Ivermectin-Activated, Cation-Permeable Glycine ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Ivermectin-activated, cation-permeable glycine receptors for the chemogenetic control of neuronal excitation Robiul Islam, Angelo Keramidas, Li Xu, Nela Durisic, Pankaj Sah, and Joseph W Lynch ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00168 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 12, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Ivermectin-activated, cation-permeable glycine receptors for the chemogenetic control of neuronal excitation Robiul Islam1, Angelo Keramidas1, Li Xu1, Nela Durisic1, Pankaj Sah1 & Joseph W. Lynch1,2* 1

Queensland Brain Institute, The University of Queensland, Brisbane, QLD 4072, Australia

2

School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia

*

Corresponding author: Email: [email protected]

Abstract The ability to control neuronal activation is rapidly advancing our understanding of brain function and is widely viewed as having eventual therapeutic application. Although several highly effective optogenetic, optochemical genetic and chemogenetic techniques have been developed for this purpose, new approaches may provide better solutions for addressing particular questions and would increase the number of neuronal populations that can be controlled independently. An early chemogenetic neuronal silencing method employed a glutamate receptor Cl- channel engineered for activation by 1-3 nM ivermectin. This construct has been validated in vivo. Here we sought to develop cation-permeable ivermectin-gated receptors that were either maximally Ca2+-permeable so as to induce neuro-excitotoxic cell death or minimally Ca2+-permeable so as to depolarize neurons with minimal excitotoxic risk. Our constructs were based on the human α1 glycine receptor Clchannel due to its high conductance, human origin, high ivermectin sensitivity (once mutated) and because pore mutations that render it permeable to Na+ alone or Na+ plus Ca2+ are well characterized. We developed a Ca2+-impermeable excitatory receptor by introducing the F207A/P2’∆/A-1’E/T13’V/A288G mutations and a Ca2+-permeable excitatory receptor by introducing the F207A/A-1’E/A288G mutations. The latter receptor efficiently induces cell death and strongly depolarizes neurons at nanomolar ivermectin concentrations.

Keywords. chloride channel, pharmacogenetic, chemogenetic, excitotoxicity, ligand-gated ion channel, glycinergic

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 32

INTRODUCTION

The ability to control the activity of defined neuronal populations in vivo is rapidly advancing our understanding of brain function. In addition to facilitating our understanding of the relationship between specific behaviours and electrical activity in defined neuronal circuits, these technologies have the potential to treat human neurological disorders caused by aberrant levels of neuronal activity1-3. Such disorders potentially include motor neuron disease, Parkinson’s disease, addiction, anxiety, epilepsy, posttraumatic stress, depression and chronic pain states. Several methods of remotely controlling neuronal function in vivo have been developed, with optogenetics4, optochemical genetics5,6 and chemogenetics7 proving the most successful to date. Optogenetics involves recombinantly expressing prokaryotic light-activated ion channels in neurons of interest. As both cation- and anion-permeable constructs have been developed, this technology permits individual neurons in intact circuits to be activated or inhibited by light with millisecond precision. Optochemical genetic approaches, which involve tagging endogenous or recombinantly expressed ion channels with photo-switchable ligands, also allow neurons to be activated or inhibited by light with millisecond precision. Chemogenetics involves recombinantly expressing receptors that are selectively activated by molecules that are otherwise biologically inert. Although a range of chemogenetic approaches have been developed, the most successful to date is the ‘DREADDs’. These are modified muscarinic G protein-coupled receptors with low sensitivity to their endogenous ligand and high sensitivity to clozapine-N-oxide, a biologically inert molecule8. When expressed in neurons, one excitatory M3-based DREADD construct couples via endogenous Gq to activate an excitatory ion flux of as yet unknown molecular origin or ionic composition9, whereas inhibitory M2- and M4-based DREADDs couple via endogenous Gi to activate an inwardly rectifying potassium conductance10. Although powerful, these techniques are not suited to all experimental situations. For example, optogenetics is not ideal for silencing anatomically large regions or diffuse neuron populations, and may induce cell-damaging side effects such as heat11. DREADDS must couple to endogenous proteins to be functional, and these may not be strongly expressed in all neuron types of interest. Novel methods of controlling neuronal activity will provide new options for addressing particular experimental circumstances, and will increase the range of options available for independently controlling distinct neuronal populations. An early chemogenetic neuronal silencing method employed a C. elegans heteromeric αβ glutamate receptor (GluR) chloride channel that was engineered for selective activation by ivermectin12. Concentrations of ivermectin in the brain following oral or intraperitoneal delivery can reach 1-3 nM13, which is sufficient to activate these receptors and thereby inhibit activity in the ACS Paragon Plus Environment

2

Page 3 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


neurons that recombinantly express the construct. One advantage of this approach is that ivermectin is a safe anthelminthic drug approved for human use14, thus rendering it a potential candidate for eventual human therapeutic use. However, the original GluR construct met with limited success, due most likely to a combination of insufficient ivermectin sensitivity, suboptimal expression efficiency, and the fact that the receptor is a heteromultimer requiring two different subunits. Two sets of second generation ivermectin inhibitory receptors were developed to overcome these limitations. Frazier and colleagues enhanced the ivermectin sensitivity of the original C. elegans αβ GluR via the L9’F mutation and improved its surface expression efficiency via mutations that removed an endoplasmic reticulum retention signal and an intracellular dimerization motif15. In an independent approach, our laboratory developed a modified human homomeric α1 GlyR as an ivermectin-gated silencing receptor16. We enhanced its ivermectin sensitivity via the A288G mutation17 and eliminated glycine sensitivity via the F207A mutation18. The α1F207A/A288G GlyR exhibited an ivermectin sensitivity similar to that of the C. elegans αβ GluR 13, although its larger unitary conductance (90pS), homomeric expression and human origin may render it more suited for eventual clinical use. GluRs and GlyRs are both anion-permeating members of the pentameric ligand-gated ion channel (pLGIC) family. This family also includes the cation-permeating nicotinic acetylcholine receptor (nAChR) and 5-hydrotryptamine type-3 receptor (5-HT3R), and the anion-permeating GABA type-A receptor (GABAAR). pLGICs comprise five subunits arranged symmetrically around a central pore. Each subunit comprises a large extracellular N-terminal domain with neurotransmitter-binding sites located at subunit interfaces. This is coupled to a transmembrane αhelical bundle comprising four membrane-spanning helices, termed TM1-TM4. A TM2 domain contributed by each of the five subunits lines the central pore, surrounded by the TM1, TM3 and TM4 domains that provide a barrier between the hydrophilic central ion-conducting pathway and the hydrophobic cell membrane. The recently elucidated cryo-EM structure of the α1 GlyR with ivermectin bound19 depicts these features and shows ivermectin binding in the intersubunit cavity between TM3 and TM1 (Figure 1A, B). The A288 sidechain lines the entry to this cavity (Figure 1B). In the present study we sought to develop cation-permeable ivermectin-gated GlyRs that are either maximally permeant to Ca2+ so that they might induce neuro-excitotoxic cell death in selected neuron populations or minimally permeant to Ca2+ so that they depolarize neurons with minimal excitotoxic risk. It has long been recognized that cation- and anion-permeable pLGICs exhibit systematic differences in the identity of TM2 domain residues at the -2’, -1’, 13’ and 19’ positions20 (Figure 1C, D). By mutating subsets of these residues in the GlyR towards their counterparts in cationACS Paragon Plus Environment

3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 32

permeable pLGICs, it has proved possible to generate GlyRs that are permeable to either Na+ only or to Na+ plus Ca2+

21-23

. The mutant receptors that successfully formed cation-permeable GlyRs,

together with their Cl-:Na+ and Ca2+:Na+ permeability ratios and unitary conductances, are shown in Figure 1D. Here we describe the development of cation-permeable ivermectin-gated GlyRs based on these constructs.

RESULTS AND DISCUSSION

Fluorescence imaging of anion influx To simplify the description of constructs, we have abbreviated the three most commonly used mutations as follows: P-2’∆ as ‘D’, A-1’E as ‘E’, and T13’V as ‘V’. All other mutations are shown in standard notation separated by forward slashes. Thus, the quadruple mutant α1P-2’D/A1’E/T13’V/A288G

GlyR is abbreviated as α1DEV/A288G GlyR.

In this study we sought to develop Na+-permeable constructs that were either maximally or minimally permeant to Ca2+. We investigated the α1E, the α1DE and α1DEV GlyRs as potentially minimally Ca2+-permeant constructs and the α1DE/R19’E GlyR as a potentially maximally Ca2+permeant construct (Figure 1D). As a preliminary screen, we evaluated their glycine- and ivermectin-sensitivities using a YFP-based anion influx assay. Averaged glycine and ivermectin dose-response relationships of the wild type (WT) control α1WT GlyR and the four constructs listed above are shown in Figure 2A and B, respectively, with averaged EC50 and maximum percentage quench values summarized in Table 1. We also investigated the α1DEV/A288G GlyR which we expected to exhibit an enhanced ivermectin sensitivity due to the A288G mutation. As shown in Figure 2A and summarized in Table 1, all five constructs exhibited significantly reduced sensitivities to glycine, with the α1DE/R19’E and α1DEV/A288G GlyRs exhibiting no response to glycine at all. We inferred that this was due to a lack of glycine sensitivity rather than to a lack of functional expression or anion permeability because all five tested constructs induced a significant anion influx when activated by ivermectin (Figure 2B). As expected, the α1DEV/A288G GlyR was more sensitive than the αWT GlyR to ivermectin, although the other constructs were modestly less sensitive than the αWT GlyR (Table 1). We also investigated the effects of the F207A mutation, which eliminates glycine binding, by comparing the glycine and ivermectin sensitivities of the α1E/A288G and α1F207A/E/A288G GlyRs. The results, summarized in Figure 2C and Table 1, indicate that the F207A mutation successfully eliminated glycine sensitivity without significantly impairing ivermectin sensitivity.


4

Page 5 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fluorescence imaging of Ca2+ influx We characterized the same constructs using a Fluo-4-AM-based Ca2+ imaging assay to compare their relative Ca2+ permeabilities in response to glycine or ivermectin. The results of these experiments are shown in Figure 3 with averaged results summarized in Table 2. As expected, the α1WT GlyR induced no detectable Ca2+ influx in response to either glycine or ivermectin (Figure 3A, B). In agreement with the original study22, the α1DE and α1DE/R19’E GlyRs exhibited a significant glycine-activated Ca2+ influx, whereas the α1DEV and α1DEV/A288G GlyRs did not (Figure 3A). The α1E GlyR, with as yet unknown Ca2+ permeability, also exhibited a significant glycinemediated Ca2+ influx (Figure 3A). The constructs exhibited broadly similar Ca2+ influx profiles in response to ivermectin activation (Figure 3B), with the α1DEV and α1DEV/A288G GlyRs inducing less Ca2+ influx than the others. From these experiments we confirmed that the α1DEV and α1DEV/A288G GlyRs are Ca2+impermeant whereas the remaining cation-permeable constructs (i.e., those that do not incorporate T13’V) are able to efficiently flux Ca2+. We also found that the A288G and F207A mutations do not significantly impair the expression efficiency or cation permeability of these GlyR constructs. However, as predicted, the F207A mutation eliminates glycine sensitivity and the A288G enhances the ivermectin sensitivity.

Electrophysiological characterization in HEK293 cells We next turned to whole cell patch-clamp recording to evaluate the ivermectin EC50 values and maximum current magnitudes (Imax) of the constructs of interest. Because ivermectin activates GlyRs irreversibly, its dose-response relationship was assessed by applying successively higher ivermectin concentrations. Sample recordings illustrating this procedure are displayed in Figure 4A. Averaged dose-response relationships for the original set of constructs (α1WT, α1E, α1DE, α1DE /R19’E and α1DEV GlyRs) are displayed in Figure 4B, with mean results summarized in Table 3. In agreement with the fluorescence measurements (Tables 1 and 2), their ivermectin sensitivities were relatively low. The ivermectin sensitivities of the same four constructs incorporating the A288G mutation were also tested, with dose-responses shown in Figure 4C and averaged results summarized in Table 3. Although the A288G mutation enhanced the ivermectin sensitivity of the α1E/A288G and α1DE/A288G GlyRs it had no significant effect on the α1DE/R19’E or α1DEV GlyRs. This may be due to the R19’ and T13’ residues lying close to, or even protruding into, the ivermectin binding site24,25. Mutations to these residues may thus limit the ability of the A288G mutation to enhance the ivermectin binding affinity. However, all A288G-containing constructs yielded robust Imax values (Table 3).


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 32

Of the constructs examined so far, the Ca2+-permeable α1E/A288G GlyR exhibited the lowest ivermectin EC50 and the largest ivermectin-induced Imax values (Table 3). We thus investigated it further by incorporating the F207A mutation, to reduce its sensitivity to glycine. A comparison of α1E/A288G and α1F207A/E/A288G GlyRs is shown in Figure 4D with mean results summarized in Table 3. The F207A did not significantly impair ivermectin EC50 or Imax values although, as shown in Figure 4A, it completely eliminated glycine sensitivity. The α1F207A/E/A288G GlyR is thus our best candidate for the Ca2+-permeable excitatory construct. We also compared the effect of the F207A mutation on the Ca2+-impermeable α1DEV GlyR. As shown in Figure 4D and Table 3, the α1F207A/DEV/A288G GlyR exhibited a significant enhancement in ivermectin sensitivity over the α1DEV GlyR, although Imax values were significantly reduced (Table 3). Nonetheless, the Ca2+impermeable α1F207A/DEV/A288G GlyR is comparable in overall performance to the Ca2+-permeable α1F207A/E/A288G GlyR. We next sought to confirm the Na+:Cl- permeability ratios of the main constructs of interest using electrophysiology. Whole cell recordings were first obtained in approximately symmetrical NaCl concentrations, with the intracellular solution containing (in mM): 145 NaCl, 2 CaCl2, 2 MgCl2, 10 HEPES and 10 EGTA (pH 7.4 adjusted with NaOH) and the extracellular solution containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4 adjusted with NaOH). We refer to the later solution as 1NaCl. We also employed a 0.5NaCl solution, which was identical in composition to the extracellular solution except that the NaCl concentration was halved. We quantified the reversal potentials of glycine- and ivermectin-activated currents by applying either voltage steps from -80 to +80 mV in 10 mV increments, or voltage ramps from -80 to +80 mV (results from both approaches were pooled) in both 1NaCl and 0.5NaCl solutions. An example of a control experiment on the α1WT GlyR is shown in Figure 5A. The reversal potential for both agonists was right-shifted in 0.5NaCl, indicating Cl- selectivity (Figure 5A). The same experiment performed on the α1DE GlyR revealed a reversal potential shift in the negative direction, confirming its cation permeability (Figure 5B). The mean reversal potentials for all tested constructs in 1NaCl and 0.5NaCl are presented in Table 4. These values have been corrected for the liquid junction potential by adding +3.6 mV to all reversal potentials recorded in 0.5NaCl26. The experimentally determined +14.5 mV reversal potential for the α1WT GlyR was close to the Cl- equilibrium potential of +17.5 mV. In the presence of 0.5NaCl, a perfectly Na+-selective channel should have a reversal potential near -17.5 mV whereas a channel equally permeant to Na+ and Cl- should have a reversal potential of 0 mV. Most of the constructs we tested fell within this reversal potential range (Table 4), consistent with relatively low Na+:Cl- selectivity ratios as previously determined (e.g., Figure 1D).


6

Page 7 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As Ca2+ permeability is difficult to quantify by reversal potential measurements27, we relied on Ca2+ imaging experiments to validate the Ca2+ permeabilities of our two final constructs: the putatively

Ca2+-impermeable

α1F207A/DEV/A288G

GlyR

and

the

putatively

Ca2+-permeable

α1F207A/E/A288G GlyR. The receptors were activated by either 10 mM glycine or 10 µM ivermectin under conditions as outlined above (Figure 3), and their responses were compared to those of the highly Ca2+-permeable α1E GlyR and to the poorly Ca2+-permeable α1DEV/A288G GlyR (Figure 6A, B). These results confirm that the α1F207A/E/A288G GlyR exhibits high Ca2+ permeability whereas the α1F207A/DEV/A288G GlyR does not.

Cell surface expression We compared the cell surface expression levels of 4 constructs (the α1E/A288G, α1F207A/E/A288G, α1DEV/A288G and α1F207A/DEV/A288G GlyRs) relative to the α1WT GlyR via immunohistochemistry on non-permeabilized, fixed HEK293 cells. Sample images for each construct are shown in Figure 7AE with averaged results presented in Figure 7F. Although the α1DEV/A288G GlyR exhibited significantly reduced expression, the expression levels of the α1F207A/E/A288G and α1F207A/DEV/A288G GlyRs were not significantly different to α1WT GlyRs.

Cell death We next determined whether the Ca2+-permeable constructs could efficiently induce excitotoxic cell death. Different constructs were individually expressed in HEK293 cells together with GFP as a transfection marker, and plated into 384 well plates at a density of 3000 cells/well. One day later, ivermectin was added at a defined concentration (with or without 5 µM strychnine) for a period of 12 h. We then quantified the number of fluorescent cells remaining using DetecTiff image analysis software28. Examples of experiments using indicated GlyRs are shown in Figure 8A, with averaged data for all tested constructs presented in Figure 8B. Each histogram in panel A shows the total number of adherent (i.e., healthy) green fluorescent cells in each well. Each experimental condition was tested in 24 adjacent wells. We also investigated the effect of 5 µM strychnine as it was routinely added to the culture medium to aid the survival of cells expressing constructs incorporating the A288G mutation. Note, however, that ivermectin-activated currents are not significantly inhibited by strychnine at concentrations up to 10 µM29. As seen in Figure 8A and B, ivermectin, at concentrations up to 10 µM had no effect on the number of viable, adherent fluorescent HEK293 cells expressing α1WT GlyRs, irrespective of the presence of strychnine. We also tested the anion-permeant α1A288G GlyR due to its elevated sensitivity to glycine and ivermectin17. Even in the absence of strychnine, ivermectin concentrations up to 10 µM had no ACS Paragon Plus Environment

7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 32

effect on the number of viable, adherent fluorescent cells expressing these GlyRs (Figure 8B). In contrast, the Ca2+-permeable α1E GlyR caused the loss of relatively few cells at ivermectin concentrations ≥ 0.1 µM (Figure 8A, B). As expected, cells were ablated at much lower ivermectin concentrations in the ivermectin-sensitive α1E/A288G and α1F207A/E/A288G GlyRs (Figure 8A, B). Indeed, 1 nM ivermectin (the lowest concentration tested) ablated around 80 % of fluorescent cells expressing the α1F207A/E/A288G GlyRs. It is likely that many of the surviving cells expressed EGFP but not the α1F207A/E/A288G GlyR. In contrast, the highly ivermectin-sensitive α1F207A/DEV/A288G GlyR was far less effective in mediating cell death, most likely because of its impermeability to Ca2+ (Figure 8A, B).

Electrophysiological characterization in adenoviral-infected neurons In a final series of experiments, cultured embryonic rat cortical neurons were transduced via an adeno-associated virus with α1F207A/E/A288G GlyRs plus EGFP as an infection marker. Whole-cell voltage-clamp recordings were performed at least two weeks later when virtually all neurons exhibited bright green fluorescence. Figure 9A shows the typical response to ivermectin at successively increasing concentrations of 0.1, 1 and 10 µM of an infected neuron voltage clamped at -70 mV. At 0.1 µM, ivermectin application not only induced a robust inward current in the voltage-clamped neuron but also enhanced the frequency of spontaneous synaptic currents, indicating that it also activated at least one presynaptic neuron. Given that anionic and cationic currents are both excitatory under our recording conditions, the later effect is important as it demonstrates neuronal excitation in intact neurons. At a 0.1 µM concentration, 7/7 neurons that exhibited robust ivermectin-gated currents and robust spontaneous synaptic activity also showed a dramatic increase in synaptic activity. Indeed, the mean increase in the frequency of synaptic events at 0.1 µM ivermectin was 211 ± 17 % (n = 7 cells, p < 0.0001). In contrast, uninfected neurons (which may contain native GlyRs) were much less responsive to applied ivermectin (e.g., Figure 9B).

As in our previous study16, we never observed an ivermectin-activated current at a

concentration < 3 µM in uninfected neurons. Furthermore, neurons from uninfected cultures that exhibited robust spontaneous synaptic activity never exhibited a change in synaptic frequency following 3 µM ivermectin exposure (Figure 9B).

CONCLUSION

We have described the development of a line of novel chemogenetic receptors, based on the human α1 GlyR. These receptors were designed with the aim of either exciting or ablating neurons ACS Paragon Plus Environment

8

Page 9 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


expressing these receptors. Excitation was achieved by developing a receptor with high Na+ and low Ca2+ permeability, whereas the second receptor was designed to have a high Ca2+ permeability, which would elevate cytosolic calcium and thus should trigger an apoptopic cascade. Conversion of the anion permeable α1 GlyR was achieved by mutating the pore selectivity region to provide a cation permeable receptor. A range of constructs was tested and of these the α1F207A/DEV/A288G GlyR proved to be the optimal Ca2+-impermeant construct. Using an intracellular Ca2+ imaging assay, we confirmed that it was virtually impermeant to Ca2+, in agreement with a previous study on the α1DEV GlyR that relied on the measurement of reversal potentials shifts when the Ca2+ concentration was varied21. Here we modified the α1DEV GlyR by adding the A288G and F207A mutations, resulting in a significant enhancement in ivermectin sensitivity (EC50 500 nM) coupled with the elimination of glycine sensitivity and no change in cell surface expression efficiency. For the Ca2+-permeant receptor, the α1F207A/E/A288G GlyR proved to be the optimal construct with robust ivermectin-induced Ca2+ permeability as assessed by Fluo-4-based Ca2+ imaging. As with the Ca2+-impermeant receptor, addition of the A288G and F207A mutations produced a receptor that was activated by ivermectin with an EC50 of 400 nM but was completely insensitive to glycine. Cell surface expression levels were similar to those of unmutated receptors. When these constructs were expressed in HEK293 cells, 1 nM ivermectin applied for 12 h resulted in the death of ~80% of cells. Furthermore, in cortical neurons expressing this construct, 100 nM ivermectin induced inward currents and enhanced synaptic activity consistent with depolarisation of synaptically connected neurons. Sustained activation of Ca2+-permeable NMDA-type glutamate receptors is known to result in excitotoxicity that can contribute to neuronal damage in stroke and neurodegenerative disorders 30,31

. Given that NMDA receptors and Ca2+-permeable GlyRs exhibit comparable fractional Ca2+

influx rates21,22,32, sustained activation of the α1F207A/E/A288G GlyR should induce a sufficient Ca2+ influx to induce excitotoxic cell death in neurons. Neurodegenerative disorders are often mediated by cell death that is caused by activation of a pathological Ca2+ influx pathway. However, it is hard to distinguish whether the Ca2+ influx is a primary or secondary pathogenic event33. By providing a defined source of Ca2+ that can be controlled independently of the pathological Ca2+ influx pathway, the Ca2+-permeable receptor we have developed here could thus provide insights into the pathogenesis of neurodegenerative disorders. Such a receptor should also be useful for selectively ablating specific populations of neurons for studying neural circuit function. One of our original motivations for using the GlyR as a template for developing chemogenetic constructs was its high (90 pS) unitary conductance. However, the cation-permeating constructs developed here most likely exhibit unitary conductances of around 5 pS (Figure 1D). In spite of ACS Paragon Plus Environment

9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 32

this, the peak whole cell current magnitudes for both the α1F207A/E/A288G and the α1F207A/DEV/A288G GlyRs were 53 and 35 % that of the α1WT GlyR current magnitude, respectively (Table 3), implying that their reduced in unitary conductance is largely compensated by an increased open probability. The conductance of our constructs is still likely to be many times higher than that of channelrhopsin-2 (~50 fS34), a widely used excitatory optogenetic receptor. It is difficult to compare our Ca2+-permeable construct with other previously developed chemogenetic constructs as little attention has been paid to the fractional Ca2+ permeability of the cationic conductances induced by other excitatory constructs. For example, a widely used M3-based DREAD construct couples via the G protein, Gq, to activate an depolarizing ion flux in neurons that appears to be initiated by inhibition of an M-type K conductance9. Thus, no Ca2+-permeable influx pathway is activated by this method, although Ca2+ influx may occur in response to the subsequent neuronal depolarization. Our constructs have a relatively low sensitivity to ivermectin in terms of neuronal depolarisation. As the ivermectin concentration in the brain following oral or intraperitoneal delivery typically reaches only 1-3 nM13, it is likely that stereotaxic injection would be needed to administer ivermectin to the required concentration in behaving animals. Our constructs may be more suited to acute brain slice or cultured neuron experiments. However, as cell death requires only 1 nM ivermectin (Figure 8), it is feasible that orally ingested ivermectin could reach the brain in a high enough concentration for this purpose. In conclusion, we have developed new excitatory chemogenetic receptors based on the human α1 GlyR that are either highly permeable to Ca2+ (the α1F207A/E/A288G GlyR) or minimally permeable to Ca2+ (the α1F207A/DEV/A288G GlyR). They may be useful for investigating the origins of neurodegenerative disorders and for inducing the acute activation of defined populations of neurons in vitro.

METHODS

Mutagenesis and expression of plasmid DNA in HEK293 cells All constructs were based on the human α1 GlyR cDNA in the pIRES plasmid vector. Sitedirected mutagenesis was performed using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) and the mutations were confirmed by DNA sequencing. HEK293 cells were cultured at 37 ºC in a humidified 5 % incubator in Dulbecco’s Modified Eagle Medium supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) and 10 % fetal calf serum. We added 5 µM strychnine to the culture medium of cells that expressed GlyRs incorporating the A288G mutation,


10

Page 11 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


as the high glycine sensitivity of these receptors otherwise rendered them susceptible to activation by glycine in the culture medium. For electrophysiology experiments, the empty pEGFP plasmid vector (Clontech, Mountain View, CA, USA) was co-expressed as a transfection marker. Cells were co-transfected with GlyR and EGFP cDNAs in a 10:1 ratio via a Ca2+ phosphate precipitation protocol. During transfection, cells were incubated at 37 ºC in a humidified 3 % incubator. Around 18-24 h post transfection, cells were washed twice with divalent cation-free phosphate buffered saline and returned to the culture medium. Cells were used for patch clamp recording over the following 24-72 h. For anion influx and Ca2+ imaging experiments, cells were transfected via a Ca2+ phosphate precipitation protocol with GlyR cDNA alone (for Ca2+ imaging experiments) or together with YFPI152L in the pcDNA3 vector in a 1:1 ratio (for anion imaging experiments). Following incubation at 37 ºC in a humidified 3 % incubator for 18-24 h, cells were washed twice with divalent cation-free phosphate buffered saline and immediately plated into 384 well plates at 3000 cells per well. Cells were used in experiments between 24-72 h after plating.

Neuronal culture and adenoviral infection Our methods for generating cortical neuronal cultures have recently been described in detail35. Briefly, euthanasia of timed-pregnant rats was performed via CO2 inhalation, in accordance with procedures approved by the University of Queensland Animal Ethics Committee (approval number: QBI/203/13/ARC). E18 rat embryos were surgically removed, and the cortical neurons were dissected out. These were then mechanically dissociated, counted and plated onto 12 mm poly-Dlysine-coated coverslips in 4-well plates at ~80,000 neurons per well. Neurons were cultured for at least one week before infection with 1 µl AAV pAM-Gly(F207A/A-1’E/A288G)-T2A-EGFP. Electrophysiological recordings commenced a minimum of 2 wk later.

Patch clamp electrophysiology Cells were viewed using an inverted fluorescent microscope and currents were recorded using the whole-cell patch-clamp configuration. Cells expressing recombinant GlyRs were identified by their green fluorescence. Cells were perfused by the standard extracellular solution containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES/ NaOH and 10 glucose (pH 7.4 adjusted with NaOH). For HEK293 cell recordings we employed an intracellular solution consisting of (mM): 145 CsCl, 2 CaCl2, 2 MgCl2, 10 HEPES and 10 EGTA (pH 7.4 adjusted with CsOH; osmolarity ≈ 290 mOsm). Unless otherwise indicated, HEK293 cell recordings were performed at a holding potential of -40 mV. These solutions were employed in all HEK293 experiments except for those where the Na+:Cl- permeability ratios were investigated (see Figure 5 and related text). The ACS Paragon Plus Environment

11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 32

intracellular and extracellular solutions used in those experiments are provided in the Results section. For neuronal recordings we employed the standard extracellular solution (see previous paragraph) and an intracellular solution consisting of (mM): 135 KMeSO4, 8 NaCl, 2 Mg2-ATP, 10 HEPES, 0.3 Na3-GTP and 0.3 EGTA (pH 7.3 adjusted with KOH; osmolarity ≈ 290 mOsm). Neuronal recordings were performed at a holding potential of -70 mV. Patch pipettes were fabricated from borosilicate hematocrit tubing (Hirschmann Laborgerate, Eberstadt, Germany) and heat polished. Pipettes had a tip resistance of 1-2 MΩ. Membrane currents were recorded using an Axopatch 200B amplifier and a Digidata 1440 analog-to-digital converter under control of pClamp10 software (Molecular Devices). Currents were filtered at 500 Hz and digitized at 2 KHz. Solutions were applied to cells via gravity induced perfusion via parallel microtubules and manual control of this system was achieved via a micromanipulator with a solution exchange time of < 250 ms. Experiments were conducted at room temperature (20-22 oC).

Anion influx imaging experiments Immediately before commencement of these experiments the culture media in each well of a 384 well plate was replaced with 15 µl of standard extracellular solution (see above). The agonist (glycine or ivermectin) was dissolved in a similar solution in which the NaCl had been entirely replaced by 140 mM NaI. To activate GlyRs, a total of 60 µM of NaI-containing solution (plus the required concentration of agonist) was added to each well. Cells in each well were imaged immediately before NaI injection and again 10 s after NaI injection. The percentage fluorescence change induced by added glycine or ivermectin is defined as [(Ffinal/Finit) - 1]×100 % where Finit and Ffinal are the initial and final values of fluorescence, respectively36,37. In determining dose-response relationships, each agonist concentration was applied to cells in two adjacent wells and a single data point was taken as the average quench observed in all fluorescent cells. We typically observed 200400 fluorescent cells/well. Ca2+ imaging experiments A 50 µg aliquot of the membrane-permeant fluorescent Ca2+ indicator, Fluo-4-AM (ThermoFisher Scientific, New York, USA), was mixed vigorously by vortexing and ultrasonication with 48 µl of dimethyl sulfoxide (DMSO) and 2 µl of 20% pluronic acid. This mixture was diluted in the standard extracellular solution to a final Fluo-4-AM concentration of 10 µM. Culture medium was removed from the cells in 384 well plates and 25 µl of 10 µM Fluo-4-AMcontaining solution was added to each well and incubated at 37 ºC in a humidified 5 % incubator for


12

Page 13 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30 – 60 min. After this time, the Fluo-4-AM-containing solution was replaced by 40 µl standard extracellular solution. The whole procedure was carried out in the dark. The cells were then imaged. To activate GlyRs, a total of 40 µM of standard extracellular solution (plus the required concentration of agonist) was added to each well. A control image of the cells was taken immediately before the addition of agonist, and a test image was taken 8 s after the addition of glycine or 30 s after the addition of ivermectin. The percentage fluorescence increase induced by added glycine or ivermectin is defined as (Ffinal/Finit)×100 %. In determining dose-response relationships, each agonist concentration was applied to cells in two adjacent wells and a single data point was taken as the average quench observed in all fluorescent cells. Anion and Ca2+ imaging equipment Fluorescence experiments were performed using an automated imaging system with a robotcontrolled liquid handling system constructed in the laboratory. The 384-well plates were placed onto a motorized stage (Prior ProScan II, GT Vision, Hagerstown, MD, USA) of an Olympus IX51 inverted microscope and cells were imaged with an Olympus 10× objective (UPlanFLN, N.A. 0.30). Illumination for both YFP and Fluo-4-AM experiments was provided by an Osram 100 W mercury short arc lamp (HBO 103/2), passing through an Olympus YFP dichroic mirror (86002V2 JP4 C76444). Emitted fluorescence passed through a magnifier lens (diopter 8, mineral glass), and was then imaged by a Photometrics CoolSNAP CF monochrome camera (Roper Scientific GmbH, Ottobrun, Germany) and digitized onto a personal computer. The final resolution of the images was 696 ×520 pixels. Liquid-handling was performed with an LC PAL autosampler (CTC Analytics, Zwingen, Switzerland) using a 100 µl syringe. A suite of LabView 8.2.1 software (National Instruments Corp, Austin, Texas, USA) routines was used to control hardware, image acquisition, storage, image analysis and data quantification. Image analysis was performed using Detectiff software28.

Immunohistochemistry HEK293 cells expressing indicated GlyRs were fixed with 4% paraformaldehyde in phosphate buffered saline and then incubated with an N-terminal polyclonal α1 GlyR-specific antibody (AB15012, Merck Millipore, Darmstadt, Germany) for 2 h. After washing with phosphate buffered saline, AlexaFluor555-conjugated secondary antibody (A-31572, ThermoFisher, Waltham, USA) was applied for 1 h. The primary and secondary antibodies were diluted in blocking buffer containing 0.5% BSA according to manufacturers’ recommendations (1:250 and 1:500 respectively). Cells were imaged the same day by confocal microscope (LSM 510 Meta, Zeiss, Germany). For comparative intensity analysis, the microscope settings were held constant for all ACS Paragon Plus Environment

13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 32

measurements. The level of surface expression was quantified by measuring fluorescence intensity profile along the membrane of the cells in ImageJ software.

Data analysis Results are expressed as mean ± SEM of three or more independent experiments. The Hill equation was used to calculate the saturating current magnitude (Imax) and half-maximal concentration (EC50) values for glycine or ivermectin activation. All curves were fitted using a nonlinear least squares algorithm (Sigmaplot 11.0; Jandel Scientific, San Rafael, CA, USA). Statistical significance was generally determined by paired or unpaired Student’s t-test or one-way ANOVA, as appropriate, with p < 0.05 representing significance. The exception is Figure 7F where we employed the non-parametric Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparison test as some data sets did not satisfy normality.

AUTHOR INFORMATION

Corresponding author * Telephone: (+617) 33466375. Fax: (+617) 33466301. Author contributions Participated in research design: R.I., A.K., P.S. and J.W.L. Provided reagents: L.X. Conducted experiments and performed data analysis: R.I., N.D., L.X. Interpreted results and wrote manuscript: R.I., A.K., N.D., P.S. and J.W.L. Funding Funding for this research was received from the Australian Research Council (LP120100297) and the National Health and Medical Research Council (APP1058542 and APP1060707). Notes The authors declare no competing financial interest.

ABBREVIATIONS 5-HT3R, 5-hydrotryptamine type-3 receptor cation channel; DMSO, dimethyl sulfoxide; E, E-1’A; EC, extracellular; EC50, half maximal concentration; GABAAR, GABA type-A receptor chloride channel; GluR, glutamate receptor chloride channel; GlyR, glycine receptor chloride channel; Imax, saturating current magnitude; nAChR, nicotinic acetylcholine receptor cation channel; P, P-2’∆; pLGIC, pentameric ligand-gated ion channel; TM, transmembrane; V, T13’V; WT, wild type.


14

Page 15 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE LEGENDS

Figure 1. Structural and functional basis of construct development. (A) View of the α1 GlyR from within the plane of the membrane with extracellular (EC) and transmembrane (TM) domains indicated. One subunit is coloured blue, the five TM2 domains are coloured yellow and the key mutated residues F207 (in the EC domain) and A288 (in the TM domain) are shown as green spheres. This model is based the zebrafish α1 GlyR cryo-EM structure with glycine and ivermectin bound (PDB access code: 3JAF)19. (B) Top-down view of the same structure with EC domains removed and a single bound ivermectin molecule shown. (C) Three of the five TM2 domains are shown from the structure depicted in A with the P-2’, A-1’, T13’ and R19’ ion-permeation control residues shown as spheres. (D) Amino acid sequence alignment of the TM2 domains of the cationpermeant human α7 nAChR and 5-HT3RA and the anion-permeant C elegans α GluR, human α1 GABAAR and human α1 GlyR. Ionic selectivity determinants that systematically vary between cation- and anion-permeant pLGICs are highlighted in green, and numbered according to the standard TM2 system. The TM2 sequences of the cation-permeant mutant α1 GlyRs that were described previously by Barry, Keramidas and colleagues21-23 are also shown together with their published Cl-:Na+ permeability ratios (PCl/PNa), Ca2+:Na+ permeability ratios (PCa:PNa) and their single channel conductances.

Figure 2. Glycine- and ivermectin-sensitivities of GlyR constructs as determined by YFP-based anion influx assay. (A) Mean glycine dose-response relationships for indicated constructs. (B) Mean ivermectin dose-response relationships for indicated constructs, with a sample experiment shown in the inset below for the α1DE GlyR. (C) Effect of the F207A mutation on the glycine and ivermectin sensitivities of the α1E/A288G and α1F207A/E/A288G GlyRs. Mean EC50 and maximum % fluorescence quench values for all experiments in this Figure are summarized in Table 1.

Figure 3. Glycine- and ivermectin-sensitivities of GlyR constructs as determined by Fluo-4-AMbased Ca2+ influx assay. (A) Mean glycine dose-response relationships for indicated constructs with a sample experiment shown on the right for the α1DE GlyR. (B) Mean ivermectin dose-response relationships for indicated constructs, with a sample experiment shown on the right for the α1DE GlyR. Mean EC50 and maximum % fluorescence increase values for all experiments in this figure are shown in Table 2.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 32

Figure 4. Ivermectin sensitivities of GlyR constructs as determined by electrophysiology. (A) Sample recordings from the α1E/A288G and α1F207A/E/A288G GlyRs indicating typical current responses to the application of glycine (filled bars) and ivermectin (unfilled bars). (B) Averaged doseresponse relationships for the original set of constructs (α1WT, α1E, α1DE, α1DE/R19’E and α1DEV GlyRs). (C) Averaged dose-response relationships for the same constructs incorporating the A288G mutation. (D) Averaged dose-response relationships for the α1E/A288G and α1DEV GlyRs without and with the F207A mutation. Mean ivermectin EC50 and Imax values for all constructs displayed in this Figure are summarized in Table 3.

Figure 5. Determination of GlyR reversal potentials in 1NaCl and 0.5NaCl solutions. Voltage ramps were applied from -80 to +80 mV to channels activated by 10 mM glycine (left panel) or 20 µM ivermectin (right panel). Leakage currents, as determined by applying ramps to baseline currents recorded in the absence of agonist, were digitally subtracted. (A) In α1WT GlyRs, switching from 1NaCl to 0.5NaCl induced a rightward shift in reversal potential, indicating anion permeability. (B) In α1DE GlyRs, switching from 1NaCl to 0.5NaCl induced a leftward shift in reversal potential, indicating Na+ permeability. Averaged reversal potentials for these and all other tested constructs in 1NaCl and 0.5NaCl are summarized in Table 4. Figure 6. Relative Ca2+ permeabilities of our final GlyR constructs as determined by Ca2+ imaging. (A) The mean percentage increase in Fluo-4-AM fluorescence in response to a 10 s application of 10 mM glycine is compared for two control constructs (the highly Ca2+-permeable α1E GlyR and the poorly Ca2+-permeable α1DEV/A288G GlyR) plus the two test constructs, the α1F207A/E/A288G GlyR and the α1F207A/DEV/A288G GlyR. The four constructs were screened in parallel in a separate series of experiments to those shown in Figure 3. Displayed results represent means ± SEM from four independent experiments. (B) A similar experiment, except that 10 µM ivermectin was applied for 10 s. In both panels, *p

Ivermectin-Activated, Cation-Permeable Glycine ... - ACS Publications

Recommend Documents