ABSTRACT

Ion channels play pivotal role in the physiological and pathological function of immune cells. As immune cells represent a functionally diverse population, subtype-specific functional studies, such as single-cell electrophysiology require proper subset identification and separation. Magnetic-activated cell sorting (MACS) techniques provide an alternative to fluorescence-activated cell sorting (FACS), however, the potential impact of MACS-related beads on the biophysical and pharmacological properties of the ion channels were not studied yet. We studied the aforementioned properties of the voltage-gated Kv1.3 K⁺ channel in activated CD4⁺ T-cells as well as the membrane capacitance using whole-cell patch-clamp following immunomagnetic positive separation, using the REAlease® kit. This kit allows three experimental configurations: bead-bound configuration, bead-free configuration following the removal of magnetic beads, and the label-free configuration following removal of CD4 recognizing antibody fragments. As controls, we used FACS separation as well as immunomagnetic negative selection. The membrane capacitance and of the biophysical parameters of Kv1.3 gating, voltage-dependence of steady-state activation and inactivation kinetics of the current were not affected by the presence of MACS-related compounds on the cell surface. We found subtle differences in the activation kinetics of the Kv1.3 current that could not be explained by the presence of MACS-related compounds. Neither the equilibrium block of Kv1.3 by TEA⁺ or charybdotoxin (ChTx) nor the kinetics of ChTx block are affected by the presence of the magnetics beads on the cell surface. Based on our results MACS is a suitable method to separate cells for studying ion channels in non-excitable cells, such as T-lymphocytes.

摘要

离子通道在免疫细胞的生理和病理功能中起着举足轻重的作用。由于免疫细胞代表功能多样的群体，亚型特异性功能研究，如单细胞电生理学，需要适当的亚群识别和分离。磁激活细胞分选 (MACS) 技术提供了荧光激活细胞分选 (FACS) 的替代方法，但是，尚未研究 MACS 相关珠子对离子通道的生物物理和药理特性的潜在影响。我们研究了激活 CD4 ^{+ 中}电压门控 Kv1.3 K ⁺通道的上述特性使用 REAlease® 试剂盒在免疫磁性阳性分离后使用全细胞膜片钳检测 T 细胞以及膜电容。该试剂盒允许三种实验配置：珠结合配置、去除磁珠后的无珠配置和去除 CD4 识别抗体片段后的无标记配置。作为对照，我们使用 FACS 分离以及免疫磁性负选择。Kv1.3 门控的膜电容和生物物理参数、稳态激活的电压依赖性和电流的失活动力学不受细胞表面 MACS 相关化合物的影响。我们发现 Kv1.3 电流的激活动力学存在细微差异，无法通过 MACS 相关化合物的存在来解释。⁺或 charybdotoxin (ChTx) 以及 ChTx 阻断的动力学都受到细胞表面存在的磁珠的影响。根据我们的结果，MACS 是一种分离细胞的合适方法，用于研究非兴奋性细胞（如 T 淋巴细胞）中的离子通道。