L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated 伪1(D219N) Subunits of GABAA Receptors

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• 作者：Dong-Yun Han ; Bo-Jhih Guan ; Ya-Juan Wang ; Maria Hatzoglou ; Ting-Wei Mu
• 刊名：ACS Chemical Biology
• 出版年：2015
• 出版时间：September 18, 2015
• 年：2015
• 卷：10
• 期：9
• 页码：2135-2148
• 全文大小：671K
• ISSN：1554-8937

文摘

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 伪1 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 伪1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of 伪1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 渭M, 24 h), an L-type calcium channel blocker, substantially increases the 伪1(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 伪1(D219N)尾2纬2 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 伪1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the 伪1(D219N) subunit and 尾2 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 伪1(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鈥揵rain 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 伪1(D219N) subunit trafficking deficiency.