An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity...

An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*

The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity. Several neuronal silencing methods have been developed, with the bacterial light-activated halorhodopsin and the invertebrate allatostatin-activated G protein-coupled receptor proving the most successful to date. However, both techniques may be difficult to implement clinically due to their requirement for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain following systemic administration. However, the limitations of this method include poor functional expression, possibly due to the requirement to coexpress two different subunits in individual neurons, and the nonhuman origin of GluClR. Here, we describe the development of a modified human α1 glycine receptor as an improved ivermectin-gated silencing receptor. The crucial development was the identification of a mutation, A288G, which increased ivermectin sensitivity almost 100-fold, rendering it similar to that of GluClR. Glycine sensitivity was eliminated via the F207A mutation. Its large unitary conductance, homomeric expression, and human origin may render the F207A/A288G α1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue appears essential for exquisite ivermectin sensitivity.


Several methods for silencing neuronal electrical activity in behaving animals have been developed that could eventually be useful for treating neurological disorders including Parkinson disease, addiction, epilepsy, depression, and chronic pain states F ). Generally, these techniques involve overexpressing an exogenous receptor in molecularly or spatially defined populations of neurons and applying a specific stimulus to activate this receptor and thereby silence the neurons.

The two most successful strategies to date have proved to be bacterial halorhodopsin and the Drosophila allatostatin receptor. Halorhodopsin is a light-activated inward chloride pump, which, when exogenously expressed in neurons, hyperpolarizes neurons rapidly and reversibly upon the application of intense yellow light ). Although this has been employed successfully to correlate neuronal activity with behavior (4 ), it is limited by 1) the requirement for strong overexpression and 2) the necessity to deliver intense light to neurons in vivo. The recent development of more efficient light-driven outward proton pumps may address the first limitation


Molecular Biology

The human α1, α2, α3, and β GlyR subunit cDNAs were each subcloned into the pcDNA3.1 plasmid vector. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA), and the successful incorporation of mutations was confirmed by DNA sequencing.

HEK-293 Cell Culture and Transfection

HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing Serum Supreme (Lonza, Walkersville, MD) and penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO) and split onto coverslips in 3-cm dishes. On the following day, cells were transiently transfected with the relevant GlyR cDNAs via a calcium phosphate method. These cells were also transfected with empty pEGFP vector (Clontech, Mountainview, CA, USA) as a fluorescent transfection marker. In experiments investigating α1β heteromeric GlyRs, α1 and β were transfected in a 1:4 ratio.

Neuron Dissociation, Culture, and Transfection

Experiments were performed on P0–P3 male Wistar rats in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes with approval from the Animal Ethics Committee for the University of Queensland. Animals were anesthetized with isoflurane and decapitated. Brains were immediately removed, and hippocampi were dissected out. Neurons were dissociated by incubation in 0.1% trypsin/EDTA (Invitrogen) for 7 min at 37 °C and were washed in HEPES-buffered minimum essential medium with 0.014% trypsin inhibitor (type I-S from soybean; Sigma-Aldrich). After diluting the cells in neurobasal A medium (Invitrogen) supplemented with 2% B27 (Invitrogen) and 0.5 mm l-glutamine, 2 × 105 cells were added to each well of 4-well dishes (BD Falcon). Each well contained a sterilized glass coverslip treated with poly d-lysine (Invitrogen), on which the cells grew. After 1 h, this medium (conditioned medium) was removed, and 0.5 ml of medium was added, consisting half of conditioned medium and half of neurobasal A, 2% B27, penicillin/streptomycin, 0.5 mm l-glutamine, and 10 ng/ml basic fibroblast growth factor (Invitrogen) (growth medium). After 3 days, cells were washed with phosphate-buffered saline, and 0.5 ml of medium was added, consisting of 20% conditioned medium and 80% fresh growth medium. On days P7–P10, cells were washed in phosphate-buffered saline and incubated for 1 h in fresh growth medium. Transfections were performed after 4–7 days in vitro, with recordings performed 2 days later.


To identify the most appropriate clone on which to base a silencing receptor, we compared the glycine and ivermectin sensitivities of human α1, α1β, α2, and α3 GlyRs. Sample glycine- and ivermectin-gated currents in HEK-293 cells expressing α1 GlyRs are shown in Fig. 1, A and B, respectively. Because ivermectin currents are irreversible (18 ), we generated ivermectin dose-response relationships by successively applying higher ivermectin concentrations. Averaged glycine and ivermectin dose-response curves for α1, α1β, α2, and α3 GlyRs are shown in Fig. 1, C and D. The parameters of best curve fit for these and all other mutant GlyRs examined in this study are summarized in Table 1. As glycine and ivermectin sensitivity did not vary dramatically among the four wild type (WT) constructs tested, we employed the α1 GlyR (the standard model system for structure-function studies) as a starting point for developing an improved ivermectin-activated silencing receptor.

An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*

As there are no clues to date concerning the location of the GlyR ivermectin binding site, we generated and screened a cDNA library comprising ∼1600 randomly mutated α1 GlyR clones and searched for mutations that affected ivermectin sensitivity. Library mutation frequency was selected so that each clone contained an average of 1–2 amino acid mutations. The methods for generating this library and screening it in HEK-293 cells for ivermectin sensitivity have recently been described in detail (

). The screen identified one mutation, T258S, that significantly increased GlyR ivermectin sensitivity. This residue is well known to contribute to the lining of the channel pore (

) and to form the pore binding site for several GlyR-active pharmacological modulators (

). Electrophysiological analysis in HEK-293 cells confirmed that the T258S mutation significantly increased receptor sensitivity to both glycine (Fig. 2, A and E) (Table 1) and ivermectin (Fig. 2, B and F and Table 1). Indeed, the T258S mutation reduced the ivermectin EC50 from ∼1.7 μm to 90 nm. As the T258S mutant GlyR was activated significantly by 10 nm ivermectin (Fig. 2, B and F), we considered it a potential candidate silencing receptor. We therefore introduced the F207A glycine binding site mutation (

) to eliminate sensitivity to the endogenous agonist glycine. As shown in Fig. 2C, a 10 mm concentration of glycine activated no detectable current in the F207A mutant GlyR, although robust ivermectin-gated currents were observed (Fig. 2C). Unfortunately, this mutant receptor showed a significant decrease in ivermectin sensitivity relative to the WT α1 GlyR (Fig. 2F and Table 1). Thus, it is not surprising that the double mutant T258S/F207A GlyR also exhibited a significantly reduced ivermectin sensitivity relative to the T258S GlyR (Fig. 2, D and F, and Table 1). Indeed, as the ivermectin sensitivity of the double mutant was not significantly different to WT, this construct was discarded as a candidate silencing receptor. Nevertheless, these results imply a long range allosteric coupling between T258S and the ivermectin binding site similar to that previously shown between the residue corresponding to L259S and the

An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*


The silencing receptor developed here exhibits a similar ivermectin sensitivity to the C. elegans αβ heteromeric GluClR ). Because that receptor is activated by concentrations of ivermectin that reach the brain following systemic injection ), we expect the F207A/A288G α1 GlyR to respond similarly. However, our human GlyR-based receptor has three main advantages over the GluClR-based silencing receptor. First, it has a larger single channel conductance ), implying a high inhibitory conductance per expressed receptor. Second, it expresses as a homomer, obviating the requirement to recombinantly express two different cDNAs in each target neuron. Both these measures are likely to result in an improved silencing efficiency in vivo. Finally and perhaps most importantly, the silencing construct described here is derived from a human receptor, making it a promising candidate for clinical applications. Furthermore, as the A288T mutant α1 GlyR exhibits normal WT glycine sensitivity but is insensitive to ivermectin, it should be useful as a control for exogenous receptor expression. With the halorhodopsin, allatostatin, and ivermectin-GluClR approaches, it is currently not possible to control for the expression per se of the silencing construct.

Credited to JBC


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