Abstract
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 alpha1 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 alpha1 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.
Highlights
The halorhodopsin and allatostatin approaches are not ideal for human therapy due to their use of exogenous nonhuman receptors and the inconvenience of applying stimuli directly to the target neurons. The second of these issues can be addressed by a third silencing approach, which employs a Caenorhabditis elegans ␣ heteromeric glutamategated chloride channel receptor (GluClR)3 mutated to abolish sensitivity to glutamate while retaining low nanomolar sensitivity to ivermectin [12, 13]
GluClR silencing is slowly reversible, requiring days, as opposed to minutes for allatostatin and milliseconds for lightactivated proton pumps. Use of this method has not yet spread beyond the originating laboratory [15], possibly due to poor functional expression stemming from the requirement to coexpress two different subunits in target neurons
To identify the most appropriate clone on which to base a Electrophysiological experiments on neurons were per- silencing receptor, we compared the glycine and ivermectin formed to those on HEK-293 cells, and only the sensitivities of human ␣1, ␣1, ␣2, and ␣3 glycine receptor chloride channel (GlyR)
Summary
Molecular Biology—The human ␣1, ␣2, ␣3, and  GlyR subunit cDNAs were each subcloned into the pcDNA3.1 plasmid vector. To establish GlyR sensitivity to ivermectin, current magnitude in response to each ivermectin concentration, I, was normalized to maximal ivermectin-gated current magnitude, Imax These I/Imax values were averaged, and reported data points represent means Ϯ S.E. from three or more experiments. To identify the most appropriate clone on which to base a Electrophysiological experiments on neurons were per- silencing receptor, we compared the glycine and ivermectin formed to those on HEK-293 cells, and only the sensitivities of human ␣1, ␣1, ␣2, and ␣3 GlyRs. Sample glydifferences are noted here. Transfected neurons were iden- cine- and ivermectin-gated currents in HEK-293 cells expresstified by GFP fluorescence and the presence of ing ␣1 GlyRs are shown, A and B, respectively. Indicated current magnitudes were recorded at 10 mM glycine or 30 M ivermectin
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