Letter pubs.acs.org/chemneuro
A Chemogenetic Receptor That Enhances the Magnitude and Frequency of Glycinergic Inhibitory Postsynaptic Currents without Inducing a Tonic Chloride Flux Robiul Islam,† Yan Zhang,† Li Xu,† Pankaj Sah,† and Joseph W. Lynch*,†,‡ †
Queensland Brain Institute and ‡School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia S Supporting Information *
ABSTRACT: The gene transfer-mediated expression of inhibitory ion channels in nociceptive neurons holds promise for treating intractable pain. Chemogenetics, which involves expressing constructs activated by biologically inert molecules, is of particular interest as it permits tunable neuromodulation. However, current chloridepermeable chemogenetic constructs are problematic as they mediate a tonic chloride influx which over time would deplete the chloride electrochemical gradient and reduce inhibitory efficacy. Inflammatory pain sensitization can be caused by prostaglandin E2-mediated inhibition of glycinergic inhibitory postsynaptic currents in spinal nociceptive neurons. We developed a highly conducting (100 pS) inhibitory chemogenetic construct based on a human glycine receptor (α1Y279F,A288G) with high ivermectin sensitivity. When virally infected into spinal neurons, 10 nM ivermectin increased the magnitude and frequency of glycinergic postsynaptic currents without activating a tonic chloride flux. The construct should thus produce analgesia. Its human origin and the well-established biocompatibility of its ligand suggest it may be suited to human use. KEYWORDS: Chloride channel, pharmacogenetic, chemogenetic, pain, gene transfer, ligand-gated, glycine receptor
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inhibitory neurotransmission in the spinal cord and brainstem.11 Nonsynaptic GlyRs are distributed more broadly throughout the brain. Human GlyRs are functionally expressed as homomers of α1−3 subunits or as heteromers of α and β subunits. The majority of adult glycinergic postsynaptic densities exclusively incorporate α1β GlyRs.11 However, glycinergic synapses on pain sensory neurons in the spinal cord dorsal horn are unique in that they incorporate comparable numbers of α1β and α3β GlyRs.12 Chronic inflammatory pain sensitization can be caused by the prostaglandin-mediated inhibition of α3β GlyRs which diminishes the magnitude of inhibitory postsynaptic currents (IPSCs) and thus disinhibits these neurons.13 This mechanism implies that therapies that enhance glycinergic IPSCs in these synapses should be analgesic.12,14 An early chemogenetic receptor comprised a Caenorhabditis elegans heteromeric αβ glutamate receptor (GluR) chloride channel engineered for selective activation by low nanomolar ivermectin concentrations.15 Its success was limited by its low conductance and the difficulty in ensuring adequate and equivalent expression levels of both subunits. We subsequently generated a homomeric version based on the human α1 GlyR that exhibited a similar ivermectin sensitivity to the GluCl-based
hronic pain is a serious and growing problem in most societies with nearly 30% of adults suffering in any 6 month period. Its annual cost in the United States was estimated at $600 billion in 2012, exceeding the combined cost of cancer, heart disease, and diabetes.1 There are few effective treatments for chronic pain, with most pharmacological agents exhibiting limited efficacy and dose-limiting side effects. As noted in a recent review, “despite substantial financial investment by the pharmaceutical industry over several decades, there has been little progress in developing new ef f icacious and safe analgesics.”2 New therapeutic paradigms are thus clearly warranted. There is an emerging view that the virus-mediated gene transfer of inhibitory ion channels into spatially targeted nociceptive neurons holds promise for treating chronic pain.3−6 Optogenetic and chemogenetic constructs are considered particularly promising in this respect as their inhibitory efficacy can be tuned according to need.6,7 Optogenetics involves expression of light-activated ion channels in desired neurons, thus enabling the neurons to be activated or inhibited within milliseconds by light.8 However, its implementation in vivo requires the implantation of optical stimulation devices which presents a range of technical obstacles.6,9 Chemogenetics,10 which involves expressing ion channels activated by biologically inert molecules delivered orally or by injection, provides an alternative option. Glycine receptor (GlyR) chloride channels are members of the pentameric ligand-gated ion channel family that mediate © XXXX American Chemical Society
Received: November 8, 2016 Accepted: December 13, 2016 Published: December 13, 2016 A
DOI: 10.1021/acschemneuro.6b00382 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Letter
ACS Chemical Neuroscience
Figure 1. Glycine and picrotoxin sensitivities of the α1, α1β, α1Y279F,A288G, and α1Y279F,A288Gβ GlyRs. (A) The left-hand panels display examples of currents activated by the indicated glycine concentrations in cells expressing α1, α1β, α1Y279F,A288G, and α1Y279F,A288Gβ GlyRs. In this and all subsequent figures, horizontal bars represent the period of glycine application. The right-hand panels show the effect of 30 μM picrotoxin on currents activated by an empirically determined EC40 glycine concentration. (B) Averaged glycine concentration−response relationships for the α1, α1β, α1Y279F,A288G, and α1Y279F,A288Gβ GlyRs. Mean parameters of best fit to the Hill equation are given in Table 1. (C) Mean percentage of current remaining at each subtypes in the presence of 30 μM picrotoxin. ***p < 0.001 by Mann−Whitney U test.
leading to a reduction or even a polarity reversal of glycinergic signaling.19 Thus, tonic activation of chloride-permeable chemogenetic receptors may actually exacerbate pain. A similar concern applies to chloride-permeable optogenetic receptors.7,20 To circumvent this problem, we sought to develop a GlyRbased receptor that is not directly activated by ivermectin but instead exhibits potent ivermectin-mediated enhancement of
construct, but had a larger unitary conductance (90 vs 25 pS) and robust homomeric expression.16 This construct has been validated in vivo in a range of behavioral paradigms.17,18 The inhibitory ivermectin receptors developed so far work by inducing a tonic chloride flux. Because the chloride concentration in adult neurons is low, small increases in the tonic influx rate can drastically impair the chloride electrochemical gradient, B
DOI: 10.1021/acschemneuro.6b00382 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Letter
ACS Chemical Neuroscience Table 1. Glycine and Ivermectin Concentration−Response Properties glycine
a
ivermectin
GlyR
EC50 (μM)a
nH
Imax (nA)
n
EC50 (μM)a
n Ha
Imax (nA)
n
α1 α1β α1Y279F,A288G α1Y279F,A288Gβ
37.1 ± 1.6 26.1 ± 2.8 309 ± 15*** 215 ± 5**
2.1 ± 0.2 1.4 ± 0.5 2.8 ± 0.6 2.7 ± 1.4
11.4 ± 3.2 8.7 ± 2.5 9.1 ± 3.0 8.4 ± 2.7
12 6 9 7
1.35 ± 0.07 4.7 ± 0.6* 0.18 ± 0.01** 0.16 ± 0.02**
1.6 ± 0.1 0.8 ± 0.03* 3.6 ± 0.1* 2.2 ± 0.5*
8.7 ± 1.3 6.9 ± 1.4 5.3 ± 0.5 5.2 ± 1.4
7 5 6 4
*p < 0.05, **p < 0.01, and ***p < 0.001 relative to corresponding α1 GlyR value by Mann−Whitney U test.
Figure 2. Effects of ivermectin on α1, α1β, α1Y279F,A288G and α1Y279F,A288Gβ GlyRs. (A) Sample recordings from the α1, α1β, α1Y279F,A288G, and α1Y279F,A288Gβ GlyRs indicating typical current responses to the application of successively increasing ivermectin concentrations. (B) Averaged ivermectin concentration−response relationships for the four GlyRs. Mean parameters of best fit to the Hill equation are given in Table 1. (C) Examples of ivermectin potentiation of EC2 (30 μM) glycine-gated responses in α1Y279F,A288G GlyRs. Ivermectin was applied for 5 s intervals at the indicated concentrations prior to each glycine application. (D) Averaged ivermectin potentiation dose−response relationships normalized to the saturating glycine-gated current. All data points were averaged from 9 cells with a mean ivermectin potentiating EC50 of 44.7 ± 8.5 nM. (E) A subthreshold (1 μM) glycine concentration is not converted into an activating concentration by 30 nM ivermectin in α1Y279F,A288G or α1Y279F,A288Gβ GlyRs. (F) Averaged results from the experiment in (E). n = 9 for α1Y279F,A288G GlyRs and n = 3 for α1Y279F,A288Gβ GlyRs; ***p < 0.001 by Mann−Whitney U test.
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currents activated by synaptically relevant glycine concentrations. We reasoned that if these receptors integrated into glycinergic postsynaptic densities, they might respond to ivermectin by enhancing the magnitude or duration of glycinergic IPSCs without tonically activating a chloride conductance. Here we describe the functional properties and in vitro performance of a construct designed for this purpose.
RESULTS AND DISCUSSION
Synaptically released glycine reaches a maximum of 1−3 mM in the synaptic cleft,21,22 whereas the tonic extrasynaptic glycine concentration remains low at ∼1 μM.23 We excluded constructs that were activated by either low (
