Involvement of Dominant-negative Spliced Variants of the Intermediate Conductance Ca2+-activated K+ Channel, KCa3.1, in Immune Function of Lymphoid Cells

摘要

The intermediate conductance Ca2+-activated K+ channel (IKCa channel) encoded by KCa3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of KCa3.1 (human (h) KCa3.1b) from the human thymus, which were lacking the N-terminal domains of the original hKCa3.1a as a result of alternative splicing events. hKCa3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hKCa3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hKCa3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking KCa3.1 variants from mice and rat lymphoid tissues (mKCa3.1b and rKCa3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hKCa3.1a and/or YFP-tagged hKCa3.1b isoforms showed that hKCa3.1b suppressed the localization of hKCa3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hKCa3.1b with hKCa3.1a suppressed IKCa channel activity of hKCa3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mKCa3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(?)CD8(?) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mKCa3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of KCa3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IKCa channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.