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UBC Theses and Dissertations

Characterization of voltage-dependent calcium channels in T lymphocytes Kotturi, Maya Fay


In T lymphocytes, sustained calcium (Ca²⁺) influx through Ca²⁺channels localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies intimated that L-type voltage-dependent Ca²⁺ channels (VDCCs) play a role in Ca²⁺ mobilization during T lymphocyte activation. However, the function of VDCCs in these non-excitable cells is still poorly understood. In order to explore the role of L-type VDCCs in T lymphocytes, molecular and pharmacological analyses were employed to identify L-type VDCCs, and to define their contribution to Ca²⁺ influx pathways in T lymphocytes. In human T lymphocytes, two novel splice isoforms of the channel-forming OCIFsubunit of retinal L-type VDCC were identified. It was found that both of the α[sub iF]-subunit splice isoforms contain unique structural features, distinct from the α[sub iF]-subunit originally isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. Through cDNA cloning with a human spleen library, the complete cDNA sequence of one of the 0Ci F-subunit splice variants was isolated for future functional studies. The mRNA expression of the α[sub iF]-subunit splice isoforms appeared to be regulated by T cell receptor (TCR)-induced activation in the human Jurkat T cell leukemia line, and to a lesser extent in human peripheral blood T lymphocytes (PBTs). In addition, the 0CiF-subunit protein was detected in Jurkat T cells and human PBTs. To further investigate the contribution of Ca²⁺ influx through L-type VDCCs, the effects of the 1,4-dihydropyridine (DHP) L-type Ca²⁺ channel agonist, (+/-) Bay K 8644, and antagonist, nifedipine, on Jurkat T cells, human PBTs and mouse splenocytes were assessed. It was found that treatment of T lymphocytes with (+/-) Bay K 8644 increased intracellular Ca²⁺ and induced the activation of phospho-extracellular regulated kinase 1/2 (Erkl/2), whereas nifedipine blocked Ca²⁺ influx, the activity of Erkl/2 and nuclear factor of activated T cells, interleukin-2 (IL-2) production and IL-2 receptor expression. Nifedipine also significantly suppressed splenocyte proliferation in an in vitro mixed lymphocyte reaction. Since patients receive nifedipine for the treatment of cardiovascular diseases and other clinical disorders, it was important to further examine the potential immunosuppressive effects associated with nifedipine administration. It was found that nifedipine inhibited the proliferation of male antigen (H-Y)-specific TCR-transgenic CD8⁺ T cells in transplanted male mice in vivo. Finally, a study exploring the effects of nifedipine administration on circulating T lymphocytes in renal disease patients showed T cells from renal patients secreted less IL-2 compared to T cells isolated from a healthy individual. This suggested that nifedipine therapy may act as an immunosuppressant. The results demonstrate that alternative splicing of the human retina [sub iF]-subunit has led to the expression of structurally unique [sub iF]-subunits in T lymphocytes. 2_|_ Furthermore, the pharmacological studies indicate that L-type Ca²⁺ channels play a significant role in the Ca²⁺influx pathways mediating T lymphocyte activation and proliferation in vitro and in vivo.

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