Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA317

Poster Communications

Epithelial Na+ channel's ability to mediate arterial shear force responsiveness depends on glycosylated asparagines and the extracellular matrix

F. Knoepp2,1, J. Baldin1,3, D. Barth1,3, Z. Ashley1,3, M. Fronius1,3

1. Physiology, University of Otago, Dunedin, Otago, New Zealand. 2. Excellence Cluster Cardio-Pulmonary System, University Giessen, Giessen, Germany. 3. HeartOtago, University of Otago, Dunedin, New Zealand.

Healthy arteries sense and respond to shear force and impairment of this response is a hallmark of vascular dysfunction. The mechanisms how arteries sense shear force are poorly understood. The epithelial Na+channel (ENaC) formed by α β and γ subunits is an emerging candidate for this role. ENaC's open probability is regulated by shear force (1) and the expression and function was reported in endothelial cells (2). Aim of our research is to reveal how ENaC senses shear force to regulate arterial blood pressure (BP) regulation. We hypothesise that ENaC N-glycans attached to asparagines interact with the extracellular matrix (ECM) to mediate shear force responsiveness. Whole cell currents of human α β γ ENaC in response to shear force were recorded in Xenopus oocytes. Extracellular asparagines of α ENaC were replaced against alanines by site directed mutagenesis. Pressure myography on freshly isolated carotid arteries from C57BL/6 mice was performed to characterise ENaC-mediated effects on arterial diameter in response to shear force. In oocytes and carotid arteries ECM components (hyaluronic acid/heparan sulfate) were enzymatically degrade by hyaluronidase or heparanase. Further, endothelial specific adenoviral transduction of wildtype (n=14), mutated α ENaC (n=14) or empty vector (n=15) was performed in C57BL/6 mice. Mean BP of these mice was measured by the tail-cuff method before and after viral transduction. Two glycosylated asparagines at position 312 and 511 in the extracellular domain of α ENaC contribute to shear force sensing. Replacement of these asparagines individually decreased the shear force currents in Xenopus oocytes by approx. 40% (n≥15, P≤0.01). Degradation the ECM of hyaluronidase or heparanase impaired shear force mediated effects of expressed ENaC in oocytes and ENaC-mediated effects in carotid arteries. In mice overexpressing wild type α ENaC caused an increase in BP from 115±2 to 157±5 mmHg (n=14, P<0.001). Transduction of α ENaC with replaced asparagines showed a markedly decreased rise in BP (114±2 to 124±2 mmHg; n=14, P<0.01). No change in BP was observed in mice transduced with an empty vector (before 112±2; after 115±1 mmHg, n=15, P=0.9). The ability of ENaC to sense shear force depends on glycosylated asparagine of α ENaC and an intact ECM. Replacement of glycosylated asparagines in the finger and palm domain of ENaC impair ENaCs ability to response to shear force and prevent an increase of arterial BP. These observations support our hypothesis that the N-glycans attached to the asparagines provide a connection to the ECM to form a shear force sensitive complex that contributes to BP regulation.

Where applicable, experiments conform with Society ethical requirements