23 22 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Macromolecular Crystallography Group Beamlines I03, I04 and I23 Understanding the flowof potassium ions across cell membranes Related publication: LolicatoM., Natale A. M., Abderemane-Ali F., Crottès D., Capponi S., Duman R.,Wagner A., Rosenberg J. M., GrabeM. &Minor D. L. K 2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions. Sci. Adv. 6 , eabc9174 (2020). DOI: 10.1126/sciadv. abc9174 Publication keywords: K 2P potassium channel; K 2P 2.1 (TREK-1); C-type gate; Selectivity filter T he flow of potassium ions across the cell membrane is regulated by K 2P potassium channels, controlling electrical activity in the brain, heart and nervous system. These channels have important roles in pain, migraine, depression, anaesthetic responses, arrhythmias, hypertension and lung injury responses. K 2P channels regulate ion flow using a selectivity filter (C-type) gate. However, previous structural studies have not captured the gating mechanisms, and they remain poorly understood. Currently, there are no drugs available that selectively target K 2P potassium channels. To develop newways to control their function, we need to understandmore about the gating mechanisms and their roles in human biology. In work published in Science Advances , researchers from Diamond Light Source, University of California, San Francisco and the University Pittsburgh, USA, combined X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics and electrophysiology to uncover the changes that occur in K 2P C-type gating. At Diamond, the team used the Long-Wavelength Macromolecular Crystallography (MX) beamline (I23) to observe changes in potassium ions within the channel directly. I23 is the only beamline in the world optimised to measure signals from potassium ions directly, and its unique capabilities were central to these studies. The results show that K 2P gating involves pinching and dilation of two key elements of the channel selectivity filter gate. These structural changes are accompanied by the loss of potassium ions. Including a small molecule activator (ML335) suppressed these changes and demonstrated how stabilisation of the selectivity filter gate facilitates ion flow through the channel. These studies show that small molecule activators bind to and stabilise the K 2P selectivity filter gate, preventing the pinching and dilation conformational changes and loss of potassium ions that lead to channel inactivation. These findings open a path to develop newK 2P channel- directed drugs to treat pain, ischemia (restricted blood flow), depression and lung injury. K 2P potassium channels respond to diverse physical and chemical inputs that include pressure, temperature, pH, signalling lipids, phosphorylation and volatile anaesthetics 1 . Their actions control electrical activity in the nervous, cardiovascular and immune systems by regulating the flow of potassium ions across the cell membrane through the action of their selectivity filter (C-type) gate 2,3 . K 2P s are dimers of subunits that each contain two conserved elements, SF1 and SF2, that form the selectivity filter (Fig. 1). These channels have important roles in pain, migraine, depression, anaesthetic responses, arrhythmias, hypertension and responses to lung injury. Yet, despite much effort, K 2P s have remained the most poorly understood potassium channel class and suffer from a poor pharmacological profile that limits our ability to investigate their biological functions. Selectivity filter C-type gating occurs in many potassium channel classes and has a hallmark sensitivity to potassium due to interactions between the permeant potassium ions and selectivity filter. Structural studies of homotetrameric potassium channels have uncovered various selectivity filter rearrangements attributed to C-type gating, but whether C-type gating involves selectivity filter pinching, dilation or more subtle structural changes has remained unclear. Further, prior K 2P structural studies have failed to identify selectivity filter conformational changes that could explain how K 2P C-type gating occurs despite showing conformational changes in the channel transmembrane helices that impact activity. This lack of a structural framework has left open questions regarding the extent to which K 2P C-type gating mechanisms resemble homotetrameric channels and whether the innate heterodimeric K 2P selectivity filter architecture confers unique properties to their C-type gates. Crystal structures of the thermo- andmechano-sensitive K 2P , K 2P 2.1 (TREK- 1), alone and complexed with a small molecule activator of the C-type gate, ML335 4 , under a series of seven different potassium concentrations revealed unprecedented conformational changes in the K 2P selectivity filter. These structures show that under low potassium concentrations (<50 mM [K + ]) both elements of the K 2P 2.1(TREK-1) selectivity filter, SF1 and SF2 , undergo potassium-dependent structural rearrangements that are accompanied by the loss of the two potassium ions in the outer portions of the selectivity filter (Fig. 1). These conformational changes pinch SF1 at the extracellular mouth of the pore and dilate the exterior portion of SF2 and unfold the linker that connects SF2 to transmembrane helix 4 (SF-M4 loop). Hence, K 2P s use both types of mechanisms that have been proposed to control the function of potassium channel selectivity filter C-type gates, pinching and dilation. Binding of the small molecule activator ML335 to a site behind the selectivity filter, the K 2P modulator pocket 4 , completely suppresses these conformational changes and the loss of potassium ions (Fig. 1). Measurement of anomalous scattering from potassium ions using long wavelength crystallographic methods 5 enabled by the unique capabilities of beamline I23 provided essential structural information about the changes in the occupancy of potassium ions in the selectivity filter. These studies showed that under high potassium conditions (200 mM [K + ]) all four potassium- binding sites were occupied regardless of the presence of the activator ML335. By contrast, all four selectivity filter potassium ion binding sites remained occupied under low potassium conditions (1 mM [K + ]) only when ML335 was bound (Fig. 1). These observations were corroborated by extensive molecular dynamics studies and single channel electrophysiology measurements that established that ML335 stabilisation of the filter enhances ion flow and channel open probability. These studies also identified a key functional role for the uniquely long SF2-M4 loop. This channel element unfolds when SF2 dilates, is stabilised by the binding of ML335, and connects the C-type gate with gating cues sensed by other parts of the channel 2 . These findings show that asymmetric order-disorder transitions enabled by the K 2P heterodimeric architecture are at the heart of K 2P gatingmechanisms. They support a model in which the K 2P C-type gate transits between an inactive state in which there is a high degree of selectivity filter disorder having low potassium ion occupancy to a rigidified conductive state inwhich all potassium binding sites are occupied 4 . Selectivity filter mobility is influenced by diverse classes of physical and chemical signals as well as agents that stabilise the SF2-M4 loop such as ML335. Such findings pave the way for the development of new chemical or biologic K 2P modulators that target critical parts of the channel and highlight the potential of the SF2-M4 loop as a key control point for pharmacological intervention. K 2P - directed agents based on these findings may provide new avenues for treating physiological problems in which K 2P channels are important such as pain, migraine, ischemia and lung injury. References: 1. Feliciangeli S. et al. The family of K 2P channels: Salient structural and functional properties. J. Physiol. 593 , 2587–2603 (2015). DOI: 10.1113/ jphysiol.2014.287268 2. Bagriantsev S. N. et al. Multiple modalities converge on a common gate to control K 2P channel function. EMBO J. 30 , 3594–3606 (2011). DOI: 10.1038/emboj.2011.230 3. Piechotta P. L. et al. The pore structure and gating mechanism of K 2P channels. EMBO J. 30 , 3607–3619 (2011). DOI: 10.1038/emboj.2011.268 4. Lolicato M. et al. K 2P 2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. Nature 547 , 364–368 (2017). DOI: 10.1038/ nature22988 5. Wagner A. et al. In-vacuum long-wavelength macromolecular crystallography. Acta Crystallogr. Sect. D Struct. Biol. 72 , 430–439 (2016). DOI: 10.1107/S2059798316001078 Funding acknowledgement: National Institutes of Health NIMH R01-MH093603 (D.L.M). Corresponding author: Dr Daniel Minor, Cardiovascular Research Institute, University of California, San Francisco, USA, USA, Lawrence Berkeley National Laboratory, USA,
[email protected] Figure 1: Structural changes in the K 2P 2.1 (TREK-1) C-type selectivity filter gate. (Centre) overall structure of K 2P 2.1 (TREK-1) (solid) highlighting SF2 and surrounding structures (wire frame), ML335 (black, space filling), and potassium ions (purple spheres). Orange arrows show K + ion flow through the channel. Grey bars indicate the membrane. (Left) SF1 structures in low [K + ] showing the conductive conformation with ML335 (top) and pinched, inactive conformation (bottom). (Right) SF2 structures in low [K + ] showing the conductive conformation with ML335 (top) and dilated, inactive conformation (bottom). K + ions are shown as purple spheres. ML335 is shown in space filling representation. Select selectivity filter residues are indicated.