Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles.

BACKGROUND: Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles--nanoparticles--in biolistic transfections to determine if they are a suitable alternative to microparticles. RESULTS: Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles. CONCLUSIONS: We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The roles of aromatic residues in the glycine receptor transmembrane domain.

BACKGROUND: Cys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1-M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues. RESULTS: Here we show that many aromatic residues in the M1, M3 and M4 α-helices of the glycine receptor are involved in the function of the receptor. The residues were explored by creating a range of mutant receptors, characterising them using two electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using currently available structural information on the open and closed states of the receptor. For 7 residues function was ablated with an Ala substitution: 3 Tyr residues at the extracellular end of M1, 2 Trp residues located towards the centers of M1 and M3, and a Phe and a Tyr residue in M4. For many of these an alternative aromatic residue restored wild-type-like function indicating the importance of the π ring. EC50s were increased with Ala substitution of 8 other aromatic residues, with those in M1 and M4 also having reduced currents, indicating a role in receptor assembly. The structure shows many potential interactions with nearby residues, especially between those that form the M1/M3/M4 interface, and we identify those that are supported by the functional data. CONCLUSION: The data reveal the importance and interactions of aromatic residues in the GlyR M1, M3 and M4 α-helices, many of which are essential for receptor function

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation.

GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6'M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6'M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6' residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.This project was supported by the Wellcome Trust (grant 81925 to SCRL). SCRL is a Wellcome Trust Senior Research Fellow in Basic Biomedical Studies.This is the final published version, which first appeared at http://www.sciencedirect.com/science/article/pii/S0965174814001684

Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP.

Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor.Supported by grants from the Wellcome Trust (81925) and the MRC to S.C.R.L.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/cn500369

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.This project was supported by the Wellcome Trust Grant 81925 to S.C.R.L. S.C.R.L. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Studies. M.A. is funded by a Yousef Jameel Scholarship. D.A.W. was funded by an MRC studentship.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/bi5008035

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors.

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.This work was supported by grants from the NIH (NS 34407 to DAD) and the Wellcome Trust (WT 81925 to SCRL), and an NIH/NRSA training grant (GM07616 to MR).This is the final version of the article. It first appeared from American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.69437

A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC-the 5-HT3A receptor-unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening

5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems.

Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined.Supported by grants from Universidad Nacional del Sur (UNS), Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT), and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) to CB, and The Wellcome Trust (81925) and the MRC (MR/L021676) to SCRL.This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/acschemneuro.5b0008

The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors.

VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [(3)H]VUF10166 displayed saturable binding with a Kd of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 10(7) M(-1) min(-1)) and dissociation (0.01 min(-1)) rates that yielded a similar Kd value (0.16 nM). At 5-HT3AB receptors two association (6.15 × 10(-7), 7.23 M(-1) min(-1)) and dissociation (0.024, 0.162 min(-1)) rates were seen, yielding Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand-receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.This is the final version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S002839081400291