. Expression of CDK6 and ␤-actin was not affected by LY-294002. Expression of the cyclin kinase inhibitor p16 INK4a was induced by the PI3K inhibitor, whereas steady-state levels of p21 CIP1/WAF1 were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation of AKT and p70S6K1, but not extracellular regulated kinase 1/2. The G 1 cell cycle arrest induced by LY-294002 was restored by the expression of active forms of AKT and p70S6K1 in the cells. Our study shows that PI3K transmits a mitogenic signal through AKT and mammalian target of rapamycin (mTOR) to p70S6K1. The mTOR inhibitor rapamycin had similar inhibitory effects on G 1 cell cycle progression and on the expression of cyclin D1, CDK4, CDC25A, and retinoblastoma phosphorylation. These results indicate that PI3K mediates G 1 progression and cyclin expression through activation of an AKT/mTOR/p70S6K1 signaling pathway in the ovarian cancer cells. phosphatidylinositol 3-kinase; cyclin-dependent kinases; retinoblastoma protein OVARIAN CANCER IS THE LEADING cause of death from gynecological malignancy and the fourth most common cause of cancer death among American women (40). Recent observations indicate that the gene encoding the p110␣ catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is increased in copy number in ϳ40% of primary ovarian cancer cells and several ovarian epithelial carcinoma cell lines. The PI3K inhibitor LY-294002 has been shown to inhibit growth of an ovarian cancer cell line in vitro (48). PI3K is a heterodimeric enzyme composed of 110-kDa catalytic and 85-kDa regulatory subunits (3). PI3K phosphorylates the D3 hydroxyl of phosphoinositides and produces phosphatidylinositol-3-phosphate. PI3K

The Long QT Syndrome is a cardiac disorder associated with ventricular arrhythmias that can lead to syncope and sudden death. One prominent form of the Long QT syndrome has been linked to mutations in the HERG gene (KCNH2) that encodes the voltage-dependent delayed rectifier potassium channel (I Kr ). In order to search for HERG-interacting proteins important for HERG maturation and trafficking, we conducted a proteomics screen using myc-tagged HERG transfected into cardiac (HL-1) and non-cardiac (human embryonic kidney 293) cell lines. A partial list of putative HERG-interacting proteins includes several known components of the cytosolic chaperone system, including Hsc70 (70-kDa heat shock cognate protein), Hsp90 (90-kDa heat shock protein), Hdj-2, Hop (Hsp-organizing protein), and Bag-2 (BCL-associated athanogene 2). In addition, two membrane-integrated proteins were identified, calnexin and FKBP38 (38-kDa FK506-binding protein, FKBP8). We show that FKBP38 immunoprecipitates and co-localizes with HERG in our cellular system. Importantly, small interfering RNA knock down of FKBP38 causes a reduction of HERG trafficking, and overexpression of FKBP38 is able to partially rescue the LQT2 trafficking mutant F805C. We propose that FKBP38 is a co-chaperone of HERG and contributes via the Hsc70/Hsp90 chaperone system to the trafficking of wild type and mutant HERG potassium channels.

Loss of function mutations in the hERG (human ether-ago-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.

The purpose of this study was to determine the changes of pulmonary function and autonomic cardiovascular control after an ultramarathon and their relation to performance. Eight entrants to the Canadian National Championship 100-km running race participated in the study. Pulmonary function and 30-s maximum voluntary ventilation (MVV30s) tests were conducted one day before the race and within 5 minutes of race completion. Heart rate and blood pressure data were collected 30 min before and 5 min after the race as well as during a 10-min stand test one day prior to the race. During the race, beat-by-beat R-R interval data were collected over the first and last 20 km. The results showed that MVV30s and MVV30s tidal volumes were reduced postrace (p < 0.001). Prerace supine total harmonic variation (p < 0.01) and prerace MVV values (10 s to 30 s) (p < 0.05) were correlated with race finish time. The changes in pulmonary function and MVV30s values from pre- and postrace were not significantly correlated to race performance. We conclude that maximal sustainable ventilatory power and dynamic autonomic cardiovascular control are important factors in determining overall performance in an ultramarathon.

EA. Regulation of cell surface expression of functional pacemaker channels by a motif in the B-helix of the cyclic nucleotide-binding domain. Am J Physiol Cell Physiol 295: C642-C652, 2008. First published July 9, 2008 doi:10.1152/ajpcell.00062.2008.-Previous studies have suggested that a portion of the cyclic nucleotide-binding domain (CNBD) of the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) "pacemaker" channel, composed of the A-and B-helices and the interceding ␤-barrel, confers two functions: inhibition of channel opening in response to hyperpolarization and promotion of cell surface expression. The sequence determinants required for each of these functions are unknown. In addition, the mechanism underlying plasma membrane targeting by this subdomain has been limitedly explored. Here we identify a four-amino acid motif (EEYP) in the B-helix that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. This motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly.pacemaker channel function; protein export; trafficking; hyperpolarization-activated cyclic nucleotide-gated channel HYPERPOLARIZATION-ACTIVATED cyclic nucleotide-gated "pacemaker" channels (HCN1-4) contribute to the regulation of spontaneous activity and membrane potential in mammalian cardiac conduction tissue and brain (21). The number of HCN channels on the cell surface is critical to these functions, but the factors that determine their supply to this region of the cell have not been extensively studied. In general, export of plasma membrane-bound ion channels from the endoplasmic reticulum (ER) to the Golgi is limited by multiple quality control mechanisms (4,6,7,22). The export of properly folded and assembled channels from the ER is also regulated and may depend on anterograde signals (6,14,15,17,24).In HCN2 channels, the cyclic nucleotide-binding domain (CNBD), located in the COOH terminus, appears to be an important determinant of cell surface expression, in addition to its better known role as regulator of channel opening (31), based on two studies (1,20). First, complex glycosylation is abolished in HCN2 mutants lacking the CNBD, which supports its necessity for export of the channel from the ER (1). Second, we identified a subdomain of the CNBD that strongly promotes cell surface and functional expression; mutants lacking this subdomain do not generate current and are retained intracellularly (20). This same subdomain, which consists of the A and B helices and the interceding ␤-barrel, exerts tonic inhibition of channel opening in response to hyperpolarization (5). Whether the complete subdomain is required for both functions, perhaps by conferring a shared conformational change, or includes distinct regions that contribute to each function is not known.The mechanism by which the CNBD subdomain promotes cell surface expression is poorly understood. To date, it has b...