Abstract
The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b 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 mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.
Highlights
Four subtypes of small/intermediate conductance Ca2ϩ-activated Kϩ channels (KCa2.1, KCa2.3, and KCa3.1) have been identified by molecular cloning studies and are activated via a mechanism involving Ca2ϩ binding to the EF hands of calmodulin [4, 12, 13]
Flow Cytometric Analysis of Mice Thymocyte Populations—We examined whether up-regulation of mKCa3.1b and the inhibition of IKCa channel activity affected thymocyte phonotypes in Con-A(Ϫ) and Con-A(ϩ) treated with TRAM-34 using anti-CD4(ϩ)CD8(ϩ) single-positive (CD8)(Ϫ) single-positive (CD4) and anti-CD8 antibodies by flow cytometry
We identified the N terminus lacking spliced variants of the intermediate conductance Ca2ϩ-activated Kϩ channel, KCa3.1 (KCa3.1-⌬N), from lymphoid tissues of human and rodents
Summary
RNA Extraction, RT-PCR, and Real Time PCR—Total RNA extraction and RT-PCR from mice and rats were performed as reported previously [21]. Western Blot, Immunoprecipitation, and Immunocytochemistry—Protein fractions of the plasma membrane and cytoplasmic region were prepared from mouse and rat tissues as reported previously [21], and those from human tissues were purchased from the BioChain Institute (Hayward, CA). Cells and tissue homogenates from the mouse thymus were incubated with anti-KCa3.1 antibody specific to the N-terminal region (anti-KCa3.1-N, ab65985; Abcam, Cambridge, MA) overnight at 4 °C, and immunoprecipitation was performed using the Seize Classic mammalian immunoprecipitation kit according to the experimental manual supplied by Pierce. Immunocytochemical staining of KCa3.1 in mouse thymocytes was performed as shown in our previous report [21], and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) was used as a secondary antibody. Apoptosis Measurement by Annexin V Staining—Apoptotic cells were analyzed with annexin V-EGFP or -phycoerythrin apoptosis detection kit (Medical and Biological Laboratories (MBL), Nagoya, Japan) by confocal laser microscopy (A1R, Nikon, Tokyo, Japan).
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