Conformational Dynamics in the Selectivity Filter of KcsA in Response to Potassium Ion Concentration

Abstract

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 μM. Potassium ion titration studies reveal that the site-specific K_d for K⁺ binding at the key pore residue Val76 is on the order of ∼ 7 μM and that a relatively high sample hydration is necessary to observe the low-K⁺ conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K⁺ and low-K⁺ states are in slow exchange on the NMR timescale (k_ex < 500 s^− 1). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K⁺ state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.

Introduction

Potassium channels are highly conserved intrinsic membrane proteins that selectively conduct potassium ions across cell membranes near the rate of free diffusion. Although the structures of some potassium channels have been known for over 10 years,1, 2 important mechanistic questions remain open for debate. One key mechanistic question is how potassium channels achieve a very fast throughput rate while remaining highly selective for potassium over sodium. Selectivity in binding could involve a relatively high affinity for some intermediate of the process, which in turn could result in the slow kinetics of release or escape from that state. While there is indeed evidence that potassium ions bind with micromolar affinity³ inside potassium channels and with affinity more than 3 orders of magnitude better than other ions such as sodium,⁴ this does not impede the inverse passage time of a potassium ion through the channel from being ∼ 10^− 8 s.⁵ This fast conduction rate is interpreted as being diffusion limited. Therefore, the mechanisms by which diffusion-limited kinetics and high selectivity are simultaneously achieved have been of great interest.

KcsA is a 160-amino-acid potassium channel isolated from the soil bacterium Streptomyces lividans.6, 7 Because of its experimental convenience, high sequence homology, and structural similarity to key regions of mammalian potassium channels, it has become a model system for studies on the biophysical details of ion conduction and gating.8, 9, 10, 11, 12, 13, 14, 15 KcsA is a homotetrameric protein with two transmembrane (TM) helices per monomer and an ion-conducting pore in the center. The highly conserved selectivity filter motif for K⁺ channels consists of residues 74–79 (TVGYG). The earliest structures showed that the backbone carbonyl oxygens in this region specifically chelate dehydrated K⁺ inside the channel.

Crystal structures of KcsA2, 4 suggest that in the conductive state of the channel, four ion binding sites are formed by carbonyl oxygens in the pore. A potassium ion fits into each of these binding sites, thus satisfying its preference¹⁶ for an octahedral chelation environment. The ion–oxygen distance in the octahedral cages of KcsA ranges from 2.7 Å to 3.1 Å, with an average of 2.85 Å. This is close to the preferred ion–chelator distance in many biological octahedral K⁺ complexes such as valinomycin and noactin.2, 17 Sodium ions, on the other hand, prefer tetrahedral coordination environments and chelation distances of ∼ 2.4 Å when they interact with six fully flexible monodentate ligands.¹⁸ The ion binding sites in KcsA are thus optimized for potassium binding in terms of both symmetry and ion–oxygen distances. These sites are typically referred to as S1–S4. S1 resides on the extracellular side, and S4 resides on the intracellular side of the membrane. During conduction, the selectivity filter is proposed to exist in two ion configurations that may be in fast exchange: the occupancies of sites S1–S2–S3–S4 are K⁺–water–K⁺–water and water–K⁺–water–K⁺ for the two states, which will henceforth be referred to as the (1,3) and (2,4) states.¹⁶ These configurations are shown in Fig. 1a.

The crystal structures provide important support for the dominant hypothesis on the mechanism of ion selectivity: the inner ion binding sites (S2 and S3) provide perfect chelation environments for potassium, but do not accommodate many other ions, including sodium. This has been called the “snug-fit” mechanism and was first proposed by Bezanilla and Armstrong.¹⁹ The Pauling ionic radii for K⁺ and Na⁺ are 1.33 Å and 0.95 Å, respectively,⁵ and, as mentioned earlier, the preferred ion–oxygen distances differ by ∼ 0.4 Å. Thus, the proposed “snug-fit” mechanism has sometimes been cast as requiring the selectivity filter to retain a geometry with sufficient rigidity in the positions of the ligating carbonyls to discriminate between these ions. Ion flux measurements show that the relative free energy of selectivity ΔΔG_(Na–K) is on the order of 5–6 kcal/mol.20, 21 Free-energy perturbation computations22, 23 show that the intrinsic carbonyl repulsion interactions and flexibility of the channel residues are necessary to maintain the relative difference in free energy for binding potassium versus sodium. This led to an alternative hypothesis, which suggests that the carbonyl groups coordinating the ion in the filter are dynamic and that their intrinsic electrostatic and dynamic properties control ion selectivity.²⁴ The crystallographic B-factors for the selectivity filter are ∼ 15–20 Å². In principle, if the B-factors are interpreted in terms of RMS fluctuations in the atom's position,⁵ then $B=\frac{8{\pi }^2}{3}〈\text{Δ}{R}^2〉$, indicating an RMS fluctuation of ∼ 0.8 Å in the pore. While this seems large compared to the difference in atomic radii between K⁺ and Na⁺, when compared to other parts of the protein (B-factors, 30–40 Å²; RMS ∼ 1–1.3 Å), the B-factors in the pore are relatively low. Since the B-factors presumably report on both static disorder and dynamic disorder at low temperature, their relationship with dynamic disorder under functional temperatures and conditions is unclear. The snug-fit and dynamic pore hypotheses differ in their emphasis on how much flexibility the pore has and whether this is related directly to selectivity. The extent of pore plasticity must therefore be directly measured by characterizing the dynamic behavior of the residues in the selectivity filter in an appropriate environment.

Regardless of whether dynamics in the pore contribute to the microscopic mechanism for selectivity, it is clear that the selectivity filter is a center of activity during normal channel function. Characterizing its structural plasticity and dynamic behavior is crucial to understanding the transport cycle of K⁺ through the channel. NMR is a natural technique used to study protein dynamics. It serves as a powerful complement to X-ray crystallography, which cannot discriminate between static disorder and dynamic disorder. Solid-state NMR gives us the unique opportunity to study channel dynamics in a native bilayer environment. Methods used to study dynamics in the solid state have recently been applied to protein systems, and several studies25, 26, 27, 28, 29 have reported on protein dynamics at different timescales in the solid state.

Proof of the inherent plasticity of the channel emerged in a series of studies targeting structure as a function of the concentration of permeant ions.4, 30 When the ambient potassium concentration is reduced from 200 mM to 3 mM, the crystal structures suggest that an ion binds at the outer sites S1 and S4, but not at the inner sites S2 and S3. This structure can be called the (1,4) structure.⁴ In the absence of any ion in S2 and S3, the selectivity filter rearranges the key chelators V76 and G77. In the (1,4) structure, the S2 and S3 sites do not appear to be capable of chelating a potassium ion; instead, the V76 carbonyl forms a water-mediated hydrogen bond with G77 on the neighboring subunit.² The G77 C^α is twisted inward, and a new hydrogen-bond network consisting of a belt of buried water molecules surrounds the selectivity filter around S2 and S3. This structure is assumed to be nonconductive (although studies on this structure have been carried out at relatively high potassium concentrations of 3–5 mM), and it has sometimes been described as collapsed or pinched. This low-K⁺ conformation of the filter is shown in Fig. 1b.

We have chosen to initially focus on this dramatic transition between the low-K⁺ state and the high-K⁺ state in a bilayer environment. Previous characterizations11, 15 of this transition involved membrane environments that might be assumed to affect dynamics and ion affinity. We present a structural and qualitative dynamic characterization of the two limiting states of the KcsA selectivity filter as a function of permeant ion identity and concentration based on solid-state NMR spectroscopy, and we illustrate ways in which the bilayer environment is very important for detailed kinetics.