Thomas Jessica R, Lee Amy
Departments of Molecular Physiology and Biophysics, Otolaryngology-Head and Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242; Interdisciplinary Graduate Program in Neuroscience, University of Iowa, Iowa City, Iowa 52242.
Departments of Molecular Physiology and Biophysics, Otolaryngology-Head and Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242;
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot087213. doi: 10.1101/pdb.prot087213.
Voltage-gated Ca(2+) (Cav) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Cav channels are subject to diverse forms of regulation, including those involving the Ca(2+) ions that permeate the pore. High voltage-activated Cav channels undergo Ca(2+)-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Cav1 (L-type) and Cav2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Cav channel. HEK-293T cells do not express endogenous Cav channels, but Cav channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca(2+)-dependent modulation of recombinant Cav channels in HEK-293T cells.
电压门控性Ca(2+)（Cav）通道调节多种生物学过程，如肌肉收缩、基因表达和神经递质释放。Cav通道受到多种形式的调节，包括那些涉及渗透通道孔的Ca(2+)离子的调节。高电压激活的Cav通道会经历Ca(2+)依赖性失活（CDI）和易化（CDF），这可以调节诸如心律和突触可塑性等过程。Cav1（L型）通道和Cav2（P/Q型、N型和R型）通道之间的CDI和CDF略有不同。用SV40大T抗原转化的人胚肾细胞（HEK-293T）有利于研究特定类型Cav通道的CDI和CDF。HEK-293T细胞不表达内源性Cav通道，但通过瞬时转染，Cav通道可以在这些细胞中高水平地外源表达。本方案描述了如何表征和分析HEK-293T细胞中重组Cav通道的Ca(2+)依赖性调节。
