Study of membrane potential in T lymphocytes subpopulations using flow cytometry

BACKGROUND: Ion channels are involved in the control of membrane potential (ψ) in a variety of cells. The maintenance of ψ in human T lymphocytes is essential for T-cell activation and was suggested to depend mostly on the voltage-gated Kv1.3 channel. Blockage of Kv1.3 inhibits cytokine production and lymphocyte proliferation in vitro and suppresses immune response in vivo. T lymphocytes are a heterogeneous cell population and the expression of Kv1.3 varies among cell subsets. Oxonol diBA-C4-(3) was used to determine ψ by flow cytometry. The presence of distinct T cell subsets was evaluated by immunophenotyping techniques and the contribution of Kv1.3 channels for the maintenance of ψ was investigated using selective blockers. RESULTS: The distribution of ψ in T lymphocytes varied among blood donors and did not always follow a unimodal pattern. T lymphocytes were divided into CD3(+)/CD45RO(- )and CD3(+)/CD45RO(+ )subsets, whose peak channel values of ψ were -58 ± 3.6 mV and -37 ± 4.1 mV, respectively. MgTX (specific inhibitor of Kv1.3 channels) had no significant effect in the ψ of CD3(+)/CD45RO(- )subsets but depolarized CD3(+)/CD45RO(+ )cells to -27 ± 5.1 mV. CONCLUSION: Combination of optical methods for determination of ψ by flow cytometry with immuophenotyping techniques opens new possibilities for the study of ion channels in the biology of heterogeneous cell populations such as T lymphocyte subsets.