The first arginine in DII/S4 and in DIV/S4 within the skeletal muscle sodium channel and the L-type calcium channel genie CACNA1S appear to be critical for normal function. In all four cases, Arg to His mutations result in a disease phenotype. The identification of a mutation within the skeletal muscle sodium channel resulting in hypokalemic periodic paralysis represents a novel finding.
Missense mutations of the human skeletal muscle voltage-gated Na channel (hSkM1) underlie a variety of diseases, including hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita, and potassium-aggravated myotonia. Another disorder of sarcolemmal excitability, hypokalemic periodic paralysis (HypoPP), which is usually caused by missense mutations of the S4 voltage sensors of the L-type Ca channel, was associated recently in one family with a mutation in the outermost arginine of the IIS4 voltage sensor (R669H) of hSkM1 (Bulman et al., 1999). Intriguingly, an arginine-to-histidine mutation at the homologous position in the L-type Ca 2ϩ channel (R528H) is a common cause of HypoPP. We have studied the gating properties of the hSkM1-R669H mutant Na channel experimentally in human embryonic kidney cells and found that it has no significant effects on activation or fast inactivation but does cause an enhancement of slow inactivation. R669H channels exhibit an ϳ10 mV hyperpolarized shift in the voltage dependence of slow inactivation and a twofold to fivefold prolongation of recovery after prolonged depolarization. In contrast, slow inactivation is often disrupted in HyperPP-associated Na channel mutants. These results demonstrate that, in R669H-associated HypoPP, enhanced slow inactivation does not preclude, and may contribute to, prolonged attacks of weakness and add support to previous evidence implicating the IIS4 voltage sensor in slow-inactivation gating.
Microsomal glutathione S-transferase-II (GST-II) has recently been discovered and characterized as a member of the 5-lipoxygenase-activating protein (FLAP)/5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC 4 ) synthase gene family, which also includes microsomal glutathione S-transferase-I (GST-I) as a distant member of this gene family. This new enzyme is unique as it is the only member of this family capable of efficiently conjugating reduced glutathione to both 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA 4 ) and 1-chloro-2,4-dinitrobenzene. Although microsomal GST-II has been demonstrated to display both general glutathione S-transferase (GST) and specific LTC 4 synthase activities, its biological function remains unknown. In this study, we investigated the physiological location of microsomal GST-II as well as the relative importance of this enzyme versus LTC 4 synthase for the production of LTC 4 in various human tissues and cells that have been previously demonstrated to possess LTC 4 synthase activity. As determined by Western blot, microsomal GST-II was predominantly expressed in human liver microsomes, human endothelial cell membranes, and sparsely detected in human lung membranes. In contrast, LTC 4 synthase was prevalent in human lung membranes, human platelet homogenates, and human kidney tissue. Concomitant to the formation of LTC 4 , microsomal GST-II also produces a new metabolite of LTA 4 , a postulated LTC 4 isomer. This isomer was used to distinguish between microsomal GST-II and LTC 4 synthase activities involved in the biosynthesis of LTC 4 . Based on the relative production of LTC 4 to the LTC 4 isomer, microsomal GST-II was demonstrated to be the principal enzyme responsible for LTC 4 production in human liver microsomes and human endothelial cells and played a minor role in the formation of LTC 4 in human lung membranes. In comparison, LTC 4 synthase was the main enzyme capable of catalyzing the conjugation of reduced glutathione to LTA 4 in human lung membranes and human platelet homogenates. Therefore, microsomal GST-II appears to be an integral component in the detoxification of biological systems due to its marked presence in human liver, in accordance with its known GST activity. Microsomal GST-II, however, may also be pivotal for cysteinyl leukotriene formation in endothelial cells, and this could change our current understanding of the regulation of leukotriene biosynthesis in inflammatory disorders such as asthma.
Bisphenol A (BPA) is used in the production of polycarbonate plastics and epoxy resins for baby bottles, liners of canned food, and many other consumer products. Previously, BPA has been shown to reduce the activity of several antioxidant enzymes, which may contribute to oxidative stress. However, the underlying mechanism of the BPA-mediated effect upon antioxidant enzyme activity is unknown. Antioxidant and phase II metabolizing enzymes protect cells from oxidative stress and are transcriptionally activated by Nrf1 and Nrf2 factors through their cis-regulatory antioxidant response elements (AREs). In this work, we have assessed the effect of BPA on the Nrf1/2-ARE pathway in cultured human embryonic kidney (HEK) 293 cells. Surprisingly, glutathione and reactive oxygen species (ROS) assays revealed that BPA application created a more reduced intracellular environment in cultured HEK 293 cells. Furthermore, BPA increased the transactivation activity of ectopic Nrf1 and Nrf2 and increased the expression of ARE-target genes ho-1 and nqo1 at high (100-200 μM) BPA concentrations only. Our study suggests that BPA activates the Nrf1/2-ARE pathway at high (>10 μM) micromolar concentrations.
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