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Shear Force Activation of Epithelial Na+ Channel (ENaC) Is Modulated by N-Glycans of the β ENaC Subunit

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Mechanobiology (ISMB 2022)

Part of the book series: Springer Series in Biophysics ((BIOPHYSICS,volume 25))

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Abstract.

Canonical epithelial Na+ channels (ENaC) formed by three subunits (α, β and γENaC) are activated by shear force. Recent evidence highlights the role of N-glycans attached to asparagines (N) of α ENaC and the extracellular matrix for shear force activation of ENaC, in agreement with the force from filament concept. Although β and γ ENaC also contain multiple putative glycosylation sites in their extracellular domains, their role for shear force activation of ENaC is unknown and was addressed in this study.

ENaC subunits were expressed in Xenopus oocytes and ENaC currents were measured by two-electrode-voltage-clamp. Shear force was applied by a fluid flow generated via a pressurised bath perfusion system. N-glycosylation sites of β (11 extracellular putative N-glycosylation motifs) and γ ENaC (five glycosylation motifs) were disrupted by site-directed mutagenesis to remove the putative N-glycans.

The replacement of individual extracellular glycosylated N of γ ENaC did not affect ENaC’s ability to respond to shear force. In β ENaC two asparagines were identified that affected the shear force-activated current. Replacement of N99 and N378 did result in channels that providing a stronger shear force response in comparison with the wild type channel. While the replacement of N99 did also impair amiloride sensitive current suggesting reduced cell membrane expression. Channels lacking N378 did have increase shear force current, but basic biophysical properties assessed by single channel recordings were unchanged.

The increased shear force response observed with N378 identifies a new role for N-glycans for ENaC’s ability to respond to shear force. It might be speculated that N-glycans attached to the asparagine at position 378 of β ENaC facilitate inter-subunit interactions that stabilise the channel. Impairment of this interaction might make the channel more responsive to shear force.

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Acknowledgements.

The study was supported by government funding administered by the Royal Society of New Zealand (Marsden Fund; UOO1505) and a University of Otago Research Grant. In addition, the contribution of J-P. B. and D. B. was supported by a University of Otago PhD Scholarship and through AIM Funds from the Department of Physiology, University of Otago.

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Authors and Affiliations

  1. Department of Physiology, University of Otago, Dunedin, New Zealand

    Jan-Peter Baldin, Daniel Barth & Martin Fronius

  2. HeartOtago, University of Otago, Dunedin, New Zealand

    Jan-Peter Baldin, Daniel Barth & Martin Fronius

  3. Excellence Cluster Cardio-Pulmonary Institute (CPI), University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus Liebig University, Giessen, Germany

    Fenja Knoepp

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  1. Jan-Peter Baldin
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  2. Daniel Barth
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  3. Fenja Knoepp
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  4. Martin Fronius
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Correspondence to Martin Fronius .

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Editors and Affiliations

  1. Lowry Packer Building, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia

    Boris Martinac

  2. Lowy Packer Building, Level 6, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia

    Charles D. Cox

  3. School of Biomedical Sciences, UNSW Sydney, Sydney, NSW, Australia

    Kate Poole

  4. Baker Heart and Diabetes Institute, Melbourne, VIC, Australia

    Sara Baratchi

  5. EMBL Australia, Single Molecule Science node, School of Biomedical Sciences, UNSW Sydney, Sydney, NSW, Australia

    Daryan Kempe

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Baldin, JP., Barth, D., Knoepp, F., Fronius, M. (2024). Shear Force Activation of Epithelial Na+ Channel (ENaC) Is Modulated by N-Glycans of the β ENaC Subunit. In: Martinac, B., Cox, C.D., Poole, K., Baratchi, S., Kempe, D. (eds) Mechanobiology. ISMB 2022. Springer Series in Biophysics, vol 25. Springer, Cham. https://doi.org/10.1007/978-3-031-45379-3_2

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