Establishment and optimization of drug screening model for N-type voltage-gated calcium channels in Xenopus laevis oocyte expression system
10.16438/j.0513-4870.2024-0156
- VernacularTitle:非洲爪蟾卵母细胞表达系统中N-型电压门控钙离子通道新药筛选模型的建立与优化
- Author:
Yuan QIN
;
Cheng CUI
;
Xiao-peng ZHU
;
Dong-ting ZHANGSUN
;
Jin-peng YU
;
Su-lan LUO
- Publication Type:Research Article
- Keywords:
voltage-gated calcium channel;
two-electrode voltage clamp;
italic>Xenopus laevis oocyte;
electrophysiology;
gating characteristics
- From:
Acta Pharmaceutica Sinica
2024;59(7):2002-2011
- CountryChina
- Language:Chinese
-
Abstract:
N-type voltage-gated calcium (Ca2+) channels (N-type VGCC, CaV2.2) mediate Ca2+ influx in response to action potential at the presynaptic terminal, and play an important role in synaptogenesis, neurotransmitter release and nociceptive signal transduction. It is a new target for the development of drugs for the treatment of neuralgia (chronic pain) and other major diseases. Due to the difficulty of calcium channel expression in vitro and the detection of channel current, there is a great lack of new drug screening models. In this study, we established and optimized the electrophysiological drug screening model using Xenopus laevis oocytes for the recombinant expression of CaV2.2 in vitro (this study were reviewed and approved by the Ethics Committee of Guangxi University, approval number: GXU-2023-0249). Firstly, the linear plasmids encoding cDNA of major subunit α1B and auxiliary subunits α2δ1 and β3 of rat CaV2.2 were used as templates for in vitro transcription to generate their related mRNA (cRNA), after which three kinds of cRNA were injected into Xenopus laevis oocytes at the mass ratio of 2∶1∶1 for expression. The two-electrode voltage clamp (TEVC) technique was used to detect the inward current produced by CaV2.2. At the same time, the expression conditions of CaV2.2 were optimized, and its gating function was characterized from the aspects of channel activation and inactivation. The results showed that 3-5 days after cRNA microinjection, stable CaV2.2-mediated barium ion (Ba2+) currents were successfully detected. The interference of endogenous potassium channels and Ca2+-activated chloride channels can be eliminated by tetraethylammonium hydroxide (TEAOH) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM) treatment. The maximum potential for CaV2.2 activation is 0 mV, and the current reverses to be outward when the membrane potential is greater than +50 mV. By fitting the steady-state activation and inactivation curves, the half-maximal activation potential and half-maximal inactivation potential of CaV2.2 are identified as -15.9 and -60.2 mV. In this study, a stable CaV2.2 expression system was established based on Xenopus laevis oocytes. The in vitro expression system can provide a new way for the screening of CaV2.2 active compounds or lead drugs.
