Implications for Channel Expression During B Cell +K
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K+ Channel Expression during B Cell Differentiation: Implications for Immunomodulation and Autoimmunity This information is current as Heike Wulff, Hans-Günther Knaus, Michael Pennington and of September 24, 2021. K. George Chandy J Immunol 2004; 173:776-786; ; doi: 10.4049/jimmunol.173.2.776 http://www.jimmunol.org/content/173/2/776 Downloaded from References This article cites 85 articles, 43 of which you can access for free at: http://www.jimmunol.org/content/173/2/776.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology :K؉ Channel Expression during B Cell Differentiation Implications for Immunomodulation and Autoimmunity1 Heike Wulff,2* Hans-Gu¨nther Knaus,† Michael Pennington,‡ and K. George Chandy§ Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel ex- pression during differentiation of human B cells from naive to memory cells. Naive and IgD؉CD27؉ memory B cells express small -numbers of the voltage-gated Kv1.3 and the Ca2؉-activated intermediate-conductance IKCa1 channel when quiescent, and in crease IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast, quiescent class-switched memory B cells express high levels of Kv1.3 (ϳ2000 channels/cell) and maintain their Kv1.3high expression after activation. Consistent with their channel phenotypes, proliferation of naive and IgD؉CD27؉ memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Downloaded from Therefore, specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders. The Journal of Immunology, 2004, 173: 776–786. wo Kϩ channels in T lymphocytes, the voltage-gated cytosolic Ca2ϩ levels in the time frame required for optimal acti- Kv1.3 channel (also known as KCNA3 (HUGO nomen- vation. Coordinated activity of Ca2ϩ and protein kinase C-depen- clature)) and the Ca2ϩ-activated IKCa1 channel (also dent signaling pathways leads to new gene expression culminating T http://www.jimmunol.org/ known as KCNN4 (HUGO nomenclature); KCa3.1 (International in cell proliferation. The relative contributions of Kv1.3 and Union of Pharmacology nomenclature) (1)), regulate Ca2ϩ signal- IKCa1 in regulating this process depend on their expression levels ing by controlling the membrane potential of lymphocytes (2–7). in the different lymphoid subsets. During activation, inositol 1,4,5-triphosphate generation induces Kv1.3 and IKCa1 channels are expressed in T lymphocytes in a ϩ ϩ the release of Ca2 from internal stores. Depletion of these Ca2 distinct pattern that depends upon the state of activation and dif- 3 stores causes calcium-release-activated calcium (CRAC) chan- ferentiation. In the quiescent state, naive, central memory (TCM) 2ϩ ϳ ϳ nels to open in the membrane, and the resulting Ca influx sus- and effector memory (TEM) T cells express 250 Kv1.3 and 20 ϩ ϩ tains elevated levels of cytosolic Ca2 (8–11). Ca2 influx IKCa1 channels per cell. The potent Kv1.3 blocker Stichodactyla through CRAC channels is reduced at depolarized potentials, and helianthus toxin (ShK) suppresses the proliferation of these cells, by guest on September 24, 2021 ϩ consequently, membrane depolarization attenuates the Ca2 signal whereas the selective IKCa1 blocker TRAM-34 is ineffective (14). ϩ (10–13). The driving force for Ca2 entry is restored by a coun- Activation induces differential expression of Kϩ channels in the ϩ terbalancing efflux of K ions and membrane hyperpolarization three T cell subsets, leading to an altered channel phenotype and brought about by the opening of Kv1.3 channels in response to consequently to altered responsiveness to Kv1.3 and IKCa1 block- ϳ membrane depolarization, and of IKCa1 channels as a conse- ers. Naive and TCM cells up-regulate IKCa1 to 500/cell upon ϩ quence of elevated cytosolic Ca2 . The tightly coupled interplay activation via transcriptional mechanisms, and as a consequence, ϩ between the K channels and CRAC channels maintains enhanced these subsets escape further Kv1.3 inhibition and become sensitive to IKCa1 blockade (14, 15). TEM cells, in contrast, up-regulate Kv1.3, and their proliferation is sensitive to Kv1.3 blockers (15). *Department of Medical Pharmacology and Toxicology, University of California, Davis, CA 95616; †Institute for Biochemical Pharmacology, Medical University Inns- The discovery that the majority of myelin-reactive T cells in pa- bruck, Innsbruck, Austria; ‡Bachem Bioscience, Inc., King of Prussia, PA 19406; and tients with multiple sclerosis (MS), an autoimmune disease of the §Department of Physiology and Biophysics, University of California, Irvine, CA CNS, are Kv1.3high T cells that can be suppressed by Kv1.3 92697 EM blockers (15), has raised interest in the therapeutic potential of Received for publication February 6, 2004. Accepted for publication May 10, 2004. Kv1.3 blockers in autoimmune disorders. This idea has been suc- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cessfully tested in experimental autoimmune encephalomyelitis, an with 18 U.S.C. Section 1734 solely to indicate this fact. animal model for the disease (16). 1 This work was supported by grants from the National Multiple Sclerosis Society (to An increasing body of evidence emphasizes the importance of K.G.C.), the Juvenile Diabetes Research Foundation (to K.G.C.), National Institutes memory B cells in the pathogenesis of autoimmune disorders (17– of Health (MH59222; to K.G.C.), Rockefeller Brothers Fund (to K.G.C.), Fonds zur Fo¨rderung der Wissenschaftlichen Forschung (Fonds zur Fo¨rderung der Wissen- 19). As in the T lineage, ion channels may play functionally impor- schaftlichen Forschung P14954-PHA; to H.-G.K.), and the Cancer Research Coordi- tant roles in B cells and may serve as therapeutic targets for autoim- nating Committee of the University of California (to H.W.). mune disorders. Four human B cell subsets can be distinguished by 2 Address correspondence and reprint requests to Dr. Heike Wulff, Department of the expression of IgD and CD27, a member of the TNFR family Medical Pharmacology and Toxicology, School of Medicine, University of Califor- (20–22). Mature naive B cells express IgD but not CD27. Acquisition nia, Davis, One Shields Avenue, Tupper Hall Room 1311, Davis, CA 95616. E-mail ϩ ϩ address: hwulff@ucdavis.edu of CD27 following somatic hypermutation results in a CD27 IgD 3 Abbreviations used in this paper: CRAC, calcium-release-activated calcium; TCM, memory B cell subset, and then during Ig class switching, replace- ϩ Ϫ central memory T; TEM, effector memory T; ShK, Stichodactyla helianthus toxin; ment of surface IgD with other Ig isotypes yields CD27 IgD mem- 2ϩ ϩ MS, multiple sclerosis; PB, peripheral blood; KCa,Ca -activated K current; KV, voltage-gated Kϩ channel; GC, germinal center; MZ, marginal zone; DTH, delayed- ory B cells. These class-switched memory B cells are a major source type hypersensitivity. of pathogenic IgG autoantibodies that contribute to tissue damage in Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 777 MS (17, 19), type-1 diabetes (23), and rheumatoid arthritis (24). A T271) and PE-conjugated mouse anti-human IgD mAb (IA6-2; both BD minor population of IgDϪCD27Ϫ B cells exists in most donors, but Pharmingen). Cells were washed, put on poly-L-lysine-coated coverslips, their functional role has yet to be defined (21). kept in the dark at 4°C for 10–30 min to attach, visualized by fluorescence microscopy, and patch-clamped in the whole-cell configuration. For ex- Mature naive B cells were reported over a decade ago to express periments on CD27ϩIgAϩ and CD27ϩIgGϩ class-switched B cells, we ϩ KV and KCa currents with properties resembling those of Kv1.3 stained CD19 cells with PE-conjugated mouse anti-human CD27 mAb ϩ Ј and IKCa1. Studies with nonspecificK channel inhibitors sug- (M-T271; BD Pharmingen) and FITC-conjugated F(ab )2 of rabbit anti- gested a functional role for these channels in B cell mitogenesis human IgA or anti-human IgG (both DakoCytomation, Carpinteria, CA). ϩ Kv1.3 currents were elicited by repeated 200-ms pulses from a holding (25–29). Because the changes in K channel phenotype are func- potential of Ϫ80 to 40 mV applied every 30 s unless otherwise stated. tionally important in the T cell lineage, we were interested in de- Kv1.3 currents were recorded in normal Ringer solution with a Ca2ϩ-free ϩ termining whether a parallel switch in K channel expression ac- pipette solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, companies differentiation from naive to memory cells in the B cell and 2 mM MgCl2 (pH 7.2; 300 mOsm).