# Molecular dissection of acute and chronic menthol interactions

> **NIH NIH F31** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2021 · $28,855

## Abstract

Project Summary
Tobacco use is the leading cause of cancer. Menthol cigarettes make up approximately 27% of the total cigarette
market in the United States and over 80% of African American smokers use menthol cigarettes. Menthol
cigarettes are more difficult to quit than conventional cigarettes. Menthol has a neurobiological effect on the
nicotinic acetylcholine receptors (nAChRs). At micromolar concentrations, menthol acts as a negative allosteric
modulator of the α4β2 nAChR, the most abundant nAChR in the brain and the one that plays the largest role in
nicotine addiction. At nanomolar concentrations applied for over 24 hr., menthol causes the upregulation of α4*
and α6* nAChRs in midbrain dopaminergic neurons. Most importantly, menthol enhances nicotine reward
through its ability to enhance dopamine neuron excitability. Ultimately, studying the effects, functions, and
mechanisms of chronic and acute menthol has tremendous clinical relevance.
Recently, I found that incorporating a L9’A mutation into the second transmembrane helix (TM2) in the α4β2
nAChR causes menthol to dramatically lose function as a negative allosteric modulator. This was observed
whether the L9’A mutation was incorporated in the α4 or β2 subunit. Spurred by these results, I want to determine
how acute menthol is functioning as an allosteric modulator. By investigating other nAChRs, using other residues
at the 9’ position, and mutating other sites on TM2, I will determine how menthol acutely effects the nAChRs.
These experiments will utilize noncanonical amino acid mutagenesis and two-electrode voltage clamp
electrophysiology in Xenopus laevis oocytes to elucidate this chemical mechanism. Additionally, nanomolar
menthol over the course of at least 24 hr. upregulates the α4* and α6* nAChRs in midbrain dopaminergic
neurons. The Lester lab is a leading contributor to the field of “inside-out pharmacology” and menthol’s effects
on α4* and α6* nAChRs falls under this category. I am interested in whether this effect is specific to menthol and
α4* and α6* nAChRs or if chronic application of other cooling agents could elicit this response or if other ion
channels are affected by chronic exposure to cooling agents. Total internal reflection fluorescence (TIRF)
microscopy will be used to observe ion channel concentration on the cell surface as a measure of upregulation.
Finally, the way menthol upregulates α4* and α6* nAChRs is not clear. We know that menthol acts on the
endoplasmic reticulum exit sites (ERES) and on the cycling between the Golgi and the endoplasmic reticulum,
but what is the first event affected by menthol is not known. The mechanism for nicotine-induced upregulation
have been probed extensively by the Lester lab and I will do the same for menthol. By using RNA sequencing,
biorthogonal noncanonical amino acid tagging, mass spectrometry, and particle tracking microscopy, I hope to
find the earliest step in the nAChR exocytic pathway that is affected by menthol.

## Key facts

- **NIH application ID:** 10101644
- **Project number:** 5F31DA046122-03
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Stephen Nicholas Grant
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $28,855
- **Award type:** 5
- **Project period:** 2019-03-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10101644

## Citation

> US National Institutes of Health, RePORTER application 10101644, Molecular dissection of acute and chronic menthol interactions (5F31DA046122-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10101644. Licensed CC0.

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