L‑type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA_A Receptors

Gamma-aminobutyric acid type A (GABA_A) 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 GABA_A 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 ³⁵S 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.