Investigating the pathophysiological mechanisms linked to 2 membrane proteins: the choline transporter 1 and the kidney anion exchanger 1
Creators: Rizvi, Midhat
Publisher: name: University of Alberta Library
Year: 2023

<p>Many diseases are caused by inherited mutations. Using molecular genetics and other laboratory techniques, they can be studied in vitro and in vivo. Congenital myasthenic syndromes is one example, for which I created an in-vitro model of the heterozygous mutations in the choline transporter (CHT1), I294T and D349N, that result in a severe form of the disease in a patient. Congenital myasthenic syndrome is a rare neuromuscular disorder and the phenotypes range from muscle weaknesses to lethality, therefore it is necessary to study them to understand this heterogeneity. I generated the two mutations via site-directed mutagenesis, introduced them into human embryonic kidney cells via lentiviral transfection and characterized each mutant. I hypothesized that the CHT1 mutations found in the patient cause a partial loss of choline transport. We found that CHT1-I294T is less abundant than WT-CHT1, has appropriate localization, a shorter half-life, and some residual activity at low concentration of choline. Because there was residual activity, we incubated cells with MKC-231, STS, and CFTR rescue molecules to assess their effect on CHT1 expression. None of the treatments were effective at increasing CHT1-I294T mutant expression. In contrast, D349N-CHT1 is more abundant than WT, has appropriate localization but is a complete loss of function mutation. Based on the residual activity of I294T mutant, the treatment for the patient was altered and his condition improved. <br>
Another example of diseases that can be caused by inherited mutations is distal renal tubular acidosis, (dRTA), which is characterized by metabolic acidosis, the inability to acidify the urine, hypokalemia, decreased bone density, difficulty to thrive and eventually chronic kidney disease. One of the key proteins involved in acid base balance in the collecting duct is the kidney anion exchanger 1 (kAE1) which, when mutated, can result in dRTA. Recently, a mouse model of dRTA that carry the kAe1 R607H knock-in mutation (R589H human equivalent), displayed a decrease in alpha intercalated cells and abnormal localization of the v-H+-ATPase. In the remainder of the A-ICs, there was accumulation of p62 and ubiquitin, both markers of autophagy. This motivated us to look further into autophagic cell death as a potential factor in the progression of dRTA. Using both in vitro and in vivo models, we uncovered some mechanisms that may explain the molecular events occurring in dRTA. In this study, we used mIMCD cells expressing kAE1 WT, R295H, S525F (both are unpublished dRTA causing mutations) and previously reported R589H, as well as R607H mice. The lab previously found that mutant mIMCD cells were unable to acidify intracellularly to the same extent as WT mIMCD cells. As cytosolic pH has an effect on autophagy by regulating the mammalian target of rapamycin (mTOR), we hypothesized that this may be the mechanism underlying altered autophagy in dRTA. Using immunoblot, we observed a decrease in autophagy in R295H and S525F mutant cells that was not appropriately upregulated when autophagy was induced via starvation. Using lysosomal staining, we identified that the S525F mutant cells also have an increase in the number of lysosomes. In the R589H mutant cells we observed an increase in autophagy flux that could not be appropriately suppressed. This mutant also displayed enlarged lysosomes, also supporting abnormal autophagy. Unfortunately, some of these methods were unreliable in the mouse model but initial findings indicate that autophagy is altered in the R607H knock-in mice as well.</p>