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Characterizing a Mutant Na + /HCO 3 - Cotransporter in a Patient With pRTA Nayem Haque 1 ; Michael Duffey 1 ; Mark Parker 1 1 Jacobs School of Medicine & Biomedical Sciences, Department of Physiology & Biophysics • Using frog X. laevis oocytes, perform two- electrode voltage clamp (TEVC) to compare the transport activity of A744T with wildtype NBCe1-A. • Quantify levels of surface expressed protein using biotinylation & western blot. • Our blood experiences a tremendous acid insult that comes from diet, exercise and cellular metabolism. Blood pH is maintained within a narrow range of 7.35 to 7.45. • Bicarbonate plays an important role in maintain blood pH and counteracting acidity. • NBCe1-A is a Na + /HCO 3 - cotransporter on the basolateral side of the proximal tubule. • Defects in NBCe1-A are associated with proximal renal tubular acidosis (pRTA). Symptoms include growth, intellectual and motor defects. • A mutant NBCe1-A is reported in a patient with pRTA 1 . The mutation involves an amino acid change from alanine at position 744 to threonine (A744T) Two-Electrode Voltage Clamp Data Western Blot Analysis 1. Transfer gel into WB sandwich case. Run at 30 V for one hour. Cathode Sponge 3M Paper Nitrocellulose Gel Anode 3. Drip ECL solution to catalyze HRP reaction. Image using Interactive Chemi. Horseradish Peroxidase 2° Antibody 1° Antibody EGFP I would like to thank Bianca Q., Aniko M., and other members of the Parker lab for helping me carry out these experiments. Special thanks to Dr. Mark P. and Dr. Michael D. for providing the resources and guidance needed to complete this project. 1 Khan, AO, Basamh, OS. Pediatric Primary Calcific Band Keratopathy With Or Without Glaucoma From Biallelic SLC4A4 Mutations. Ophthalmic Genetics. 2018; 425-427. • Western blot for EGFP-tagged NBCe1 revealed a significant reduction in both total and surface NBCe1-A expression in the mutant as compared to wildtype NBCe1. • Electrophysiology data indicates a significant reduction in the bicarbonate transport activity of the mutant compared to wildtype NBCe1. Residue A744 resides in a small nonpolar pocket within NBCe1. The A744T mutant, which introduced a large polar threonine into this region, may kink the overall three-dimensional structure of NBCe1 and is therefore not well tolerated. Creating mutants with small nonpolar residues will help to determine if A744 is absolutely required for proper NBCe1 function. • Perform immunocytochemistry on MDCKI cells transfected with NBCe1. This experiment will be used to identify if surface expression of the mutant is also decreased in a mammalian system. • Repeat these experiments with A744V and A744G mutants to see if these amino acids are tolerated at position 744. • Virtually all bicarbonate is generated and recycled in the kidneys. On the basolateral side of the proximal tubule, NBCe1-A works to reabsorb bicarbonate back into blood. Model 1. Inject oocytes with Water, NBCe1-A A744T and NBCe1-WT RNA 3-5 Days 2. Split oocytes for TEVC experiments and use 15 oocytes from each group for western blot. 3a. Run TEVC 3b. Homogenize oocytes. Extract small fraction for total protein analysis. OC725 BATH 4b. Extract surface proteins with Neutravidin column. Run gel and proceed with western blot. Total & Surface NBCe1-A Lanes 2. Probe PVDF membrane with Mouse anti-EGFP 1° antibody and Goat anti-mouse 2° antibody. NBCe1-A Luminol 3-amino phthalate HRP Figure 1. Current-voltage (I-V) relationship obtained from water-injected oocytes (A), single oocytes expressing NBCe1-A WT (B), and oocytes expressing mutant NBCe1-A (C), with corresponding average bicarbonate dependent conductance (BDC) taken between -20 mV and +20 mV (D). Oocytes were exposed to bicarbonate free ND96 solution and subsequently exposed to 5% CO 2 / HCO 3 - solution (BIC) for 2 minutes before voltage clamping. In panel D, means not marked with the same number of asterisks are significantly different from each other using t-tests (P < 0.025). Western Blot Analysis A B 35 48 63 75 100 135 180 245 kDa Marker WT WT WT H 2 O A744T A744T A744T WT WT WT H 2 O A744T A744T A744T Figure 2. Western blot of total (A) and surface (B) EGFP-tagged NBCe1 from solubilized oocyte protein extracts expressing NBCe1-A WT, A744T mutant, or oocytes injected with H 2 O. Molecular weight of NBCe1 is 116 kDa as a core glycosylated monomer (m). Glycosylated monomeric and dimer NBCe1 is denoted (gm) and (d) on the blots, respectively. The combined density of the three bands representing total EGFP- tagged transporter is significantly lower in the A744T mutant as compared to WT (Average reduction of 90 ±2%; n = 3 batches of 15 oocytes). There was no visible band for the biotinylated mutant in panel B. m gm d NBCe1-A Na + 3 HCO 3 - A744T Wildtype Intracellular Extracellular Gel Lane Sample 1 Marker 2-4 NBCe1-A WT 5 Water Injected 6-8 NBCe1-A A744T
