figure 8 ) is identified for the canonical structure 1KC4 with a closed inner gate. It is shown [ 35 , 36 ] that opening the inner gate leads to the perturbations on the backbones of the SF. Therefore, the inner gate should be included in this dynamical network as well.…”

“…These studies led to the suggestion of four channel’s states with an open or closed inner gate and a conducting or non-conducting SF [ 4 , 11 ]. One of the hypotheses [ 6 , 11 , 34 ] suggests that the activation by opening the inner gate simultaneously alters the SF via allosteric coupling [ 35 , 36 ]. This coupling leads to a slow (on a time scale of seconds) collapse of the SF to a non-conducting configuration.…”