Abstract
Mechanotransduction describes the ability of a living organisms to sense mechanical forces that are caused through changes in the environment. It is a complex mechanism in which mechanical forces are converted into a cellular signal. Ion channels are important molecules for mechanotransduction. Epithelial sodium channel (ENaC) is one channel of the DEG/ENaC superfamily, mainly expressed in the lung, kidney and vasculature and is known to be important for blood pressure regulation. In the kidney and vasculature ENaC are continuously exposed to mechanical forces such as shear force (SF) caused by the urine or blood flow. Canonical ENaC is made up of three homologous subunits known as α, β, and γ which form a heterotrimeric channel. Till today, little is known about how single subunits and different combinations of these ENaC subunits respond to SF. Previous studies on the mechanosensitive channels of the nematode Caenorhabditis elegans (C. elegans) demonstrated that a linkage between the extracellular domain of the channel and the extracellular matrix (ECM) is important for sensing mechanical forces. The underlying molecular mechanism governing how ENaC senses SF remains unknown. To answer this question research in the Fronius lab derived a model, where N-linked glycans of glycosylated asparagines are tethered to the extracellular matrix to facilitate SF sensation and mechanotransduction. Therefore the aims of my PhD thesis were:
1) To investigate whether individual subunits of ENaC respond to SF. Electrophysiological experiments were performed by expressing (α, β, γ, or δ-subunit) as homotrimeric and co-expressing (αβ, αγ, βγ, δβ, or δγ-subunits) as heterotrimeric ENaC in Xenopus laevis oocytes followed by the determination of channel activity in response to SF.
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2) To investigate whether N-linked glycans of glycosylated asparagines of the β- and γ-ENaC subunits are important for sensing SF. N-glycans of all ENaC subunits were enzymatically removed by PNGaseF and glycosylated asparagines removed in the β- and γ-subunits.
The main findings from aim 1 were that homotrimeric α- , β- , δ-ENaC expressed in oocytes can form functional channels that respond to SF, whereas γ-ENaC cannot. The co-expression of two ENaC subunits αβ- , αγ- , βγ- , δβ- , and δγ-ENaC can form heterotrimeric functional channels that respond to SF. However, this finding was accompanied by decreased amiloride-sensitive currents compared to wild-type control channels (αβγ- or δβγ-ENaC), indicating an impaired trafficking/maturation of these channels.
The β-ENaC subunit was observed to consistently weaken the SF response when co-expressed with α- , δ- and γ-ENaC, identifying, for the first time, a modulatory role of β-ENaC subunit for SF activation.
The presence of the γ-ENaC subunit was observed to constantly increase the SF response when co-expressed with α- and δ- ENaC subunit. This increased response to SF was noted in the heterotrimeric αγ- and δγ-ENaC when compared to control αβγ- or δβγ-ENaC. These findings support the notion that γ-ENaC subunit enhances the ability of ENaC to sense SF. This was further confirmed by the elimination of the decreased SF response of homotrimeric β-ENaC, when the β-subunit was co-expressed with the γ-subunit.
In the second part of my thesis (aim 2) I observed that removal of N-glycans by enzymatic degradation resulted in a reduction in the measured SF response. This showed that N-glycans play an important role for SF activation of ENaC. In contrast to my hypothesis, removal of a single or multiple glycosylated asparagines
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in the β-ENaC showed an increased ENaC activity in response to SF. N-glycans might have distinct functions such as providing an intra-subunit tether within the individual subunit itself, inter-subunit tether to an adjacent subunit, or an ECM-tether that connects the channel to the ECM. This finding confirmed that in addition to the role of trafficking and maturation of ENaC, N-glycans have a new distinct role in modulating/enhancing channel activity in response to SF.
In conclusion, these results show that single subunits of ENaC expressed in oocytes as homotrimeric or heterotrimeric channels can form functional channels that respond to SF. This indicates that the β- and γ-ENaC subunit are important for the SF response. Changes in one of the subunits may affect the overall ability of ENaC to respond to SF. Furthermore, N-glycans are also important for this process. N-glycans of the β- and γ-ENaC subunit might have distinct functions that are involved in stabilisation, gating kinetics, trafficking and SF sensation of ENaC. This could be a new role for N-glycans in mechanotransduction and a new mechanism for how ENaC senses SF.