Figure 17. Illustration of an LAPS as a microphysiometer: (a, b) principle and structure of the LAPS, (c) proton release in cellular metabolism. Panel a reprinted with permission from ref 309.

Unbalanced production of proinflammatory cytokines and type I interferons in immune responses may lead to immunopathology; thus, the mechanisms that ensure the beneficial production of proinflammatory cytokines and type I interferons are of particular importance. Here we demonstrate that the phosphatase SHP-1 negatively regulated Toll-like receptor-mediated production of proinflammatory cytokines by inhibiting activation of the transcription factor NF-kappaB and mitogen-activated protein kinase. Simultaneously, SHP-1 increased the production of type I interferon mediated by Toll-like receptors and the helicase RIG-I by directly binding to and inhibiting activation of the kinase IRAK1. Our data demonstrate that SHP-1 contributes to immune homeostasis by balancing the production of proinflammatory cytokines and type I interferons in the innate immune response.

The Toll-like receptor 3 (TLR3) and TLR4-signaling pathway that involves the adaptor protein TRIF activates type I interferon (IFN) and proinflammatory cytokine expression. Little is known about how TRIF pathway-dependent gene expression is regulated. SH2-containing protein tyrosine phosphatase 2 (SHP-2) is a widely expressed cytoplasmic tyrosine phosphatase. Here we demonstrate that SHP-2 negatively regulated TLR4- and TLR3-activated IFN-beta production. SHP-2 inhibited TLR3-activated but not TLR2-, TLR7-, and TLR9-activated proinflammatory cytokine IL-6 and TNF-alpha production. SHP-2 inhibited poly(I:C)-induced cytokine production by a phosphatase activity-independent mechanism. C-terminal domain of SHP-2 directly bound TANK binding kinase (TBK1) by interacting with the kinase domain of TBK1. SHP-2 deficiency increased TBK1-activated IFN-beta and TNF-alpha expression. TBK1 knockdown inhibited poly(I:C)-induced IL-6 production in SHP-2-deficient cells. SHP-2 also inhibited poly(I:C)-induced activation of MAP kinase pathways. These results demonstrate that SHP-2 specifically negatively regulate TRIF-mediated gene expression in TLR signaling, partially through inhibiting TBK1-activated signal transduction.

Voltage-gated potassium channels control cardiac repolarization, and mutations of K ؉ channel genes recently have been shown to cause arrhythmias and sudden death in families with the congenital long QT syndrome. The precise mechanism by which the mutations lead to QT prolongation and arrhythmias is uncertain, however. We have shown previously that an N-terminal fragment including the first transmembrane segment of the rat delayed rectifier K ؉ channel Kv1.1 (Kv1.1N206Tag) coassembles with other K ؉ channels of the Kv1 subfamily in vitro, inhibits the currents encoded by Kv1.5 in a dominant-negative manner when coexpressed in Xenopus oocytes, and traps Kv1.5 polypeptide in the endoplasmic reticulum of GH3 cells. Here we report that transgenic mice overexpressing Kv1.1N206Tag in the heart have a prolonged QT interval and ventricular tachycardia. Cardiac myocytes from these mice have action potential prolongation caused by a significant reduction in the density of a rapidly activating, slowly inactivating, 4-aminopyridine sensitive outward K ؉ current. These changes correlate with a marked decrease in the level of Kv1.5 polypeptide. Thus, overexpression of a truncated K ؉ channel in the heart alters native K ؉ channel expression and has profound effects on cardiac excitability.Mutations of the K ϩ channel genes HERG and KVLQT1 cause the autosomal dominant long QT (LQT) syndrome, presumably by interfering with the cardiac currents I Kr and I Ks (1-6). The precise biochemical mechanism by which these mutations cause prolongation of the QT interval is uncertain. A single wild-type potassium channel gene in a heterozygous-affected individual may produce an insufficient number of functional channels to support normal repolarization of the heart. Alternatively, a mutated or truncated channel polypeptide might coassemble with wild-type channel polypeptides to produce nonfunctional channels via a dominant-negative mechanism (7).Voltage-gated potassium channels form multimeric complexes by association of four ␣-subunits (8). We previously have used site-directed mutagenesis and dominant-negative techniques to study structure-function relationships and elucidate the domains that play an important role in Shaker-like potassium channel assembly (9-11). Overexpression of the N-terminal fragment and the first transmembrane segment of the rat brain potassium channel (Kv1.1N206Tag) in Xenopus oocytes inhibited the currents encoded by Kv1.1 and Kv1.5 in a dominant-negative manner. Kv1.1N206Tag also formed in vitro heteromultimeric complexes with Kv1.1 and Kv1.5 (11). Furthermore, we have shown that overexpression of Kv1.1N206Tag in GH3 cells led to the formation of heteromultimeric complexes with the native Kv1.4 and Kv1.5 potassium channel polypeptides (12). These complexes were trapped in the endoplasmic reticulum and did not reach the plasma membrane. The trapping of Kv1.1N206Tag led to its rapid degradation. These experiments elucidated the biochemical mechanisms that underlie the dominant-negative effect of a truncate...