Study of membrane potential in T lymphocytes subpopulations using flow cytometry.
BACKGROUND
Ion channels are involved in the control of membrane potential (psi) in a variety of cells. The maintenance of psi 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 psi 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 psi was investigated using selective blockers.


RESULTS
The distribution of psi 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 psi were -58 +/- 3.6 mV and -37 +/- 4.1 mV, respectively. MgTX (specific inhibitor of Kv1.3 channels) had no significant effect in the psi of CD3+/CD45RO- subsets but depolarized CD3+/CD45RO+ cells to -27 +/- 5.1 mV.


CONCLUSIONS
Combination of optical methods for determination of psi 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.
Mello de Queiroz
Fernanda
F
Molekulare Biologie Neuronaler Signale, Max-Planck-Institut für Experimentelle Medizin, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany. queiroz@em.mpg.de
Ponte
Cristiano G
CG
Bonomo
Adriana
A
Vianna-Jorge
Rosane
R
Suarez-Kurtz
Guilherme
G
D000818:Animals; D017252:CD3 Complex / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000502:physiology; D016131:Lymphocyte Subsets / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D012604:Scorpion Venoms / Q000494:pharmacology; D013601:T-Lymphocytes / Q000502:physiology*
D017252:CD3 Complex; D012604:Scorpion Venoms; C077910:margatoxin; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human