Browsing Electronic Theses and Dissertations by Author "Aditya Dilip Joshi"
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ItemStructural and functional studies of sodium/dicarboxylate cotransporters(2008-06-23) Aditya Dilip Joshi; Dr. Ana M. Pajor; Dr. Steven C. King; Dr. Simon A. Lewis; Dr. Nancy K. Wills; Dr. Krishna RajarathnamThe sodium/dicarboxylate cotransporter (NaDC1) is found on the apical membrane of the kidney proximal tubule and the small intestine. It carries various di- and tri- carboxylates such as succinate, á-ketoglutarate and citrate. NaDC1 is involved in regulating the concentration of tricarboxylic acid cycle intermediates in kidney cells and urine. Therefore, NaDC1 influences the homeostasis of citrate, which may be associated with the formation of kidney stones. The studies in this dissertation focus on understanding various structural and functional aspects of NaDC1. \r\n\r\nPrevious studies indicate importance of TM 7, 10 and 11 for substrate binding. In this study, conserved prolines from TM 7 and 10 were mutated to alanine and glycine to understand structural as well as functional importance of these residues. Alanine is a strong alpha-helix former and is less flexible whereas glycine, with no side chain, is a strong helix breaker and is more flexible. If prolines in NaDC1 are responsible for kink formation and if kink is necessary for maintaining the stability of the transporter then mutating proline to glycine will have less adverse effect compared with mutating to alanine. This study indicates that Proline 327 in TM 7 when mutated to glycine was not able to reach the plasma membrane, showed no expression as well as succinate transport activity. Proline 351 plays an important role in cell surface regulation and protein trafficking. The prolines found in TM 10 at positions 523 and 524 do not appear to have functional roles but might be important for protein stability. \r\n\r\nThe current model of NaDC1, based on hydropathy analysis, contains 11 TM but secondary structure prediction algorithms predict at least 13 TM. To differentiate between the 11 and the 13 TM models, individual cysteine residues were substituted for other amino acids in predicted extracellular and intracellular loops of rbNaDC1. The extracellular accessibility of the cysteines was determined by chemical labeling with MTSEA-biotin [N-biotinylaminoethyl methanethiosulfonate]. Based on the site-directed chemical labeling experiments and computational homology modeling a modified model of NaDC1 was constructed containing 11 TM. Further studies indicate that mutants A39C, K84C, A133C, T252C, G356C, T482C and M493C were accessible in sodium buffer but their reactivity with methanethiosulfonate and their accessibility from outside changes in different buffer conditions in presence and absence of sodium and succinate. This indicates conformational changes in the transporter during the transport cycle. This new model will provide a structural framework towards understanding the structure-function relationship of NaDC1.\r\n\r\nTo identify conformationally sensitive residues in the sodium/dicarboxylate cotransporter that are accessible from both sides of the membrane, a bacterial homolog of NaDC1, sodium/dicarboxylate symporter (SdcS) from Staphylococcus aureus was used. The eukaryotic transporter could not be used due to experimental difficulties in studying accessibility of the transporter from inside the cell. Previous studies indicate that rbNaDC1 contains structurally, functionally and conformationally important residues such as Lys-84, Asp-373, Met-493. When protein sequence of rbNaDC1 was aligned with SdcS these residues correspond to Asn-108, Asp-329 and Leu-436 of SdcS. Cysteines were substituted at these locations in cysteineless SdcS and their accessibility was tested by MTSET reagent in right-side-out vesicles (RSO) and inside-out vesicles (ISO) in different conformational conditions such as in presence and absence of Na+ and substrate. SdcS showed similar succinate affinity in ISO and RSO vesicles but the transport system was asymmetric with Vmax of SdcS RSO is four times higher than Vmax of ISO. Residue Asn-108 was accessible from outside and inside in presence of sodium only. The N108C mutant was not accessible with the conformational state in absence of sodium and showed substrate protection likely due to steric hindrance by chemical labeling. Therefore, residue Asn-108 is probably located in a transmembrane helix near a water-filled pore or in a re-entrant loop accessible from both sides of the membrane. \r\n\r\nIn conclusion, this work provides a new insight into structural and functional aspects of sodium/dicarboxylate cotransporters. As limited high-resolution structural information is available about membrane transporters these biochemical and computational studies elucidate fundamental information of structural details of sodium/dicarboxylate cotransporters as well as help us in understanding its role in cellular functions.\r\n