Abstract
Mechanosensation is the ubiquitous ability of cells to sense and respond to their mechanical environment and has been linked to the activation of ion channels. Members of the DEG/ENaC ion channel protein family including vertebrate epithelial sodium channel (ENaC) and acid-sensing ion channel (ASIC) proteins have been shown to form mechanosensitive channels. ENaC is a heterotrimeric, amiloride-sensitive sodium channel expressed in both epithelial (e.g. kidney) and non-epithelial tissue (e.g. endothelium). Amongst various factors, ENaC activity is regulated by the mechanical stimulus shear force (SF). SF is generated e.g. by the urinary flow in the kidney or blood flow in the vasculature and contributes to blood pressure regulation. The mechanism of how ENaC senses SF is not fully understood. It was suggested to be facilitated via a connection (tether) of ENaC and the extracellular matrix (ECM) and preliminary data indicated that this involves glycosylated asparagines of the channel. Therefore, one aim of this thesis was to characterise the role of N-linked glycans for SF sensation of ENaC and whether the position of the N-linked glycans within the extracellular domain of ENaC is crucial for SF sensation.
Electrophysiological experiments were employed to investigate the SF-induced activation of human ENaC. Removal of N-linked glycans from glycosylated asparagines of αENaC reduced the SF effect. In addition, N-linked glycans have to be positioned in the palm or knuckle of the extracellular domain of α and δENaC to facilitate SF sensation. Further, degradation of the ECM using hyaluronidase reduced the SF effect of ENaC supporting the role of N-linked glycans as tethers for the connection to the ECM.
Taken together, results from these experiments suggest that SF sensation of ENaC requires a connection (tether) to the ECM via N-linked glycans of glycosylated asparagines located in the knuckle- and palm domain of either α or δENaC.
ASICs form homo- or heterotrimeric acid-sensitive ion channels that are widely expressed in the central- and peripheral nervous system and in particular in specialised mechanosensitive cells. ASICs have been shown to be involved in mechanosensation however, experimental evidence showing a direct effect of ASIC regulation via mechanical stimuli is absent. Since ASICs are close relatives to ENaC proteins it was aimed to determine whether or not ASIC activity can be modulated by SF as well.
Electrophysiological recordings were performed to investigate the potential modulation of human ASIC1b, ASIC2a and ASIC3 activity by SF. The results show that the activity of ASIC1b, ASIC2a and ASIC3 are not changed by SF alone. However, high SF rates applied in conjunction with low pH resulted in a stronger activation compared with low SF rates applied in conjunction with low pH. This indicates that SF modulates pH-induced activation of AISCs. Also preliminary results indicate that the ECM is involved in this modulatory action since hyaluronidase reduced the SF effect of ASIC1b.
Taken together, ASIC activity can be modulated by SF in combination with acidic pH and ASIC SF sensation might be facilitated via a connection to the ECM similar to ENaC.
These results demonstrate that members of the DEG/ENaC family (ENaC and ASIC) are mechanosensitive ion channels which require a connection to the ECM via N-linked glycans to facilitate SF sensation. Channel N-linked glycans connected with the ECM may be a ubiquitous feature of mechanosensation.