Kuchel, P. W. | Search bioRxiv

bioRxiv2020

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like all membrane proteins, they pass through biosynthetic quality control in the endoplasmic reticulum and Golgi that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the cap region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for higher-order glycosylation in the propeller regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated protein. The higher order glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

biophysics↗

bioRxiv2018

Transmembrane exchange of fluorosugars: characterization of red cell GLUT1 kinetics using 19F NMR

We developed a novel approach for quantifying the equilibrium-exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. Our method is based on an adapted kinetic theory describing the concentration dependence of the transmembrane-exchange rates of two simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogues in human erythrocytes (red blood cells: RBCs) using 19F nuclear magnetic resonance (NMR) exchange spectroscopy (EXSY). An inosine-based glucose-free medium was shown to promote survival and stable metabolism of RBCs over the duration of the experiments (a few hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we were able to categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 towards them. Differences in the membrane permeability of the three glucose analogues were qualitatively interpreted in terms of local perturbations in the bonding of substrates to amino-acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results will apply to the design of probes of glucose transport, or inhibitors of glucose metabolism in cells including those exhibiting the Warburg effect.\n\nABBREVIATIONS

biophysics↗