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Bikash R Pattnaik, Abhishek Kumar, Pawan Shahi; Nonsense suppression: are the ribosomal selection of near-cognate amino acids functional?. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4986.
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In eukaryotic systems, translation termination is signaled through in-frame nonsense mutations UAG, UAA, and UGA codons. We recently demonstrated that both RTC14 and NB84 were capable of inducing read-through of autosomal recessive Leber Congenital Amaurosis (LCA16) nonsense mutation (c.158G>A: UAG or W53x) in KCNJ13 gene. It is believed that glutamine, tyrosine, and lysine are preferably incorporated at UAG codon readthrough. We tested the functionality of the KCNJ13 protein product (Kir7.1 channel) due to the incorporation of these preferred amino acids for LCA16 mutation.
Human Kir7.1 open reading frame was subjected to site-directed mutagenesis to generate x53E, x53S, x53Y, and x53Q clones. HEK293 cells were transfected with these clones by Trans-LT1 (Mirus Bio). After 48 to 72 hours post-transfection, the cells were either lysed for protein separation and Western blot or imaged for a fused GFP localization in the cells by live-cell confocal imaging or used for whole-cell patch-clamp electrophysiology evaluation of Kir7.1 channel function. Results were compared to wild-type and W53x, which served as experimental controls.
A full-length protein product was detected for wildtype, x53E, x53S, and x53Y but not x53Q and W53x clones. Similarly, wildtype, x53E, x53S, and x53Y protein localized to the membrane but x53Q, and W53x showed cytoplasmic distribution. Membrane potential measured -67±3, -48±5, -42±4, -30±6, -36±3 mV and current amplitude was measured at -150 mV membrane potential as channel current density, and the values are 19±4, 5±1, 2±1, 3±1, and 3±2 pA/pF for W, E, S, Y, and Q clones respectively. Since Rb+ is known to amplify Kir7.1 current, the measured inward current density of Rb+ was 80±18, 56±14, 24±5, and 32±7 pA/pF for W, E, S, and Y respectively.
Near cognate amino acids tyrosine, glutamic acid, and serine when inserted at position 53 in the Kir7.1 protein make a full-length protein product on the membrane with a sub-optimal functional channel. Whereas, glutamine, which is the preferred amino acid for readthrough makes a completely non-functional channel. Restoration of function in our previous study due to drug-induced readthrough points to the insertion of a wildtype amino acid.
This is a 2020 ARVO Annual Meeting abstract.
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