Which strains of cannabis that are a good source of α-pinene is difficult to determine. Of all of the terpenes in cannabis, the investigators at the John Hopkins University Behavioral Pharmacology Research Unit is only considering the entourage of α-pinene and THC in their observational clinical trial. This post will make a case for α-pinene being a “pro-drug” of active drugs myrtenol and verbenol with GABAA receptor family members as drug targets.
A Korean mouse sleep study 
Pine and rosemary are also sources of α-pinene according to the introduction of the feature paper.  This particular post reviews another paper from Suengmok Cho and colleagues. Some of Dr Cho’s affiliations include Division of Functional Food Research, Center for Neuroscience and Functional Connectomics, Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, and KU-KIST Graduate School of Converging Sciences and Technologies, Korea University. This post will not dive as deeply as into the sleep inducing aspect of a α-pinene as the featured publication in favor of exploring complementary structural studies.
This study used male Imprinting Control Region (ICR18–22 g) and C57BL/6N (27–30 g) mice. The reason for the use of these strains was not given. Housing conditions like temperature (23 ± 0.5°C), humidity (55 ± 2%), 12-hour dark/light cycle (lights off at 17:00) were given. The mice had free access to food and water. No mention was given on access to a means of exercise such a running wheel or the composition of the food. We can probably safely assume that they were eating a standardized chow as opposed to a Western diet addressed on some posts on this site.
Firgures2-7. Pentobarbital-Induced Sleep Test
The initial screening for hypnotic effect of (−)-α-pinene sleep was done with pentobarbital-induced sleep. Experiment was performed between 13:00 and 17:00 (military time.) This coincided with the normal time for “lights out.” The ICR mice were
- fasted for 24 hours before the experiment to minimize the drowsiness induced by food.
- (−)-α-Pinene and zolpidem were administered orally to the ICR mice (n = 10) 45 minutes before the pentobarbital injection (45 mg/kg, i.p.)
- After the injection (intraperitoneal) of pentobarbital, mice were placed in individual cages and observed for measurements of sleep latency and duration. The observers were blinded to the individual treatments.
- The mice were considered asleep if they stayed immobile and lost their righting reflexes when positioned on the back.
- The sleep latency was defined as the elapsed time from pentobarbital injection to onset of righting reflex loss. The sleep duration was defined as the difference in time between the loss and the recovery of the righting reflex.
This post will not discuss the electroencephalogram and electromyogram recordings and their interpretation. This publication is public access. The interested reader is invited to read it. The take home is that they saw something with an oral dosage that almost inevitably involved at least some liver metabolism.
Figure 8 Electrophysiological Measurements
Adult mice were humanely killed, their brains removed, and placed in an artificial cerebral spinal fled chamber containing aCSF an hour before use. Recordings were obtained using Axopatch. The small Inhibitory Post Synaptic Currents (sIPSCs) were automatically detected.
Panel B shows an average of the small inhibitory post synaptic currents, sIPSCs. Note that sIPSCs stay lower longer. Note that Zolpidem does not affect the amplitude (D) or frequency (E) of sIPSC.
Moving on to α-pinene…Like Zolpidem, the rate of return from the hyper polarized state is longer in the presence of α-pinene. This increase in τ is abolished with the GABAA inhibitor flumazenil FLU.
Note that even small amounts of α-pinene can increase the relaxation time tau (panel H)
The odd thing is that panel H looks like the GABAA receptor is saturating with α-pinene.
Cho group  chose the α1β2γ2 sub type GABAA receptor. Using a technique called homology modeling Cho et al were able to swap out the β3 sub unit for the β2 sub unit. The template X-ray structure with the β3 sub unit also contained benzamidine, sugars, and ions that had to be removed and the best model of α1β2γ2 subtype GABAA receptor. Chemical structures of (−)-α-pinene was sketched using ChemDraw program. Potential ligand binding modes were evaluated by the G score which includes interaction energies of hydrogen bond, hydrophobic, van der Waals in binding site, and ligand strain energy. The α1β2γ2 version of GABAA was chosen . 
Note that the cytosolic side of GABAA is not shown here. The docking of sleep inducing drugs, panels B and C is not shown. Shown instead are two metabolites of α-pinene: Myrtenol and Verbenol. 
Note that this site to which α-pinene is docked is not completely hydrophobic: Tyrosine 159 and threonine 142. These amino acids have hydrophilic hydroxyl -OH) groups.
Myrtenol & verbenol modulate GABAA [3,4]
The first study was a collaboration between scientists in Germany, the UK and Turkey.  These investigators were interested in which terpenes in Sideritis plants, popular in Mediterranean regions, were responsible for the sedative actions. These investigators measured currents in two permutations of GABAA channels for a very long list of terpenes. These data are for our α-pinene metabolites: myrtenol and verbenol.
The GABA concentration is 1 μM. Irel(%) is the current relative to GABA alone. Both metabolites increase Cl– currents at concentrations that are not that high, i.e. 30 μM. Table 3 data are from use of a fluorescent dye that responds to membrane potential that results from Cl– currents. Pinocarveol is a plant metabolite of pinene. pEC50 [M] is the effective concentration needed for 50% of the maximum response expressed in positive log10 units. The authors might have done this so because lower EC50 expressed in linear units denote higher affinities. The take home is that these α-pinene metabolites are almost as effective as the classical positive allosteric modulator diazepam. 
Another group expressed extra synaptic tonic α4β2δ GABAA receptors in HEK293 cells. The Alzheimer group measured miniature inhibitory postsynaptic potentials (mIPSCs) using whole-cell voltage-clamp recordings from granule cells in the dentate gyrus of mouse hippocampal brain slices. 
In hippocampal slices four parameters were examined. Changes (Δ) were reported in figure 3 as follows:
- Amplitiude (%Δ) Both verbenol and myrtenol increased amplitude by about 20-40%, respectively 
- Area. (%Δ) was increased by both compounds by about 30% 
- Rise time (%Δ) was not significantly changed by either compound. 
- Decay time constant (%Δ) was also not significantly changed by either compound. 
Correlating with structural studies
The Cho group proposed an α-pinene binding site in the second benzodiazepam binding site.  Diazepam has two sites on the GABAA. One is on the extracellular surface between the the cytosolic side between the α and γ sub units. The other is at the interface between the outer leaflet of the cell membrane and the extra cellular environment 
Alprozolam, aka Xanax, an agonist
classical BZDs including act specifically through GABAA receptors containing α1/2/3/5, but not α4/6 sub unit
- diazepam (DZP, Valium)
- alprazolam (ALP, Xanax)
.. Alp also also at the interface between the α and γ sub units.The amino acid numbering seems to be slightly off as well. Nonetheless we can see the proposed pinene site is very similar to the ALP site: Try209/Y210, Phe99/F100, Thr206/S205 (similar amino acids), His101/H102, Phe77/F77. The curious difference is in Threonine 142.
Thr142, an antagonist, and a partial agonist
The authors claimed that the ALP and DZP-bound structures demonstrate that BZDs with a pendant phenyl ring do not share the binding mode of the GABAA antagonist flumzenil and partial agonist bretazenil.  These small molecules bind “deeper” and “higher” from the BZD pocket floor delineated by the side chain of γ2 Asn60. This is possibly due to additional hydrogen bonds between the imidazole nitrogen and ester carbonyl of FLZ/BRZ which are not present in ALP/DZP structures. ) Masiulis et al also speculate involvement of the γ2 hydroxyl group of Thr1420 The above images come from their extended data. 
Summary and “Conclusions”
The Cho group showed a positive effect of α-pinene on mouse sleep. The also showed an effect of α-pinene on expressed GABAA receptors.  Their molecular modeling suggested involvement of threonine 142 of the γ subunit.  They did not however address possible metabolites of α-pinene  being the biologically active agents in modulating mouse sleep. Reasonable concentrations of α-pinene metabolites myrtenol and verbenol modulate the α1β2γ2 variation of GABAA expressed in HEK293 cells.  Myrtenol seems to be more potent than verbenol.  These same metabolites modulate the α4β2δ variation of GABAA with mytenol being more effective than verbenol.  The binding site of prosed by Cho group  agrees well with the alprazolam site.  The GABAA partial agonist bretazanil utilizes threonine 142 of the γ subunit. 
The GABAA family of receptors seem to be extremely promiscuous. α-Pinene may more likely be a pro-drug for myrtenol and/or verbenol. If so, the presence of α-pinene in inhaled cannabis has implications. The effect of THC may be felt long before α-pinene is converted to its active metabolites.
- Yang H, Woo J, Pae AN, Um MY, Cho NC, Park KD, Yoon M,Kim J, Lee CJ, Cho S (2016) α-Pinene, a major constituent of pine tree oils, enhances non-rapid eye movement sleep in mice through GABAA-benzodiazepine receptors. Mol Pharmacol90:530-539. PMC free article
- Schmidt L, Göen T. Human metabolism of α-pinene and metabolite kinetics after oral administration. Arch Toxicol. 2017 Feb;91(2):677-687.
- Kessler A, Sahin-Nadeem H, Lummis SC, Weigel I, Pischetsrieder M, Buettner A, Villmann C. (2014) GABA(A) receptor modulation by terpenoids from Sideritis extracts. Mol Nutr Food Res. 2014 Apr;58(4):851-62. PMC free article
- van Brederode J, Atak S, Kessler A, Pischetsrieder M, Villmann C, Alzheimer C. (2016)The terpenoids Myrtenol and Verbenol act on δ subunit-containing GABAA receptors and enhance tonic inhibition in dentate gyrus granule cells. Neurosci Lett. 2016 Aug 15;628:91-7.
- Masiulis, S., Desai, R., Uchański, T., Serna Martin, I., Laverty, D., Karia, D., Malinauskas, T., Zivanov, J., Pardon, E., Kotecha, A., Steyaert, J., Miller, K. W., & Aricescu, A. R. (2019). GABAA receptor signalling mechanisms revealed by structural pharmacology. Nature, 565(7740), 454–459. PMC free article