Guaiol

Sesquiterpenes are terpenes with the formula C15H24, hence the prefix “sesqui” Latin for 15. The featured study came out of Wuertzburg, Germany. These German authors extracted terpenes from hops and chamomile. [1] The extracts were subjected to a process to simulate digestion in the human gastrointestinal tract. β-Caryophyllene became β-caryophyllenol. Unlike many Asian studies reviewed on this site, Janzen et al were not explicitly searching for a more natural sleep aid.

They expressed versions of GABA_A found in the hippocampus in human embryonic kidney cells (HEK293). They also isolated hippocampus neurons from mouse embryos. The expectation might have been discovery of positive allosteric modulators. They discovered some negative allosteric modulators in the process. This post brings in additional information about the guaiol binding site near the PIP2 binding site[2]that might explain why guaiol is a weak positive allosteric modulator in one system and a negative modulator in another. Does any of this explain guaiol association of cannabis strain anxiety? [3]

Basic Methods

• Embryos were harvested from pregnant CD1 mice.  The hippocampi of the brains of these embryos were dissected.
•  Hippocampal neurons were cultured until division 18-21, which were used for electrophysiology experiments. 
• Human embryonic kidney cells (HEK293) were transfected with DNA vectors coding for various sub unit combinations of GABA_A receptors. 
• It was usually the γ sub unit that was tagged with green fluorescence protein (GFP).  GFP fluorescence was used to identify successfully transfected cells. Currents were measured by the patch clamp technique.

Phasic vs tonic GABA_A receptors

The difference between phasic and tonic GABA_A receptors have been addressed on the GABA_A page.

The α1β2 version of GABA_A in HEK293 cells

The following data are from Figure 2 and table 1. [1] There are more combinations of the α1 and β2 subunits that what is shown in this cartoon. These GABA_A receptors have the GABA binding site between α1 and β2 going clockwise. What is missing is the benzodiazepine site between γ and α. Benzodiazepines are positive allosteric modulators of GABA_A, i.e their binding potentiates the current when GABA binds.

Note that in this case guaiol is acting as a negative allosteric modulator of GABA binding the GABA_A receptor.

Primary hippocampal neurons

This image was adapted from Figures 4 and table 2 of Janzen et al [1] We are not making any assumptions about the particular isoform of the α, β, γ, and δ subunits, hence α_xβ_xγ_x and α_xβ_xδ_x. Guaiol acts as a positive allosteric modulator in cultured neurons. Positive allosteric modulator Diazepam is shown for comparison.

Gaboxadol is a GABA_A targeting drug that intended to be used as a sleep aid but was recalled due to side effects.

The classic α₁β₂γ₃ version of GABA_A in HEK293 cells

These data were extracted from Janzen et al [1] Figure 5 and Table 3. Guaiol had no effect on the amplitude of the GABA_A current.

Janszen et al did not report the time constant τ for how long it takes to return to the resting membrane potential.

The α₄β₃δ version of GABA_A in HEK293 cells

Here again guaiol proved to be a negative allosteric modulator of GABA_A with this particular sub unit configuration.

While this concentration of guaiol is rather high and the GABA concentration is at a high enought concentration to saturate the GABA_A receptor, guaial is a negative allosteric modulator in these conditions.

The α₆β₃δ version of GABA_A in HEK293 cells

Again and again guaiol proved to be a negative allosteric modulator of GABA_A with the α₆β₃δ configuration.

In summary…

Molecular Modeling and making sense of the confusing

So far nothing of what we have seen so far makes sense. Guaiol is a negative allosteric modulator of a variety of GABA_A receptors in HEK293 cells but a positive alloseric modulator in a cultured hippocampus neuron system. [1] The authors of an X-ray structure of Janzen et al used also published some structures of the the same region of the ⍺₁ binding to a phospholipid that might be different in HEK293 cells and neurons. Janzen et al used PDB structure 5O8F to do their modeling. Guaiol and other sesquiterpenes were docked to helix ⍺₁M1 (⍺₁⁻), [1] the pregnalnolone (neurosteroid) site. Malinauskas and other authors published an cryoelectron microscope image of GABA_A in a membrane. [2] See the post on PIP2 and GABA_A post.

Janzen et al also discussed GABA_A interacting proteins that might explain their results. [1]

Bring this back to cannabis

A statistical study attempted to correlate terpene content of chemovars with the reported ability to relive anxiety. [3] Kamal et al found that guaiol was the only major terpene with a “perfect” correlation with relief of and/or creation of anxiety. Guaiol was found in three of the least effective axiolytic strains and in none of the most effective, giving a correlation coefficient of−1. [3] Gualiol also had the highest significance of any compound (p=0.003). Guaiol is not found in major quantities in most of the chemvars analyzed. It may only be a marker of other combinations that cause anxiety. [3] Perhaps chemovars that have guaiol also are more likely to express other sesqiterpenes that negatively modulate various forms of GABA_A. This site will explore these terpenes.

References

1. Janzen D, Slavik B, Zehe M, Sotriffer C, Loos HM, Buettner A, Villmann C. Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABA_A receptor function in vitro. J Neurochem. 2021 Oct;159(1):101-115 free article
2. Laverty, D., Desai, R., Uchański, T., Masiulis, S., Stec, W. J., Malinauskas, T., Zivanov, J., Pardon, E., Steyaert, J., Miller, K. W., & Aricescu, A. R. (2019). Cryo-EM structure of the human α1β3γ2 GABA_A receptor in a lipid bilayer. Nature, 565(7740), 516–520. PMC free paper
3. Kamal, B. S., Kamal, F., & Lantela, D. E. (2018). Cannabis and the Anxiety of Fragmentation-A Systems Approach for Finding an Anxiolytic Cannabis Chemotype. Frontiers in neuroscience, 12, 730. PMC free article