Conformational Dynamic Studies of Prokaryotic Potassium Channels Explored by Homo-FRET Methodologies.

Fluorescence techniques have been widely used to shed light over the structure-function relationship of potassium channels for the last 40-50 years. In this chapter, we describe how a Förster resonance energy transfer between identical fluorophores (homo-FRET) approach can be applied to study the gating behavior of the prokaryotic channel KcsA. Two different gates have been described to control the K+ flux across the channel's pore, the helix-bundle crossing and the selectivity filter, located at the opposite sides of the channel transmembrane section. Both gates can be studied individually or by using a double-reporter system. Due to its homotetrameric structural arrangement, KcsA presents a high degree of symmetry that fulfills the first requisite to calculate intersubunit distances through this technique. The results obtained through this work have helped to uncover the conformational plasticity of the selectivity filter under different experimental conditions and the importance of its allosteric coupling to the opening of the activation (inner) gate. This biophysical approach usually requires low protein concentration and presents high sensitivity and reproducibility, complementing the high-resolution structural information provided by X-ray crystallography, cryo-EM, and NMR studies.

Steady-state and time-resolved fluorescent methodologies to characterize the conformational landscape of the selectivity filter of K+ channels.

A variety of equilibrium and non-equilibrium methods have been used in a multidisciplinary approach to study the conformational landscape associated with the binding of different cations to the pore of potassium channels. These binding processes, and the conformational changes resulting therefrom, modulate the functional properties of such integral membrane properties, revealing these permeant and blocking cations as true effectors of such integral membrane proteins. KcsA, a prototypic K+ channel from Streptomyces lividans, has been extensively characterized in this regard. Here, we revise several fluorescence-based approaches to monitor cation binding under different experimental conditions in diluted samples, analyzing the advantages and disadvantages of each approach. These studies have contributed to explain the selectivity, conduction, and inactivation properties of K+ channels at the molecular level, together with the allosteric communication between the two gates that control the ion channel flux, and how they are modulated by lipids.

Anionic Phospholipids Shift the Conformational Equilibrium of the Selectivity Filter in the KcsA Channel to the Conductive Conformation: Predicted Consequences on Inactivation.

Here, we report an allosteric effect of an anionic phospholipid on a model K+ channel, KcsA. The anionic lipid in mixed detergent-lipid micelles specifically induces a change in the conformational equilibrium of the channel selectivity filter (SF) only when the channel inner gate is in the open state. Such change consists of increasing the affinity of the channel for K+, stabilizing a conductive-like form by maintaining a high ion occupancy in the SF. The process is highly specific in several aspects: First, lipid modifies the binding of K+, but not that of Na+, which remains unperturbed, ruling out a merely electrostatic phenomenon of cation attraction. Second, no lipid effects are observed when a zwitterionic lipid, instead of an anionic one, is present in the micelles. Lastly, the effects of the anionic lipid are only observed at pH 4.0, when the inner gate of KcsA is open. Moreover, the effect of the anionic lipid on K+ binding to the open channel closely emulates the K+ binding behaviour of the non-inactivating E71A and R64A mutant proteins. This suggests that the observed increase in K+ affinity caused by the bound anionic lipid should result in protecting the channel against inactivation.

Silica/Proteoliposomal Nanocomposite as a Potential Platform for Ion Channel Studies.

The nanostructuration of solid matrices with lipid nanoparticles containing membrane proteins is a promising tool for the development of high-throughput screening devices. Here, sol-gel silica-derived nanocomposites loaded with liposome-reconstituted KcsA, a prokaryotic potassium channel, have been synthesized. The conformational and functional stability of these lipid nanoparticles before and after sol-gel immobilization have been characterized by using dynamic light scattering, and steady-state and time-resolved fluorescence spectroscopy methods. The lipid-reconstituted KcsA channel entrapped in the sol-gel matrix retained the conformational and stability changes induced by the presence of blocking or permeant cations in the buffer (associated with the conformation of the selectivity filter) or by a drop in the pH (associated with the opening of the activation gate of the protein). Hence, these results indicate that this novel device has the potential to be used as a screening platform to test new modulating drugs of potassium channels.

Molecular Events behind the Selectivity and Inactivation Properties of Model NaK-Derived Ion Channels.

Y55W mutants of non-selective NaK and partly K+-selective NaK2K channels have been used to explore the conformational dynamics at the pore region of these channels as they interact with either Na+ or K+. A major conclusion is that these channels exhibit a remarkable pore conformational flexibility. Homo-FRET measurements reveal a large change in W55-W55 intersubunit distances, enabling the selectivity filter (SF) to admit different species, thus, favoring poor or no selectivity. Depending on the cation, these channels exhibit wide-open conformations of the SF in Na+, or tight induced-fit conformations in K+, most favored in the four binding sites containing NaK2K channels. Such conformational flexibility seems to arise from an altered pattern of restricting interactions between the SF and the protein scaffold behind it. Additionally, binding experiments provide clues to explain such poor selectivity. Compared to the K+-selective KcsA channel, these channels lack a high affinity K+ binding component and do not collapse in Na+. Thus, they cannot properly select K+ over competing cations, nor reject Na+ by collapsing, as K+-selective channels do. Finally, these channels do not show C-type inactivation, likely because their submillimolar K+ binding affinities prevent an efficient K+ loss from their SF, thus favoring permanently open channel states.

Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements.

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel's selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+-KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67-W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+-bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

Modulation of Function, Structure and Clustering of K+ Channels by Lipids: Lessons Learnt from KcsA.

KcsA, a prokaryote tetrameric potassium channel, was the first ion channel ever to be structurally solved at high resolution. This, along with the ease of its expression and purification, made KcsA an experimental system of choice to study structure-function relationships in ion channels. In fact, much of our current understanding on how the different channel families operate arises from earlier KcsA information. Being an integral membrane protein, KcsA is also an excellent model to study how lipid-protein and protein-protein interactions within membranes, modulate its activity and structure. In regard to the later, a variety of equilibrium and non-equilibrium methods have been used in a truly multidisciplinary effort to study the effects of lipids on the KcsA channel. Remarkably, both experimental and "in silico" data point to the relevance of specific lipid binding to two key arginine residues. These residues are at non-annular lipid binding sites on the protein and act as a common element to trigger many of the lipid effects on this channel. Thus, processes as different as the inactivation of channel currents or the assembly of clusters from individual KcsA channels, depend upon such lipid binding.

Accessibility of Cations to the Selectivity Filter of KcsA in the Inactivated State: An Equilibrium Binding Study.

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore "resting-like" under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD's of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K⁺, decreases considerably with respect to the resting channel. This is likely to cause a loss of K⁺ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K⁺ coming from the channel vestibule when K⁺ exits the cell. Moreover, binding of the nonpermeant species, Na⁺, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant.

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl ß-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.