Electrophysiological characterization of primary and immortalized immune cells using automated patch-clamp technology 2335008

Abstract Introduction Ion channels and transporters mediate key functions of innate and adaptive immune cells, including the regulation of membrane potential and Ca2+-mediated intracellular signaling pathways. Kv1.3 voltage-gated potassium channels and KCa3.1 intermediate conductance Ca2+-activated K+ channels are particularly important for regulating membrane depolarization and oscillations that facilitate intracellular calcium-release activated calcium (ICRAC) currents and activation of naïve T-cells. Methods Here, we use automated patch-clamp electrophysiology (Sophion QPatch48) to record and characterize K+ currents from both immortalized and primary B-cells and T-cells. Primary murine T-cells were collected and compared with immortalized T- and B-cell lines and stable cell lines expressing Kv1.3 and KCa3.1 channels. Recording solutions and compounds were optimized for each target ion channel of interest. Results Inactivating voltage-dependent outward currents were determined to be Kv1.3 mediated by inhibition with low concentrations of the isoform-specific blocker PAP-1. Intermediate conductance (IK) KCa3.1 channels were measured using different Ca2+ concentrations in the intracellular solutions and applying voltage ramps during the whole-cell break in period. IK currents grew in amplitude with each sweep as Ca2+ diffused into the intracellular environment, and these currents could be either enhanced with riluzole or blocked by TRAM-34, suggesting they were mediated by KCa3.1. Conclusion Our results demonstrate the utility of high-throughput automated patch-clamp for measuring ion channel composition and function across primary and immortalized immune cells. This approach permits rapid, simultaneous recording of many cells under the same conditions, allowing more accurate assessment of ion-mediated processes during immune cell activation. Funding Source n/a/ Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)