One of the first reports of a α-humulene comes from a company called Xenova in Slough Berkshire, UK.  Like many, they were looking for GABAA receptor modulators. “Recall that GABAA “receptors” are also chloride channels that, when open, lead to hyperpolarization and inhibition of neurotransmitter release.
Benzodiazepines a class of modulators that increase the frequency of Cl–channel opening without changing the conductance or the duration of opening. They may be used as tranquilizers  Some of the side effects that drove these authors to look for benzoiazepine alternatives are
- development of dependence
- development of tolerance
- anxiety from acute withdrawal
Xenova embarked on a screening program to find a natural product that inhibited the binding of funitrazepam to GABAA.  Xenova discovered a novel case of compounds from fermentation products of Acremonium strictum. Why this fungus was not fully disclosed.
Much of the Xenova publication was devoted to explaining the growth conditions for xenovulene and techniques used to determine its structure. Shown below is their structure for “xenovulene.” What they call a tricyclic moiety is circled in panel  in the figure below. Xenova authors speculated that the moiety arose from a polyketide pathway. 
Preparation of Membranes for Assay
GABAA receptors were prepared from ox cerebral cortex10). Dura and blood vessels were removed from the cerebral cortex minus the dura and blood vessels. . The tissue was homogenized and subjected to a low speed spin to remove large chunks of debris. The supernatant was subjected to a high speed spin to collect small membrane vesicles.
Binding to GABAA was measured by the ability of the ligand to dislodge the high affinity agonist [3H]-flunitrazepam. [3H] is of course radioactive hydrogen, or tritium. Non-specific [3H]-flunitrazepam binding was determined by another high affinity agonist diazepam. “Bound” [3tH]-flunitrazepam was separated from [3H]-flunitrazepam in solution by filtration. The filter papers were rinsed and placed in a scintillation counter to measure [3H] radioactive decay.  The authors did not show a binding curve but did give an IC50 of 40 nM.
A German group isolated terpenes from hops and chamomile.  This group postulated that we not only the 15 carbon sesquitepene but also the oxidation products, sesquiterpenoids need to be examined as GABAA ligads. Panel 
in the figure below was derived from Figure 1 of reference . The carbons of humulene and humulol have been labeled according to the xenovulene structure of reference  The double bond in humulene to which the “tricyclic moiety” and -OH group is added is starred. Carbon 19 in the first panel is drawn as a stick with “-CH3” on the end. The rendering in panel  assumes that the end of the stick labeled “19” is -CH3.
Digestion in this study referred to a protocol simulating the human digestive system that was used to convert humulene from a chemical vendor to humulol. Without deviating to much from the purpose of  it is assumed that transformation by the liver is not part of the process.
- α1β2γ2 is considered phasic
- α4β3δ and α6β3δ are considered tonic
Modulation of α12β2 GABAA
GABAA channels should contain a γ or δ subunit in addition to the αβ subunits. The GABA ( γ-amino butyric acid) is between α1 and β2 going clockwise.
Humulol, but not humulene, increases the amplitude of the depolarization current produced by the opening of the GABAA channel. In actual neurons there are two other components of small post synaptic currents (sIPS): frequency and recovery time, τ. In this one example it takes longer to recover from GABA induced hyperpolarization when humulene in part of the mixture. (Compare red and black traces.)
In cultured hippocampal neurons
The image below was adapted from figure 4 of the Janzen publication.  Picotoxin responses were removed from the publication image  for this post. In this particular system humulol, but not humulene was a negative allosteric modulator of the GABA response. Both the γ and δ subunits should have been present in this preparation. The “x” subscripts tell the reader that this preparation was not rigorously characterized in terms of isoforms of individual sub units.
The α1β2γ2 isoform combination of GABAA is perhaps the most common. The diazepine (DZP) site is present in this version of GABAA. Humulol, but not humulene, caused a significant decrease in maximum current.  The Alan Brain mRNA expression map was obtained from proteinatlas.org
Note the differences between the red (GABA + Humanulol) and black (GABA alone).
Unlike the α,1 subunit, α4 is not expressed in the cerebellum and brain stem according to Alan brain atlas data obtained from proteinatlas.com. Humulene and humulol are both negative allosteric modulators of α4β2δ when it is expressed in HEK293 cells. 
The negative allosteric modulator picotoxin significantly reduced the GABA current as well. (not shown) Note that “digestion” is not required for inhibitory activity in the cerebellum.
For this particular configuration the negative allosteric modulator picotoxin only slightly decreased GABA current. (p=0.0558) Humulene and humulol had no effect. 
Also note that, for the most part, α6 is only expressed in the brain stem and cerebellum. Compare this with the expression of humulene/humulol modulated α4 with opposite brain expression.
These images were obtained from the Xenova publication  and docking of humulol to the α1 subunit.  An attempt was made to render the numbering of the former to the structure in the latter.
From the 3D stucture of Xenovulene from PubChem, the additional group appears to extend vertically from the humlene structure. We can speculate that Xenovulene binds to a similar site.
Entourage with cannabinoids
A mouse study from the University of Arizona in the United States tested the hypothesis that terpenes in Cannabis sativa altered the cannabinoid tetrad behaviors. Mice were injected with
- 200 mg/kg terpene alone,
- combined with 5.6 mg/kg CB1 agonist WIN55,212-2, This compound also inhibits ATP production in a CB1 dependent manner.
- or after pre-treatment with 10 mg/kg CB1 antagonist rimonabant Hot flushes are a side effect of this compound in humans.
- or 10 mg/kg adenosine A2A receptor antagonist istradefyllene, i.p..
- 2A Win plus humulene have have an additive when it comes to how long a mouse can withstand having their tails gently inserted in 52 °C or 47 °C water. The CB1 antagoninst Rimonabant abolished the analgesic effect of humulene.
- 3A Humulene and CB1 agonist Win had an additive effect in lowering rectal temperatures. CB1 and adeonsine A2A antagonists singly partially blocked this effect.
- 4A percentage (%) of time in a “cataleptic” immobile state over the 5 min observation period. Humulene and Win had additive effects on Inhibition of adenosine A2A completely blocked the effect of humulene.The CB1 antagonist only partially relieved the immobile state.
- 5A The distance that mice roamed about a large box in 5 min was recorded with a video camera. Again, humulene and Win have an additive effect in reducing wandering. Rimonabant reversed the humulene sedative response. Blocking adenosine A2A increased wandering even with humulene.
These authors were not considering action on GABAA. Other terpenes were examined in the publication  that are not presented on this post. Personal communication with authors is that there are likely other receptors in addition to adenosine A2.A.
- Ainsworth AM, Chicarelli-Robinson MI, Copp BR, Fauth U, Hylands PJ, Holloway JA, Latif M, O’Beirne GB, Porter N, Renno DV, et al. Xenovulene A, a novel GABA-benzodiazepine receptor binding compound produced by Acremonium strictum. J Antibiot (Tokyo). 1995 Jul;48(7):568-73. PMC free article
- Janzen D, Slavik B, Zehe M, Sotriffer C, Loos HM, Buettner A, Villmann C. Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABAA receptor function in vitro. J Neurochem. 2021 Oct;159(1):101-115. PMC free article
- LaVigne, J. E., Hecksel, R., Keresztes, A., & Streicher, J. M. (2021). Cannabis sativa terpenes are cannabimimetic and selectively enhance cannabinoid activity. Scientific reports, 11(1), 8232. PMC free article