Ca2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human α1C subunit gene

AbstractL-type Ca2+ channels are important targets for drugs, such as dihydropyridines (DHPs), in the treatment of cardiovascular diseases. Channel expression is regulated by alternative splicing. It has been suggested that in the cardiovascular system tissue-specific expression of different L-type Ca2+ channel splice variants may underlie the observed differences in sensitivities to channel block by DHPs. We investigated the sensitivity of Ca2+ channel splice variants derived from the human α1C gene to the DHP isradipine. Among seven α1C channels we observed up to 10-fold differences in IC50 values for isradipine, as well as changes in the voltage dependence of DHP action

Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1

The cAMP-dependent protein kinase A (PKA) regulates various cellular functions in health and disease. In endothelial cells PKA activity promotes vessel maturation and limits tip cell formation. Here, we used a chemical genetic screen to identify endothelial-specific direct substrates of PKA in human umbilical vein endothelial cells (HUVEC) that may mediate these effects. Amongst several candidates, we identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269, driving phosphorylation-dependent degradation of ATG16L1 protein. Reducing PKA activity increased ATG16L1 protein levels and endothelial autophagy. Mouse in vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. Together these results indicate that endothelial PKA activity mediates a critical switch from active sprouting to quiescence in part through phosphorylation of ATG16L1, which in turn reduces endothelial autophagy

Cyclin-dependent kinase 18 controls trafficking of aquaporin-2 and its abundance through ubiquitin ligase STUB1, which functions as an AKAP

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure

Rem2-Targeted shRNAs Reduce Frequency of Miniature Excitatory Postsynaptic Currents without Altering Voltage-Gated Ca2+ Currents

Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca2+ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca2+ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca2+ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons

The severe presentation and poor outcomes of rheumatic heart disease in Namibia: Lessons from the REMEDY study

Background. This paper reports the baseline characteristics and outcomes of 266 Namibian patients in the Global Registry of Rheumatic Heart Disease. Objective. To describe clinical findings and outcomes in a cohort of children and adults with rheumatic heart disease in Namibia. Methods. Prospective study of all patients with rheumatic heart disease at Windhoek Central Hospital between January 2010 and November 2012. Results. A total of 266 patients were enrolled; median age was 22 years, 72.6% were <30 years old and 60.5% female. The majority (62.8%) had moderate-severe disease; 48.9% were in congestive cardiac failure. Secondary antibiotic prophylaxis was used by 34.2%. Warfarin was used by 75.3% (n=64/85) with clinical indications. Forty-seven (17.6%) had previous valve interventions, of whom 40 (15.0%) had mechanical valve replacements. Over a 2-year follow-up period 19.1% of patients died. Severe valve involvement at enrolment was independently associated with mortality (24.6% v. 5.1% in those without severe disease; hazard ratio 4.9; 95% confidence interval 1.50 - 15.98). Sixty-five (29.8%) of the 218 without previous intervention had valvular intervention after enrolment. Conclusions. In Namibia rheumatic heart disease affects young people who present with severe disease and have a high case fatality rate. Rates of secondary prevention were low. These findings have informed the National Programme for Prevention and Control of Rheumatic Heart Disease in Namibia