Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory
ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus
idiopathic generalized epilepsy (
IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with
verapamil (4 μM, 24 h), an
L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the
GABA-induced maximal
chloride current in HEK293 cells expressing α1(D219N)β2γ2 receptors to a level that is comparable to wild type receptors. Our
drug mechanism study revealed that
verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that,
verapamil treatment enhances the interaction between the α1(D219N) subunit and β2 subunit and prevents the aggregation of the
mutant protein by shifting the
protein from the
detergent-insoluble fractions to
detergent-soluble fractions. By combining (35)S pulse-chase labeling and
MG-132 inhibition experiments, we demonstrated that
verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a
dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that
verapamil increases the interaction between the
mutant protein and
calnexin and
calreticulin, two major
lectin chaperones in the ER. Moreover,
calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that
verapamil treatment enhances the
calnexin-assisted forward trafficking and subunit assembly, which leads to substantially enhanced functional surface expression of the mutant receptors. Since
verapamil is an FDA-approved
drug that crosses blood-brain barrier and has been used as an additional medication for some
epilepsies, our findings suggest that
verapamil holds great promise to be developed to ameliorate
IGE resulting from α1(D219N) subunit trafficking deficiency.