Question:

Epithelial Na⁺-channels (ENaC) are members of the DEG/ENaC-superfamily, which were found to be activated by mechanical stimuli, shear force (SF) in particular. There is growing evidence that the extracellular matrix (ECM), which is a filamentous mesh consisting of proteoglycans, glycoproteins, glycosaminoglycans and associated proteins, may be involved in detection and transmission of mechanical stimuli on ENaC. The present study characterizes the influence of the ECM in detection of SF by ENaC.

Methods:

ECM-components of Xenopus laevis oocytes were degraded using mechanical (devitellinization) and enzymatical (collagenase, hyaluronidase) treatments before injecting human αβγENaC-RNA, which led to a heterologous expression of the investigated channels. The activity of ENaC in response to SF was measured as transmembrane currents (I_M) using the two-electrode-voltage-clamp technique. SF (0,2 dynes/cm²) was applied using an individualized measurement-chamber in combination with a pressure-controlled perfusion-system. The degradation of ECM was proved by scanning- (SEM) and transmission-electron-microscopy (TEM).

Results:

Application of SF led to a reversible increase of approximately 45+8% in amiloride-sensitive I_M of αβγhENaC-expressing oocytes. Neither the mechanical excision of the surrounding vitelline-envelope alone nor solely enzymatical treatment with the matrix-degrading enzymes collagenase or hyaluronidase influenced the SF-response, although a part of the ECM was destroyed, as confirmed by SEM-, resp. REM-imaging. By contrast, enzymatical treatment with hyaluronidase in combination with mechanical devitellinization attenuated the mechanosensitive response to 18+2%.

Conclusions:

Varieties in responses to SF between ENaC-expressing oocytes with or without the surrounding ECM indicate first evidences of the ECM being involved in mediating the mechanosensitive behavior of ENaCs.