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Sci. STKE, 12 December 2006
Vol. 2006, Issue 365, p. tw418
[DOI: 10.1126/stke.3652006tw418]

EDITORS' CHOICE

Biochemistry Channel Activated by Histidine Phosphorylation

Nancy R. Gough

Science's STKE, AAAS, Washington, DC 20005, USA

Previous work had indicated that the calcium-activated K+ channel KCa3.1, which is important for T and B cell activation, was indirectly activated by phosphatidylinositol 3-phosphate [PI(3)P]. Srivastava et al. now show that KCa3.1 interacts with and is stimulated by nucleoside diphosphate kinase B (NDPK-B), which is a histidine kinase. A 14-amino-acid sequence in the C-terminal region of KCa3.1 was known to mediate the regulation by PI(3)P, and using this region in a yeast two-hybrid assay, the authors identified NDPK-B as an interacting partner. KCa3.1 and NDPK-B were also coimmunoprecipitated from transfected cells, and the endogenous proteins were coimmunoprecipitated from T cells. Overexpression of active NDPK-B, but not a kinase inactive mutant or the related protein NDPK-A, stimulated KCa3.1 channel activity in whole cells and inside-out patches. Furthermore, depletion of PI(3)P from the cells with wortmannin inhibited the KCa3.1 channel activity, and full activation by NDPK-B required the addition of PI(3)P in these patches from the PI(3)P-depleted cells. KCa3.1 has a histidine at position 358 within the C-terminal 14-amino-acid sequence that is required for PI(3)P regulation. The related channels KCa2.1, KCa2.2, and KCa2.3 have an aspartic acid, and these channels are not regulated by PI(3)P (previous work), nor were they stimulated by NDPK-B. The C-terminal domain of KCa3.1 was phosphorylated in vitro by NDPK-B. Mutation of the His358 eliminated the stimulation of channel activity by NDPK-B, and these mutant channels were resistant to the inhibitory effects of PI(3)P depletion. Finally, mRNA analysis of activated CD4+ T cells showed that the transcription of gene encoding NDPK-B, as was already known for gene encoding KCa3.1, was increased. Down-regulation of NDPK-B activity with siRNA reduced KCa3.1 channel activity in stimulated T cells without altering the activity of an unrelated K+ channel. Because KCa3.1 hyperpolarizes the membrane potential to maintain a driving force for Ca2+ entry, siRNA-mediated down-regulation of NDPK-B also decreased Ca2+ influx in stimulated T cells, thus confirming the biological importance of NDPK-B activity in regulation of T cell function.

S. Srivastava, Z. Li, K. Ko, P. Choudhury, M. Albaqumi, A. K. Johnson, Y. Yan, J. M. Backer, D. Unutmaz, W. A. Coetzee, E. Y. Skolnik, Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells. Mol. Cell 24, 665-675 (2006). [Online Journal]

Citation: N. R. Gough, Channel Activated by Histidine Phosphorylation. Sci. STKE 2006, tw418 (2006).



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