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Ca2+-activated K+ channels in human leukemic Jurkat T cells. Molecular cloning, biochemical and functional
1Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel.
Abstract:
Previous studies have demonstrated the presence of apamin-sensitive, small-conductance Ca(2+)-activated K(+) currents in human leukemic Jurkat T cells. Using a combined cDNA and reverse transcriptase-polymerase chain reaction cloning strategy, we have isolated from Jurkat T cells a 2.5-kilobase cDNA, hSK2, encoding the human isoform of SK2 channels. Northern blot analysis reveals the presence of a 2.5-kilobase hSK2 transcript in Jurkat T cells. While present in various human tissues, including brain, heart, skeletal muscle, kidney, and liver, no hSK2 mRNA could be detected in resting and activated normal human T cells. The hSK2 gene is encoded by 8 exons and could be assigned to chromosome 5 (q21.2-q22.1). The protein encoded by hSK2 is 579 amino acids long and exhibits 97% identity with its rat counterpart rSK2. When expressed in Chinese hamster ovary cells, hSK2 produces Ca(2+)-activated K(+) currents with a unitary conductance of 9.5 pS and a K(0.5) for calcium of 0.7 microm; hSK2 currents are inhibited by apamin, scyllatoxin, and d-tubocurarine. Overexpression of the Src family tyrosine kinase p56(lck) in Jurkat cells, up-regulates SK2 currents by 3-fold. While IKCa channels are transcriptionally induced upon activation of normal human T cells, our results show that in Jurkat cells SK2 channels are constitutively expressed and down-regulated following mitogenic stimulation.
Insights
Researchers identified the hSK2 gene in human leukemic Jurkat T cells, encoding small-conductance calcium-activated potassium channels (SK2). These channels are constitutively expressed in Jurkat cells but not in normal T cells.
Area of Science:
- Molecular Biology
- Immunology
- Neuroscience
Background:
- Small-conductance calcium-activated potassium (SK) channels are crucial for T cell function.
- Previous studies indicated apamin-sensitive SK currents in Jurkat T cells.
Purpose of the Study:
- To isolate and characterize the specific SK channel isoform responsible for these currents in Jurkat T cells.
- To investigate the expression pattern and regulation of this channel in both leukemic and normal T cells.
Main Methods:
- cDNA and reverse transcriptase-polymerase chain reaction (RT-PCR) cloning.
- Northern blot analysis for mRNA expression.
- Gene mapping to chromosome 5.
- Functional expression in Chinese hamster ovary (CHO) cells.
- Western blot analysis and Src family tyrosine kinase p56(lck) overexpression.
Main Results:
- Isolated a 2.5-kilobase cDNA, hSK2, encoding the human SK2 channel from Jurkat T cells.
- hSK2 mRNA is present in Jurkat cells and various human tissues but absent in normal T cells.
- Expressed hSK2 channels in CHO cells produced apamin-sensitive Ca(2+)-activated K(+) currents.
- hSK2 gene mapped to chromosome 5 (q21.2-q21.1).
- Overexpression of p56(lck) in Jurkat cells upregulated SK2 currents.
- Jurkat T cells exhibit constitutive SK2 expression, downregulated by mitogenic stimulation, contrasting with transcriptional induction in normal T cells.
Conclusions:
- Identified and characterized the hSK2 channel, a key component of Ca(2+)-activated K(+) currents in Jurkat T cells.
- Demonstrated a distinct expression and regulation pattern of SK2 channels in leukemic T cells compared to normal T cells.
- Suggests a potential role for hSK2 channels in the pathophysiology of T cell leukemia.