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. 2018 Apr 18;9(4):684-690.
doi: 10.1021/acschemneuro.7b00485. Epub 2017 Dec 27.

A Caged Enkephalin Optimized for Simultaneously Probing Mu and Delta Opioid Receptors

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Free PMC article

A Caged Enkephalin Optimized for Simultaneously Probing Mu and Delta Opioid Receptors

Matthew R Banghart et al. ACS Chem Neurosci. .
Free PMC article

Abstract

Physiological responses to the opioid neuropeptide enkephalin often involve both mu and delta opioid receptors. To facilitate quantitative studies into opioid signaling, we previously developed a caged [Leu5]-enkephalin that responds to ultraviolet irradiation, but its residual activity at delta receptors confounds experiments that involve both receptors. To reduce residual activity, we evaluated side-chain, N-terminus, and backbone caging sites and further incorporated the dimethoxy-nitrobenzyl moiety to improve sensitivity to ultraviolet light-emitting diodes (LEDs). Residual activity was characterized using an in vitro functional assay, and the power dependence and kinetics of the uncaging response to 355 nm laser irradiation were assayed using electrophysiological recordings of mu opioid receptor-mediated potassium currents in brain slices of rat locus coeruleus. These experiments identified N-MNVOC-LE as an optimal compound. Using ultraviolet LED illumination to photoactivate N-MNVOC-LE in the CA1 region of hippocampus, we found that enkephalin engages both mu and delta opioid receptors to suppress inhibitory synaptic transmission.

Keywords: Caged compounds; neuropeptides; neurophysiology; opioid receptors; potassium channels; synaptic transmission.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Residual activity of caged LE derivatives at recombinant MOR and DOR. (a) Dose–response curves for 14 in comparison to LE at MOR (top) and DOR (bottom) expressed in HEK293T cells using a functional secreted-alkaline phosphatase (SEAP) assay (n = 6–12 wells per data point). Data were normalized to the maximal response to LE and are expressed as the mean ± SEM. (b) As in (a) for 5 and 6. The same LE data is presented in both graphs.
Figure 2
Figure 2
Functional analysis of light sensitivity and photorelease kinetics in brain slices of rat locus coeruleus. (a) Potassium currents evoked by photolysis at 10 µM using a 50 ms flash of 10 or 100 mW 355 nm laser light. The traces shown are the average currents measured across multiple cells (n = 6–9 cells). Light flashes are indicated by purple arrowheads. (b) Summary of current amplitudes evoked at different power levels expressed as the mean ± SEM. (c) Rising phase of amplitude-normalized average currents evoked by 100 mW light flashes. (d) Summary of the current activation time constants expressed as the mean ± SEM.
Figure 3
Figure 3
Uncovering mixed actions of LE via MOR and DOR on synaptic inhibition with N-MNVOC-LE (6) in brain slices of mouse hippocampus. (a) Electrically evoked inhibitory postsynaptic currents (IPSCs) in a pyramidal cell immediately before (black) and 3–5 min after (gray) bath application of LE (6 µM). The traces shown are averages across 6 sweeps in a single cell. Two stimuli were applied with a 50 ms interstimulus interval. (b) Average baseline-normalized IPSC amplitude over time in response to bath application of LE (n = 5 cells) or 6 (n = 9 cells). (c) Summary of paired-pulse ratios (PPRs) measured before and after LE application. Each point represents the average across six sweeps in a single cell (p = 0.0625, Wilcoxon paired signed rank test). (d) IPSCs in a pyramidal cell immediately before (black) and after (purple) photolysis of 6 (6 µM) using a 50 ms flash of light from a 365 nm LED. The traces shown are averages across 11 trials in a single cell. (e) IPSC amplitude over time measured in the same cell shown in panel (d). Light flashes were applied every 5 min. The amplitude of the first IPSC of the pair is shown. (f) Summary of paired-pulse ratios (PPRs) measured immediately before and after photolysis. Each point represents the average PPR measured across trials in a single cell (n = 3–9 trials per cell). Asterisk (*) denotes p < 0.05 (Wilcoxon paired signed rank test). (g) Photolysis-induced IPSC suppression over time in the absence and presence of highly specific opioid receptor antagonists. After three baseline uncaging events, either the MOR antagonist CTOP (1 µM, n = 5 cells) or the DOR antagonist TIPP-Psi (1 µM, n = 5 cells) was added to the bath (drug 1), followed by the other antagonist (drug 2). The indicated slope corresponds to a line fit to the antagonist-free control data set (n = 9 cells). (h) Summary of the photolysis-induced IPSC suppression data. In addition to the data shown in (g), cells are included for which only one drug was applied. Asterisks (*) denote p < 0.05 in comparison to control (Mann–Whitney U test).
Scheme 1
Scheme 1
Caged Leucine-Enkephalin Derivativesa aChemical structures of CYLE (1), N-NB-Gly2-LE (2), N-NB-Gly3-LE (3), N-NPEOC-LE (4), CNV-Y-LE (5), and N-MNVOC-LE (6). The caging groups are indicated in red.

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