Conformational Changes and Gating at the Selectivity Filter of Potassium Channels (original) (raw)
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The Structure of the Potassium Channel: Molecular Basis of K + Conduction and Selectivity
Science, 1998
The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K + channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K + channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K + ions but not smaller Na + ions. The selectivity filter contains two K + ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive for...
Biophysical Journal, 2009
A dihedral energy correction (CMAP) term has been recently included in the CHARMM force field to obtain a more accurate description of the peptide backbone. Its importance in improving dynamical properties of proteins and preserving their stability in long molecular-dynamics simulations has been established for several globular proteins. Here we investigate its role in maintaining the structure and function of two potassium channels, Shaker K v 1.2 and KcsA, by performing molecular-dynamics simulations with and without the CMAP correction in otherwise identical systems. We show that without CMAP, it is not possible to maintain the experimentally observed orientations of the carbonyl groups in the selectivity filter in Shaker, and the channel loses its selectivity property. In the case of KcsA, the channel retains some selectivity even without CMAP because the carbonyl orientations are relatively better preserved compared to Shaker.
Journal of General Physiology, 2021
C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intrace...
A gate mechanism indicated in the selectivity filter of the potassium channel KscA
Theoretical Chemistry Accounts, 2007
Classical molecular dynamics (MD) and non-equilibrium steered molecular dynamics (SMD) simulations were performed on the molecular structure of the potassium channel KcsA using the GROMOS 87 force fields. Our simulations focused on mechanistic and dynamic properties of the permeation of potassium ions through the selectivity filter of the channel. According to the SMD simulations a concerted movement of ions inside the selectivity filter from the cavity to extracellular side depends on the conformation of the peptide linkage between Val76 and Gly77 residues in one subunit of the channel. In SMD simulations, if the carbonyl oxygen of Val76 is positioned toward the ion bound at the S3 site (gate-opened conformation) the net flux of ions through the filter is observed. When the carbonyl oxygen leaped out from the filter (gate-closed conformation), ions were blocked at the S3 site and no flux occurred. A reorientation of the Thr75-Val76 linkage indicated by the CHARMM-based MD simulations performed Berneche and Roux [(2005) Structure 13:591–600; (2000) Biophys J 78:2900–2917] as a concomitant process of the Val76-Gly77 conformational interconversion was not observed in our GROMOS-based MD simulations.
Ion selectivity in potassium channels
Potassium channels are tetrameric membrane-spanning proteins that provide a selective pore for the conduction of K + across the cell membranes. One of the main physiological functions of potassium channels is efficient and very selective transport of K + ions through the membrane to the cell. Classical views of ion selectivity are summarized within a historical perspective, and contrasted with the molecular dynamics (MD) simulations free energy perturbation (FEP) performed on the basis of the crystallographic structure of the KcsA phospholipid membrane. The results show that the KcsA channel does not select for K + ions by providing a binding site of an appropriate (fixed) cavity size. Rather, selectivity for K + arises directly from the intrinsic local physical properties of the ligands coordinating the cation in the binding site, and is a robust feature of a pore symmetrically lined by backbone carbonyl groups. Further analysis reveals that it is the interplay between the attractive ion-ligand (favoring smaller cation) and repulsive ligand-ligand interactions (favoring larger cations) that is the basic element governing Na + /K + selectivity in flexible protein binding sites. Because the number and the type of ligands coordinating an ion directly modulate such local interactions, this provides a potent molecular mechanism to achieve and maintain a high selectivity in protein binding sites despite a significant conformational flexibility.
Conformational plasticity in the KcsA potassium channel pore helix revealed by homo-FRET studies
Scientific Reports, 2019
Potassium channels selectivity filter (SF) conformation is modulated by several factors, including ionprotein and protein-protein interactions. Here, we investigate the SF dynamics of a single Trp mutant of the potassium channel KcsA (W67) using polarized time-resolved fluorescence measurements. For the first time, an analytical framework is reported to analyze the homo-Förster resonance energy transfer (homo-FRET) within a symmetric tetrameric protein with a square geometry. We found that in the closed state (pH 7), the W67-W67 intersubunit distances become shorter as the average ion occupancy of the SF increases according to cation type and concentration. The hypothesis that the inactivated SF at pH 4 is structurally similar to its collapsed state, detected at low K + , pH 7, was ruled out, emphasizing the critical role played by the S2 binding site in the inactivation process of KcsA. This homo-FRET approach provides complementary information to X-ray crystallography in which the protein conformational dynamics is usually compromised. Potassium channels are integral membrane proteins present in prokaryotic and eukaryotic organisms where they contribute to the control of potassium flow, cell volume, release of hormones and neurotransmitters, resting potential, excitability, and behavior. Under physiological conditions these molecules are highly selective, allowing the permeation of K + at near diffusion-limited rates, whereas Na + is effectively excluded from passing through 1. So far, the molecular basis of selectivity and permeation is still a matter of debate. KcsA is a proton-activated, voltage-modulated channel cloned from S. lividans and used as a prototypical protein on the biophysical studies of the K + channels due to its simple structure: four identical subunits around a central pore, each one comprising an N-terminal domain, two transmembrane segments and the cytosolic C-terminal section. The two transmembrane segments (TM1 and TM2) are connected by a pore region that contains a tilted short-helix (pore helix), two loops and an ion selectivity filter (SF) (Fig. 1A). The SF contains four putative K + binding sites delineated by the signature sequence TVGYG (Fig. 1B,C), clearly homologous to the more complex eukaryotic potassium channels and its conformational dynamics is mainly responsible for the permeation and the selectivity features of these membrane proteins 2,3. Based on the conformation of the intracellular bundle (intracellular gate or gate I) and the SF (gate II), the gating cycle of KcsA is defined by at least four kinetic states: closed/conductive (pH7, high K +), open/conductive (transient state found in high K + after the pH4 gating), open/inactivated (pH4, high K + , at steady state) and closed/inactivated (pH7, high K + , immediately after increasing the pH from 4 to 7) 4. The opening movement at gate I is allosterically coupled to changes in SF conformation, inducing the slow inactivation process, and consequently leading to a steady-state, very low open probability of the wild-type (WT) channel 5,6. The inactivation phenomenon after several milliseconds of activity occurs in a very similar way to eukaryotic channels 7,8. Additionally, X-ray crystallography data from the closed state revealed two different conformations of the SF: a conductive conformation (Fig. 1B,C) in the presence of sufficient amounts of permeant cations (e.g. K + , Rb + or Cs +), and a non-conductive or collapsed structure when in low concentration of permeant species or high quantities of blocking species such as Na + , with only the most external binding sites (S1 and S4) accessible to ion-protein interaction (Fig.
A Gate in the Selectivity Filter of Potassium Channels
bent at a conserved glycine residue acting as a hinge (Jiang et al., 2002), presents a general paradigm for 1 Department of Physiology and Biophysics such a gating mechanism. Although this is less well Weill Medical College of Cornell University understood, several lines of evidence suggest that the 1300 York Avenue opening and closing of K + channels may implicate the New York, New York 10021 selectivity filter itself (i.e., that the selectivity filter might have the ability to act as a gate by switching between conducting and nonconducting states).
Proceedings of the National Academy of Sciences of the United States of America, 2017
In many K(+) channels, prolonged activating stimuli lead to a time-dependent reduction in ion conduction, a phenomenon known as C-type inactivation. X-ray structures of the KcsA channel suggest that this inactivated state corresponds to a "constricted" conformation of the selectivity filter. However, the functional significance of the constricted conformation has become a matter of debate. Functional and structural studies based on chemically modified semisynthetic KcsA channels along the selectivity filter led to the conclusion that the constricted conformation does not correspond to the C-type inactivated state. The main results supporting this view include the observation that C-type inactivation is not suppressed by a substitution of D-alanine at Gly77, even though this modification is believed to lock the selectivity filter into its conductive conformation, whereas it is suppressed following amide-to-ester backbone substitutions at Gly77 and Tyr78, even though these s...
Structural mechanism of C-type inactivation in K+ channels
Nature, 2010
Interconversion between conductive and non-conductive forms of the K + channel selectivity filter underlies a variety of gating events, from flicker transitions (μs) to C-type inactivation (ms-s). Here, we report the crystal structure of the K + channel KcsA in its Open-Inactivated conformation and investigate the mechanism of C-type inactivation gating at the selectivity filter from channels "trapped" in a series of partially open conformations. Five conformer classes were identified with openings ranging, from 12 Å in closed KcsA (Cα-Cα distances at T112) to 32 Å when fully open. They revealed a remarkable correlation between the degree of gate opening and the conformation and ion occupancy of the selectivity filter. We show that a gradual filter backbone reorientation leads first, to a loss of the S2 ion binding site and a subsequent loss of the S3 binding site, presumably abrogating ion conduction. These structures suggest a molecular basis for C-type inactivation in K + channels. The functional behaviour of most ion channels is defined by the relationship between two coupled mechanisms: activation and inactivation gating. Activation is associated with a large hinged motion around the inner helix bundle, as observed in a number of K + channel structures 1,2,3,4,3,4,5,6 , and characterized spectroscopically in KcsA 7,8,9,10,11. Inactivation typically takes place in a stimulus independent way, though it is sometimes coupled to the activation process 12,13. In K + channels, inactivation gating can occur by two distinct molecular mechanisms: N-type inactivation, a fast, autoinhibitory process where an Nterminal particle binds to the open pore, blocking conduction 14 ; and C-type inactivation, which originates from transitions at the selectivity filter 15,16. C-type inactivation often (though not always) develops with much slower kinetics and is highly modulated by permeant ions and pore blockers 17,18,19,20,21. Coordination of K + ions at the selectivity filter establishes unique structural constraints in order to optimally select against impermeable ions while allowing for fast K + ion translocation 22,23,24. Therefore, perturbations in selectivity filter geometry can have