Faculty Bibliography

Nucleoside diphosphate kinases (NDPKs) are encoded by the Nme (non-metastatic cell) gene family. Although they comprise a family of 10 genes, NDPK-A and -B are ubiquitously expressed and account for most of the NDPK activity. We previously showed that NDPK-B activates the K(+) channel KCa3.1 via histidine phosphorylation of the C terminus of KCa3.1, which is required for T cell receptor-stimulated Ca(2+) flux and proliferation of activated naive human CD4 T cells. We now report the phenotype of NDPK-B(-/-) mice. NDPK-B(-/-) mice are phenotypically normal at birth with a normal life span. Although T and B cell development is normal in NDPK-B(-/-) mice, KCa3.1 channel activity and cytokine production are markedly defective in T helper 1 (Th1) and Th2 cells, whereas Th17 function is normal. These findings phenocopy studies in the same cells isolated from KCa3.1(-/-) mice and thereby support genetically that NDPK-B functions upstream of KCa3.1. NDPK-A and -B have been linked to an astonishing array of disparate cellular and biochemical functions, few of which have been confirmed in vivo in physiological relevant systems. NDPK-B(-/-) mice will be an essential tool with which to definitively address the biological functions of NDPK-B. Our finding that NDPK-B is required for activation of Th1 and Th2 CD4 T cells, together with the normal overall phenotype of NDPK-B(-/-) mice, suggests that specific pharmacological inhibitors of NDPK-B may provide new opportunities to treat Th1- and Th2-mediated autoimmune diseases

The calcium-activated K(+) channel KCa3.1 plays an important role in T lymphocyte Ca(2+) signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca(2+) influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1(-/-) mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1(-/-) mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor-stimulated Ca(2+) influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca(2+) influx and cytokine production were impaired in KCa3.1(-/-) Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1(-/-) protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naive CD4 T cells into rag2(-/-) recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis

The Ca(2+)-activated K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2beta (PI3K-C2beta) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2beta by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2beta siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2beta in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca(2+) influx, whereas silencing of PI3K-C2beta inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2beta colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation

The calcium activated K(+) channel KCa3.1 plays an important role in T lymphocyte Ca(2+) signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca(2+) influx. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, is required for KCa3.1 channel activation in human CD4 T lymphocytes. We now show that the mammalian protein histidine phosphatase (PHPT-1) directly binds and inhibits KCa3.1 by dephosphorylating histidine 358 on KCa3.1. Overexpression of wild-type, but not a phosphatase dead, PHPT-1 inhibited KCa3.1 channel activity. Decreased expression of PHPT-1 by siRNA in human CD4 T cells resulted in an increase in KCa3.1 channel activity and increased Ca(2+) influx and proliferation after T cell receptor (TCR) activation, indicating that endogenous PHPT-1 functions to negatively regulate CD4 T cells. Our findings provide a previously unrecognized example of a mammalian histidine phosphatase negatively regulating TCR signaling and are one of the few examples of histidine phosphorylation/dephosphorylation influencing a biological process in mammals

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by numerous fluid-filled kidney cysts. Net fluid secretion into renal cysts is caused by transepithelial transport mediated by the apical cystic fibrosis transmembrane conductance regulator chloride channel, which leads to cyst enlargement. Here we found that forskolin, a potent adenylyl cyclase agonist, stimulated anion secretion by monolayers of kidney cells derived from patients with ADPKD. TRAM-34, a specific KCa3.1 potassium channel blocker, inhibited this current, and in vitro cyst formation and enlargement by the cells cultured within a collagen gel. Net chloride secretion was enhanced by the KCa3.1 activator DCEBIO and both chloride secretion and in vitro cyst growth were inhibited by overexpression of myotubularin-related protein-6, a phosphatase that specifically inhibits KCa3.1 channel activity. Our study suggests that KCa3.1 channels play a critical role in transcellular chloride secretion and net fluid transport into the kidney cysts of patients with ADPKD by maintaining the electrochemical driving force for chloride efflux through apical chloride channels. Pharmacological inhibitors of KCa3.1 channels may provide a novel and effective therapy to delay progression to kidney failure in patients with ADPKD

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation

Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease