Lanthanide-based Resonance Energy Transfer Research Articles

Lanthanide-based resonance energy transfer biosensors for live-cell applications.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enable sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living mammalian cells. LRET biosensors are polypeptides that consist of an alpha-helical linker sequence sandwiched between a lanthanide complex-binding domain and a fluorescent protein (FP) with two interacting domains residing at each terminus. Interaction between the terminal affinity domains brings the lanthanide complex and FP in close proximity such that lanthanide-to-FP, LRET-sensitized emission is increased. A recent proof-of-concept study examined model biosensors that incorporated the affinity partners FKBP12 and the rapamycin-binding domain of m-Tor (FRB) as well as p53 (1-92) and HDM2 (1-128). The sensors contained an Escherichia coli dihydrofolate reductase (eDHFR) domain that binds with high selectivity and affinity to Tb(III) complexes coupled to the ligand trimethoprim (TMP). When cell lines that stably expressed the sensors were treated with TMP-Tb(III), TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. Much larger signal changes (>2500%) and Z'-factors of 0.5 or more were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. In this chapter, we elaborate on the design and performance of LRET biosensors and provide detailed protocols to guide their use for live-cell microscopic imaging studies and high-throughput library screening.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Position of Inactivation Particle of Shaker Kv Channels in Resting State

Shaker Kv channels inactivate rapidly to culminate the action potential and maintain the homeostasis of excitable cells. The so-called N-type inactivation is caused by the first 46 amino acids of the N-terminus of the channel, known as the inactivation peptide (IP). Numerous mutational studies have characterized N-type inactivation functionally, however, the position of the IP in the resting state and its transition during inactivation is still debated. We tracked the movement of IP during inactivation using voltage clamp fluorometry. By inserting an unnatural amino acid, 3-[(6-acetyl-2-naphthalenyl) amino]-L-alanine (Anap), which is sensitive to changes in environment, we identified the movements of ball and chain seperately. Our data suggests that N-type inactivation occurs in a biphasic movement by first releasing the IP, which then blocks the pore from the cytoplasmic side. Using Lanthanide-based Resonance Energy transfer (LRET) and transition metal FRET (tmFRET), we narrowed down the resting position of the IP. We propose that is located in the window between the channel and the T1 domain interacting with the acidic residues of the T1-S1 linker.

Movements of N‐terminus of LeuT during the Substrate Transport Cycle

LeuT from Aquifex aeolicus aeolicus is a bacterial homologue to mammalian solute carrier family 6 (SLC6) of neurotransmitter transporters. SLC6 transporters are of great pharmacological interests because of their crucial role in neurotransmitter clearance. These proteins are also targets of many clinically relevant drugs, including antidepressant drugs and drugs of abuse such as amphetamines. The amino terminus of SLC6 transporters plays an important regulatory role for function.High resolution X‐ray crystal structures of LeuT are available in different conformations. However, these structures are not able to fully explain the dynamic nature of LeuT function during the substrate transport cycle. The aim of this project is to look at the movements of the N terminus of LeuT in the proteoliposomes using spectroscopic based technique called lanthanide based resonance energy transfer (LRET).We developed a LeuT variant in which we inserted a Lanthanide Binding Tag (LBT) at position 335, positioned at the end of extracellular loop 4. Three LeuT mutants (K4C, H7C and A9C) were created in the background of the LeuT‐LBT construct. Using LRET based distance measurements we determined the distance between the LBT bound lanthanide and the fluorophore TMR attached to the cysteines in the N‐terminal of LeuT. The results did not show the expected increase in distance towards the N‐terminus, as the distance to H7C was shorter than to A9C. In contrast, iodate quenching experiments of TMR fluorescence showed no difference in accessibility to KI for all three residues.Intramolecular distances determined by LRET suggest that the fluorophore attached to H7 could be oriented towards the inner vestibule, while the KI based accessibility experiments displays a contrasting picture. In order to elucidate the dynamics of the N‐terminus and support one of the two findings, I am further planning to stabilize the transporter in an outward open conformation using tryptophan, and as an alternative strategy, stabilize LeuT‐LBT in the inward‐facing and an outward‐facing conformation using the R5A or the R30A mutants.Support or Funding InformationThis work is jointly supported by the Austrian Science Fund (FWF) Austria and Higher Education Commission (HEC) PakistanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b

Gating of the mammalian inward rectifier Kir1.1 at the helix bundle crossing (HBC) by intracellular pH is believed to be mediated by conformational changes in the C-terminal domain (CTD). However, the exact motion of the CTD during Kir gating remains controversial. Crystal structures and single-molecule fluorescence resonance energy transfer of KirBac channels have implied a rigid body rotation and/or a contraction of the CTD as possible triggers for opening of the HBC gate. In our study, we used lanthanide-based resonance energy transfer on single-Cys dimeric constructs of the mammalian renal inward rectifier, Kir1.1b, incorporated into anionic liposomes plus PIP2, to determine unambiguous, state-dependent distances between paired Cys residues on diagonally opposite subunits. Functionality and pH dependence of our proteoliposome channels were verified in separate electrophysiological experiments. The lanthanide-based resonance energy transfer distances measured in closed (pH 6) and open (pH 8) conditions indicated neither expansion nor contraction of the CTD during gating, whereas the HBC gate widened by 8.8 ± 4 Å, from 6.3 ± 2 to 15.1 ± 6 Å, during opening. These results are consistent with a Kir gating model in which rigid body rotation of the large CTD around the permeation axis is correlated with opening of the HBC hydrophobic gate, allowing permeation of a 7 Å hydrated K ion.

Nav channel binder containing a specific conjugation-site based on a low toxicity \u03b2-scorpion toxin

Voltage-gated sodium (Nav) channels play a key role in generating action potentials which leads to physiological signaling in excitable cells. The availability of probes for functional studies of mammalian Nav is limited. Here, by introducing two amino acid substitutions into the beta scorpion toxin Ts1, we have chemically synthesized a novel binder [S14R, W50Pra]Ts1 for Nav with high affinity, low dissociation rate and reduced toxicity while retaining the capability of conjugating Ts1 with molecules of interests for different applications. Using the fluorescent-dye conjugate, [S14R, W50Pra(Bodipy)]Ts1, we confirmed its binding to Nav1.4 through Lanthanide-based Resonance Energy Transfer. Moreover, using the gold nanoparticle conjugate, [S14R, W50Pra(AuNP)]Ts1, we were able to optically stimulate dorsal root ganglia neurons and generate action potentials with visible light via the optocapacitive effect as previously reported. [S14R, W50Pra]Ts1 is a novel probe with great potential for wider applications in Nav-related neuroscience research.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

The Environment Shapes the Inner Vestibule of LeuT.

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.

β1-subunit–induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

Determining Nitric Oxide‐induced Conformational Changes in Soluble Guanylate Cyclase using Lanthanide‐based Resonance Energy Transfer

Nitric Oxide (NO) is a crucial signaling molecule involved in vasodilation and regulation of blood pressure. A key target for NO signaling is soluble guanylate cyclase (sGC), a heterodimeric protein that upon NO binding converts GTP to a secondary messenger, cGMP. NO binding at the ferrous heme of the beta subunit of the sGC dimer induces a conformational change in the protein resulting in increased catalysis and increased production of cGMP. Attempts to enhance the enzymatic activity of sGC by drugs tartegeting cardiovascular disease are hindered by a lack of information on the mechanism of this allosteric regulation and the structure of the protein. One method that can provide a better understanding of conformational changes in sGC is lanthanide‐based resonance energy transfer (LRET). This technique monitors luminescence of terbium ion (Tb3+) bound to a lanthanide‐binding tag (LBT) inserted into a protein sequence. We have designed several sGC constructs with an LBT tag at different locations in the protein in addition to 6‐His‐tags, which can bind transition metals and quench luminescence of the LBT‐bound Tb3+. Rates for Tb3+ luminescence decay upon quenching by Cu2+ bound to the 6‐His‐tag were used to calculate the distance between the metal ions using the Förster equation. Our preliminary conclusions from the LRET experiments are: (i) Tb luminescence decays with a bi‐exponential time course and a rate constant for the slow phase of ~2.3 ms; (ii) significant quenching of Tb luminescence is observed in the presence of millimolar concentrations of Cu2+; (iii) a calculated distance of ~23 Å between Tb and Cu ions in construct NT19_LBT2 suggests the C and N termini lie close to one another in the protein. Future experiments will probe how these distances change upon ligand binding and the distances between the LBT tags and a candidate drug with quenching fluorophore. These decay times and distances will lead to greater understanding of sGC structure and dynamics and will aid in the design of new drugs to treat cardiovascular disease through allosteric stimulation of sGC.Support or Funding InformationNIH MARC Training Grant T34 GM08718, AHA 14GRNT2008006, NIH R01 GM117357, Ironwood Pharmaceuticals

Probing Voltage-Dependent Structural Changes of the VSD in Mammalian Nav with LRET

Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

High Affinity Nav Channel Binder with Specific Conjugation Site Derived from Scorpion Toxin with Attenuated Toxicity

Voltage-gated sodium channels (Nav) play a key role in generating action potential which leads to physiological signaling in excitable cells. However, the availability of probes for functional study of mammalian Nav is limited. Based on the beta-scorpion toxin Ts1, we have chemically synthesized a novel biologically friendly binder [S14R, W50Pra]Ts1 (Ts1-S14R) for Nav with high affinity and extremely low dissociation rate. This Ts1 analog has a S14R point mutation, corresponding to E15R in Css4, another beta-scorpion toxin, and also has a propargylglycine (Pra) mutation from W50 for bioconjugation. We identified the pharmacological properties of Ts1-S14R on rat skeletal muscle Nav channel (Nav1.4) using the cut-open oocyte voltage clamp technique and confirmed that its toxicity has been dramatically reduced. Chemical synthesis of this binder enabled us to conjugate with molecules of interest for various studies. Using the fluorescent-dye, BODIPY, conjugated binder, we examined its binding location in Nav1.4 using Lanthanide-based Resonance Energy Transfer technique (LRET). Our LRET study showed Ts1-S14R-BODIPY binds to Nav1.4 at virtually identical location as that of Ts1-BODIPY. In addition, LRET data showed that, once Ts1-S14R-BODIPY binds to Nav1.4, it stays in place for at least 40 min in toxin-free solution, indicating that Ts1-S14R-BODIPY can be employed as a stable binder for Nav1.4. Moreover, we generated Ts1-S14R conjugated with gold nanoparticle (Ts1-S14R-AuNP). We demonstrated this conjugate can induce action potentials by visible light stimulation through the opto-thermal-capacitive effect in rat dorsal root ganglia (DRG) neurons similarly to that previously reported (Carvalho-de-Souza JL et al. Neuron 2015). Ts1-S14R is therefore a novel probe with great potential for wider applications in Nav-related neuroscience research. Support: U54GM087519 and GM030376.

Effects of the Accessory Subunit γ1 on the External Architecture of BK Channel

Regulatory β and γ subunits are responsible for conferring functional diversity to BK channels but little is known about the detailed way that accessory subunits modulate the structure of the pore forming α subunit. It is known that the γ1 subunit produces a large leftward shift of the open probability vs. voltage curve in the absence of internal Ca2+ (Yan and Aldrich, 2010). To explore the external architecture of α subunit in the presence of γ1 subunit, we used lanthanide-based resonance energy transfer (LRET) as a molecular ruler to measure intra- and inter-molecular distances. We introduced a genetically encoded lanthanide binding tag (LBT) that binds Tb3+ (LRET donor) with high affinity at different positions in the α subunit (N-terminal, S0, S1 and S2) and γ1 subunit. Fluorescent probe BODIPY linked to a scorpion toxin was used as LRET acceptor. LRET sensitized emission (SE) decays were analysed using a nano-positioning system that determines the position of LBT-tagged sites with respect to the fixed acceptor near the pore axis. Interestingly, the external architecture of the BK α subunit is modified when co-expressed with the regulatory γ1 subunit indicating a conformational change of the BK voltage sensor domain of the BK channel. The largest changes was in S1 position (∼25 Angstroms) followed by S0. In addition, all γ1-LBT positions were found peripherally positioned with respect to the α subunit.Fondecyt Grant 1110430 (To R. L.)

Let’s Twist (the S4) Again

Let’s Twist (the S4) Again

Tracking Voltage-Dependent Conformational Changes of the VSD in Nav with LRET

Voltage-gated sodium channels (Nav) are fundamental for the generation and the propagation of action potentials. Mammalian Nav alpha subunits are single macromolecules organized in four different domains (DI-DIV). Each is composed of 6 transmembrane segments (S1-S6) from which S1-S4 constitute the voltage sensing domain (VSD) and with S5 and S6 constituting the pore. While Nav function has been studied extensively, the exact structural mechanisms of gating are not fully understood. Recently, the crystal structure of the prokaryotic sodium channel, NavAb, has been solved, but NavAb is a homotetrameric protein in contract to the mammalian Navs. Thus many questions were not answered by the prokaryotic channel structures. To resolve the voltage dependent conformational changes of Nav, we tracked conformational changes of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), a FRET technique that allows for precise measurement of intermolecular distances by taking advantage of the special properties of lanthanide as an energy donor. We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which holds a lanthanide (Tb3+) ion with high affinity, inserted at the top of the S4 segment in each domain. Also, we synthesized two toxins conjugated to dyes to function as acceptors: the pore-blocking small molecule tetrodotoxin conjugated with a HiLyte fluor488 (TTX-F), and the peptide β scorpion toxin Ts1, from the Brazilian scorpion Tityus serrulatus, conjugated with Alexa488 (Ts1-Alexa488). Having several donor positions (Tb3+ ions in LBT's) and two different acceptor positions (TTX-F and Ts1-Alexa488), we calculated multiple distances in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs that remained functionally active. The results provide new insight to structure-function information in mammalian Nav channels. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Searching for the Interaction Sites of the Beta1 Subunit with the Voltage-Sensing Domains of Sodium Channels Using LRET

Mammalian voltage-gated sodium channels (Nav) are composed of two subunits: a monomeric pore-forming subunit (α-subunit), that contains four domains (DI-DIV), each composed of a voltage-sensing domain (VSD, S1-S4) and a pore domain (S5-S6) and auxiliary subunits (β-subunits) that are transmembrane proteins with type I topology: containing an extracellular amino-terminus, a single transmembrane segment and an intracellular carboxyl terminus. A large body of literature has shown that interaction between α- and β-subunits results in Nav with altered gating kinetics suggesting that β-subunits may directly interact with VSD. However, aspects of the stoichiometry, arrangement and molecular interaction between α and β subunits remain unclear. In this study, we explored the location of β1 in relation to the rat skeletal muscle sodium channel α-subunit (Nav1.4) using lanthanide-based resonance energy transfer (LRET) via two strategies. 1) Four Nav1.4 constructs were designed to encode a Tb3+ binding-tag (Nav1.4-LBT) on top of the S4 of each domain (DI-LBT, DII-LBT, DIII-LBT and DIV-LBT) as energy donor. A hexa-histidine-tag was inserted in β1 (6His-β1), which binds a Cu2+ ion to act as acceptor. 2) β1 constructs were designed to encode at an extracellular site an LBT that binds Tb3+ as energy donor while the acceptor was Alexa488 conjugated to Ts1, a β scorpion toxin which binds to DII-VSD in Nav1.4. For both experiments, we used Xenopus laevis oocytes co-expressing Nav1.4 and β1 constructs injected with the cRNA at 1:1 molar ratio. In preliminary results, DIV-LBT + 6His-β1 oocytes displayed robust energy transfer between Tb3+ and Cu2+, indicating that at least one β1-subunit is located at ∼28 A of S4-DIV. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Unfolding the Structure of LeuT Employing Luminescence Resonance Energy Transfer

Background: Neurotransmitter sodium symporters (NSS) are located in the brain and retrieve neurotransmitters from the synaptic cleft to end synaptic transmission. Solute carier class six proteins (SCLC6) are of great pharmacological importance in terms of their localization and function. The crystal structures obtained from a bacterial homolog, the leucine transporter LeuTAa, in open to outward, occluded and open to inward conformations are present in frozen state with high resolution. Due to its close kinship with SLC6 proteins, LeuTAa serves as a paradigm for these transporters.Methods: In order to address the dynamicity of the substrate transport cycle in LeuTAa, we use the Lanthanide based resonance energy transfer (LRET) technique. This method is a spin-off of the fluorescence resonance energy transfer method according to Forster employing the introduction of the genetically encoded lanthanide binding tags (LBT) as donor elements. Exogenous cysteine residues labelled with cysteine specific fluorophores are used as acceptor elements. This technique is an alternative to address the movement of helices, with great resolution and has been employed successfully to examine potassium channels.Results: We screened for the functional LBT_mutants using the scintillation proximity assay. The LeuT_A335-LBT-G336 mutant displayed function in terms of its binding activity. Within this background, we generated cysteine mutants. To date, we have successfully measured the intramolecular distances in different LBT_LeuT_Cys mutants. Furthermore, we observed intramolecular distance changes from these purified proteins in detergent micelles.Conclusion: Our LRET measurements will help us to understand the transport cycle and help to complete the missing steps in substrate transport cycle of LeuTAa. Currently, we focus on the reconstitution of purified LeuTAa into liposomes and have our LRET measurements in a reconstituted system that allows to use more physiological ionic gradients.

Lanthanide based resonance energy transfer (LRET) between Ce-doped LaPO4 nanorods and coumarin 440 dye

Here, we demonstrate an efficient lanthanide based resonance energy transfer (LRET) between Ce-doped LaPO4 nanorods and coumarin 440 dye using steady state and time resolved spectroscopy. Water dispersible stable colloidal Ce-doped LaPO4 nanorods are synthesized via a facile aqueous route using a capping reagent 6-amino hexanoic acid (AHA). XRD and TEM analysis confirm the formation of uniform and discrete Ce-doped LaPO4 nanorods, having a tetragonal phase. FTIR analysis confirms that the surfaces of Ce-doped LaPO4 nanorods are functionalized with AHA. From zeta potential analysis, it is confirmed that the positively charged nanorods are attached to the negatively charged dye molecules electrostatically. Analysis suggests that both dynamic (1.04 × 104 M−1) and static quenching (3.3 × 102 M−1) processes occur in this assembled system. The prepared nanorods show an emission quantum yield of 10.2% and a long fluorescence lifetime (24.9 ns). The spectral overlap of the Ce3+ emission with the absorption of coumarin 440 dye molecules enables an efficient energy transfer from Ce-doped LaPO4 nanorods to coumarin 440 dye. The values of the overlap integral and Forster radius are found to be 2.028 × 1014 M−1 cm−1 nm4 and 27.02 A, respectively. The PL quenching and the shortening of the decay time of the donor (Ce-doped LaPO4 nanorods) confirm the energy transfer (24%) from the nanorods to the dye. Such energy transfer between LaPO4:Ce nanorods and C-440 dye could pave the way for designing new optical materials for potential applications.

External Architecture of the Large-Conductance Ca2+ and Voltage-Activated K+ (BKCa) Revealed by a Spectroscopic Ruler

BKCa channels are involved in a large variety of physiological processes and regulatory β subunits are one of the mechanisms responsible of creating BKCa channel diversity fundamental to the adequate function of many tissues. Regardless the proven importance of this channel little is known about its detailed structure. Here we disclose the external architectural intimacies of BKCa channels using Lanthanide based Resonance Energy Transfer (LRET) as a molecular ruler to measure intra and intermolecular distances. We introduced a genetically encoded lanthanide binding tag (LBT that binds Tb3+ with high affinity) at several positions of the external loops of the α and β1 subunits, and constructed a fluorescent molecule of BODIPY-FL linked to a scorpion toxin, iberiotoxin (Bodipy FL-IbTX), that was used as an acceptor for the LRET interaction with Tb3+. These functional LBT-BKCa constructs were expressed in Xenopus laevis oocytes that were voltage clamped with two microelectrodes to obtain simultaneously electrical and LRET recordings under physiological ionic conditions. Sensitized emission (SE) recordings from different LBT-BKCa positions had different kinetics indicating different relative positions for each construct. We analyzed SE records with a novel method developed by our group that determines the position of LBT-tagged sites of BKCa to obtain an external structural map, including the β1 subunit. Interestingly, when the BKCa α subunit was co-expressed with the regulatory β1 subunit, SE becomes slower, indicating a large conformational change of the BKCa channel structure. The methodology presented here gives us the first glimpses to the BKCa channel external surface structure in its different functional states with and without the β1 subunit. Supported by Fondecyt grant 1110430 and NIH grants U54GM087519 and GM030376. CINV is a Scientific Millennium Institute.

A Molecular Mechanics Model of a Closed Voltage-Gated Potassium Channel Generated from S4-S5 Linker LRET Measurements

Voltage-gated potassium ion (Kv) channels regulate action potentials of the nervous system by responding to changes in transmembrane voltage, enabling K+ transport across the membrane to restore cells to their resting potential. While several crystal structures of Kv channels have been presented in the open conformation, the closed structure remains unsolved, leaving the Kv channel gating mechanism unclear. Using lanthanide-based resonance energy transfer (LRET) measurements of the S4-S5 linker, which connects the voltage sensor to the pore domain, we modeled the Kv 1.2/2.1 chimera in the open and closed conformations. Through fully atomistic molecular dynamics simulations of the models, we find that a small 4 A displacement of the linker is sufficient to gate the channel. Additionally, we find a 9 A vertical translation of S4, and a 37° change in tilt of S4 with respect to the S4-S5 linker between the open and closed states, in agreement with previously published studies. Here, we present the first model of the closed channel derived from measurements on the cytosolic side of the channel.