SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K ϩ current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH i from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1–based mechanosensory complex in ECs.

Conflict of interest: JT is a cofounder of Principia Biopharma, which has licensed the p90RSK inhibitor FMK-MEA.

Background: The incidence of cardiovascular disease (CVD) is higher in HIV + patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for CVD. However, the molecular mechanisms that link cART and CVD are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species (ROS)-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. Methods: Peripheral whole blood from cART-treated HIV + individuals and non-treated HIV − individuals was treated with H 2 O 2 (200 μM) for 4 minutes, and p90RSK activity in CD14 + monocytes was measured. Plaque formation in the carotids were also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK specific inhibitor (FMK-MEA) or dominant negative p90RSK (DN-p90RSK), and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild type and DN-p90RSK transgenic mice. Results: Monocytes from HIV + patients exhibited higher levels of p90RSK activity and were also more sensitive to ROS than monocytes from HIV − individuals. A multiple linear regression analysis involving cART, Reynolds CV risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the two significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H 2 O 2-induced over-activation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Lastly, the data obtained from our gain-or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. Conclusions: cART increased monocyte/macrophage sensitivity to ROS in HIV + individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and pro-inflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing CVD, especially in HIV + patients treated with cART.