Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug.
Ontology highlight
ABSTRACT:
SUBMITTER: Tyagi A
PROVIDER: S-EPMC8812516 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388-400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.
| S-EPMC8253976 | biostudies-literature
Project description:Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations.
| S-EPMC8008366 | biostudies-literature
Project description:The ClC channels are members of a large protein family of chloride (Cl-) channels and secondary active Cl- transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl- ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl- channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction.
| S-EPMC1356352 | biostudies-literature
Project description:The conservation of structure across paralog proteins promotes alternative protein-ligand associations often leading to side effects in drug-based inhibition. However, sticky packing defects are typically not conserved across paralogs, making them suitable targets to reduce drug toxicity. This observation enables a strategy for the design of highly specific inhibitors involving ligands that wrap nonconserved packing defects. The selectivity of these inhibitors is evidenced in affinity assays on a cancer-related pharmacokinome: a powerful inhibitor is redesigned by using the wrapping technology to enhance its selectivity and affinity for a target kinase. In this way, the packing defects of a soluble protein may be used as selectivity filters for drug design.
| S-EPMC1963458 | biostudies-literature
Project description:Fluorescently labeled peptide blockers of ion channels are useful probes in studying the localization and functioning of the channels and in the performance of a search for new channel ligands with bioengineering screening systems. Here, we report on the properties of Atto488-agitoxin 2 (A-AgTx2), a derivative of the Kv1 channel blocker agitoxin 2 (AgTx2), which was N-terminally labeled with Atto 488 fluorophore. The interactions of A-AgTx2 with the outer binding sites of the potassium voltage-gated Kv1.x (x = 1, 3, 6) channels were studied using bioengineered hybrid KcsA-Kv1.x (x = 1, 3, 6) channels. In contrast to AgTx2, A-AgTx2 was shown to lose affinity for the Kv1.1 and Kv1.6 binding sites but to preserve it for the Kv1.3 site. Thus, Atto488 introduces two new functionalities to AgTx2: fluorescence and the selective targeting of the Kv1.3 channel, which is known for its pharmacological significance. In the case of A-AgTx2, fluorescent labeling served as an alternative to site-directed mutagenesis in modulating the pharmacological profile of the channel blocker. Although the affinity of A-AgTx2 for the Kv1.3 binding site was decreased as compared to the unlabeled AgTx2, its dissociation constant value was within a low nanomolar range (4.0 nM). The properties of A-AgTx2 allow one to use it for the search and study of Kv1.3 channel blockers as well as to consider it for the imaging of the Kv1.3 channel in cells and tissues.
| S-EPMC9312206 | biostudies-literature
Project description:Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca2+-activated K+ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K+ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF's four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K+ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K+, similar to KcsA, but that even a single K+ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K+ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK's S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.
| S-EPMC7703589 | biostudies-literature
Project description:Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.
| S-EPMC7766132 | biostudies-literature
Project description:On the basis of single-channel currents recorded from the muscle nicotinic acetylcholine receptor (AChR), we have recently hypothesized that the conformation adopted by the glutamate side chains at the first turn of the pore-lining ?-helices is a key determinant of the rate of ion permeation. In this paper, we set out to test these ideas within a framework of atomic detail and stereochemical rigor by conducting all-atom molecular dynamics and Brownian dynamics simulations on an extensively validated model of the open-channel muscle AChR. Our simulations provided ample support to the notion that the different rotamers of these glutamates partition into two classes that differ markedly in their ability to catalyze ion conduction, and that the conformations of the four wild-type glutamates are such that two of them "fall" in each rotamer class. Moreover, the simulations allowed us to identify the mm (?(1) ? -60°; ?(2) ? -60°) and tp (?(1) ? 180°; ?(2) ? +60°) rotamers as the likely conduction-catalyzing conformations of the AChR's selectivity-filter glutamates. More generally, our work shows an example of how experimental benchmarks can guide molecular simulations into providing a type of structural and mechanistic insight that seems otherwise unattainable.
| S-EPMC4128128 | biostudies-literature
Project description:K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K(+) channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na(+) or low [K(+)]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K(+) ion binding to channels with an inactivated or conductive selectivity filter is different from K(+) ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.
| S-EPMC4679001 | biostudies-literature
Project description:Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-? interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.
| S-EPMC5778891 | biostudies-literature