# Opioids inside Organelles

> **NIH NIH R21** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2020 · $246,750

## Abstract

Biological membranes are permeable to exogenous opioid drugs--both plant-derived molecules
such as morphine, and synthetic molecules of which hundreds exist. Now a genetically encoded
fluorescent biosensor technique allows us to measure opioids within neutral organelles such as
the endoplasmic reticulum (ER). We term these molecules the intensity-based opioid-sensitive
fluorescent reporter, iOpioidSnFR, family (Figure 1). Ongoing experiments, before the project
begins, will extend the iOpioidSnFR family to the major classes of µ-opioid agonists.
Aim 1 Further extends the iOpioidSnFRs for measurements within acidic organelles such as
endosomes and synaptic vesicles. Aim 1a utilizes the present circularly permutated green
fluorescent protein (cpGFP) moiety. Aim 1b develops novel circularly permuted HaloTags, which
are pH-insensitive. Aim 1c, Extends the existing measurements to measure the entry of opioids
into organelles, and their exit from organelles. Quantification involves both dynamics and steady-
state measurements.
Aim 2 tests the hypothesis that some effects of opioid drugs result after synaptic vesicles
accumulate opioids via acid trapping. The synaptic vesicles would then release the opioids upon
presynaptic stimulation. This mechanism would extend the patho-pharmacology of exogenous
opioids to their release from many types of presynaptic neurons—even those neurons that do not
release endogenous opioid peptides. Aim 2a evolves iOpioidSnFR sensitivity further, to the
required nanomolar levels. Aim 2b Identifies the most sensitive method for testing presynaptic
release.
Aim 3 tests the hypothesis that brain regions expressing µ-opioid receptors vary in the extent and
timing of organellar opioids. Aim 3a generates adeno-associated viral vectors that encode “floxed”
iOpioidSnFRs. These will be expressed under the control of vesicular GABA transporter (vGAT)
cre recombinase in suitable mouse lines. Aim 3b measures in brain slices from ventral tegmentum
area (VTA) / substantia nigro pars reticulata (SnR), periaqueductal gray (PAG), and ventral
pallidum (VP).The results will aid in the ongoing efforts to understand the cellular and molecular
basis of tolerance to µ-opioid ligands.

## Key facts

- **NIH application ID:** 9982844
- **Project number:** 5R21DA049140-02
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Henry A. Lester
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $246,750
- **Award type:** 5
- **Project period:** 2019-08-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9982844

## Citation

> US National Institutes of Health, RePORTER application 9982844, Opioids inside Organelles (5R21DA049140-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9982844. Licensed CC0.

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