GABAA and PIP2

The purpose of this post is to explain some nuances of a publication suggesting that sesquiterpenes bind to the first transmembrane M1 domain of the α₁ sub unit of GABA_A. [1] The same group that provided the X-ray crystal structure for a neurosteroid binding site that Janzen et al used later published a paper showing phosphatidyl inositol bisphosphate (PIP₂) binding to a proximal region as the proposed sesquiterpene sites. [1] The featured image shows some typical phospholipids of the inner cytoplasmic leaflet of the lipid bilayer. The phosphate (orange) and oxygens (red) on PIP2 represent a cluster of negative charges that interact with membrane proteins. Some other inner leaflet phospholipids are shown for comparison.

Terpenes and PIP₂ binding to GABA_A α₁

These images are from Figure 7B of reference [1], Figure 2b and 2a of reference [1]. Reference [1] showed sequence alignment of other GABAA ununits.

Note the close proximity of PIP2 (orange hexagons) and guaiol (yellow stars) binding sites. The exciting implications of this work suggest that changes in the PIP2 content of a membrane could determine how the GABA_A channel responds to a sesquiterpene.

Explaining α isoform sesquiterpene selectivity

These images are from panels b and c of Figure 4 from reference {2] A sequence alignment of the six classes of α sub units is also shown. Some of the sesquiterpenes are negative allosteric modulators of some α subunit combos but not others. Does this have to do with the number of PIP2 sites?

An important thing to remember is the M2 transmembrane helices form the interior of the GABA_A channel.

PIP₂ gates GABA_A channels

Etomidate is an agonist of GABA_A that does not require the presence of GABA. Etomidate binds between the alpha and beta subunits (α−β+) of β3containing GABA_A.

Laveerty et al [2] performed inside-out patch-clamp electrophysiology measurements on the same cell-line used in structural studies. Important to the first study [1], It was assumed that receptors expressed on HEK293 cells were likely pre-bound to endogenous PIP2. Poly L-lysine is a chelator of PIP2. [2]

Relating this to cannabis

Could CB1 activation of PIP2 cleaving phospholipase C open GABA_A receptors? A 2001 study demonstrated taht a CB1 antagonist and a PLC inhibitor both, separately, blocked sleep induced by anandamide injection into the brains of rats. [3] GABA_A is a popular target of hypnotic (sleep inducing) drugs. Some cannabis users might like strains with certain sesquiterpenes because they don’t make them sleepy during the day.

Are there genetic polymorphisms that could explain why some users respond differently to different chemovar/strains? UniProt.org was searched for genetic variations of human α1, α2, α4, and α6. (GRBA1,2,3,4,5 human) GBRA2/α2 had variant information than the other sub units simply because it was the the subject of an epilepsy study. [4] The EC50 concentrations as well as the Hill coefficients for the mutant α2 are presented. The Hill coefficient relates to the slope of the EC50 dose/response curve.

Note that many of the α2 regions around the PIP2 binding intracellular domains. Some of this becomes interesting when one considers the use of cannabis to treat epilepsy. Do individuals with epilepsy caused by these mutations respond to cannabis?

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. Murillo-Rodríguez E, Cabeza R, Méndez-Díaz M, Navarro L, Prospéro-García O. Anandamide-induced sleep is blocked by SR141716A, a CB1 receptor antagonist and by U73122, a phospholipase C inhibitor. Neuroreport. 2001 Jul 20;12(10):2131-6
4. Maljevic S, Keren B, Aung YH, Forster IC, Mignot C, Buratti J, Lafitte A, Freihuber C, Rodan LH, Bergin A, Hubert L, Poirier K, Munnich A, Besmond C, Hauser N, Miller R, McWalter K, Nabbout R, Héron D, Leguern E, Depienne C, Petrou S, Nava C. Novel GABRA2 variants in epileptic encephalopathy and intellectual disability with seizures. Brain. 2019 May 1;142(5):e15. Free article