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Modulation of Function, Structure and Clustering of K Channels by Lipids: Lessons Learnt from Kcsa

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Modulation of Function, Structure and Clustering of K Channels by Lipids: Lessons Learnt from Kcsa International Journal of Molecular Sciences Review Modulation of Function, Structure and Clustering of K+ Channels by Lipids: Lessons Learnt from KcsA María Lourdes Renart 1 , Ana Marcela Giudici 1, Clara Díaz-García 2 , María Luisa Molina 1 , Andrés Morales 3, José M. González-Ros 1,* and José Antonio Poveda 1,* 1 Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), and Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, Elche, E-03202 Alicante, Spain; [email protected] (M.L.R.); [email protected] (A.M.G.); [email protected] (M.L.M.) 2 iBB-Institute for Bioengineering and Bioscience, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal; [email protected] 3 Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, E-03080 Alicante, Spain; [email protected] * Correspondence: [email protected] (J.M.G.-R.); [email protected] (J.A.P.) Received: 5 March 2020; Accepted: 5 April 2020; Published: 7 April 2020 Abstract: 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. Keywords: lipid–protein interactions; C-type inactivation; membrane protein folding; ion channel clustering; ion binding; KcsA modulation 1. Introduction Ion channels are a superfamily of membrane proteins involved in a myriad of cellular processes such as the control of cell excitability, signal transduction, muscle contraction or synaptic transmission. Thus, their dysfunction is associated with numerous diseases related to the nervous, cardiovascular, respiratory, endocrine, urinary and immune systems, and has been termed channelopathies [1–3]. Our knowledge on these proteins has increased exponentially in the last decades since the atomic structure of a few ion channels has been solved, mainly through X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. KcsA, a potassium channel from the Streptomyces lividans bacteria, was the first ion channel whose atomic structure was solved [4–6]. This fact, the relative ease to purify it in relative large amounts, and its endurance to experimental conditions have made KcsA the main model to study the structure/function relationships in ion channels. This protein is formed by four identical subunits arranged around a central aqueous pore through which ions flow. Each subunit comprises cytosolic N-terminal and C-terminal domains and two α-helical transmembrane Int. J. Mol. Sci. 2020, 21, 2554; doi:10.3390/ijms21072554 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2554 2 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 19 segments (TM1 and TM2). Between the two transmembrane segments there is a short, tilted helix ions flow. Each subunit comprises cytosolic N-terminal and C-terminal domains and two α-helical spanningtransmembrane inward to aboutsegments one-third (TM1 and of the TM2). membrane Between thicknessthe two transmembrane and oriented towards segments the there centre is a of the tetramer,short, followed tilted helix by aspanning stretch ofinward amino to acids about containing one-third theof the consensus membrane sequence thickness75 TVGYGand oriented79, known as thetowards selectivity the centre filter (SF).of the This tetramer, is the followed narrowest by parta stretch of the of amino protein acids pore containing and, as its the name consensus reveals, is + highlysequence selective 75TVGYG to define79, known which kindas the of selectivity ions can passfilter through(SF). This it. is Thus,the narrowest it allows part K permeation,of the protein while preventingpore and, the as physiologically its name reveals, relevant is highly Na selectiv+ frome to flowing define which through kind the of ions pore. can Below pass through it, an aqueous it. vestibuleThus, forms,it allows shaped K+ permeation, by the TM2 while segments. preventing There, the physiologically ions are fully relevant hydrated Na and+ from become flowing partly stabilizedthrough near the thepore. inner Below mouth it, an aqueous of the narrower vestibule selectivityforms, shaped filter by the by theTM2 four segments. dipolar There, pore ions helices, whichare point fully their hydrated negatively and become charged partly C-terminal stabilized ends near toward the inner the mouth hydrated of the ions narrower (Figure 1selectivityA). However, filter by the four dipolar pore helices, which point their negatively charged C-terminal ends toward residues lining this cavity exhibit a low affinity by surrounding ions and only modestly discriminate the hydrated ions (Figure 1A). However, residues lining this cavity exhibit a low affinity by + + betweensurrounding Na and ions K .and only modestly discriminate between Na+ and K+. FigureFigure 1. Scheme 1. Scheme of theof the KcsA KcsA structure. structure. ( A(A)) CrystallographicCrystallographic structure structure of ofthe the full-length full-length KcsA KcsA in the in the closedclosed state (PDBstate (PDB entry: entry: 3EFF). 3EFF). Main Main structures structures and domainsand domains are highlighted. are highlighted. (B) Zoom (B) Zoom of the of selectivity the filterselectivity (SF) structure, filter including(SF) structure, the signatureincluding the sequence signature TVGYG. sequence The TVGYG. inactivation The inactivation triad (E71-D80-W67) triad location(E71-D80-W67) is also shown location in blue is also sticks. shown The in SF blue can sticks adopt. The a non-conductiveSF can adopt a non-conductive or collapsed or conformation collapsed at conformation at low K+ concentration (<5 mM) (PDB entry; 1K4D). Increasing the amount of the low K+ concentration (<5 mM) (PDB entry; 1K4D). Increasing the amount of the permeant cation leads permeant cation leads to a shift of the equilibrium to a conductive state, where all four K+ binding to a shift of the equilibrium to a conductive state, where all four K+ binding sites can be identified (PDB: sites can be identified (PDB: 1K4C). 1K4C). In the resting state, the TM2 segments form a cross-section close to their C-terminal through Inelectrostatic the resting interactions, state, the which TM2 effectively segments prevent form apassage cross-section of ions. However, close to theira low C-terminalintracellular throughpH electrostaticcauses the interactions, protonation which of residues effectively involved prevent in such passage interactions, of ions. triggering However, a large a low hinge-bending intracellular pH causesmotion the protonation away from the of fourfold residues symmetry involved axis in that such opens interactions, the aqueous triggering vestibule ato large the cytoplasmic hinge-bending motionmedia, away eventually from the allowing fourfold ion symmetry flow. This axis open that channel opens thestate, aqueous however, vestibule is not stable to the and cytoplasmic the media,selectivity eventually filter allowing evolves within ion flow. a few This seconds open channelfrom a resting state, however,conductive is state not stableto a non-conductive and the selectivity filterconformation evolves within known a few as secondsthe inactivated from astate, resting thus conductive determining state the time to a during non-conductive which the channel conformation is knownactive. as the Three inactivated residues, state,E71, D80 thus and determining W67, called thethe inactivation time during triad, which have the been channel identified is active. as key Three elements to stabilize this conformation [7–9]. They are located just behind the selectivity filter and residues, E71, D80 and W67, called the inactivation triad, have been identified as key elements to interact through a web of hydrogen bonds between themselves and the selectivity filter, thus, stabilize this conformation [7–9]. They are located just behind the selectivity filter and interact through a influencing its conformation (Figure 1B). Once the pH is restored to neutrality, TM2 segments cross web ofback hydrogen to the closed bonds position between while themselves the selectivity and thefilter selectivity again adopts filter, the thus, resting influencing conductive its state. conformation Thus (Figure 1B). Once the pH is restored to neutrality, TM2 segments cross back to the closed position while the selectivity filter again adopts the resting conductive state. Thus the concerted movement of these Int. J. Mol. Sci. 2020, 21, 2554 3 of 19 two gates, the TM2 crossing (inner gate) and the selectivity
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