Studies on the DIDS-binding Site of Monocarboxylate Transporter 1 Suggest a Homology Model of the Open Conformation and a Plausible Translocation Cycle*

Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys38, Lys45, Lys282, and Lys413. K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO3− groups interact with positively charged Lys282 together with Lys45 and/or Lys413. This positions one DIDS isothiocyanate group close to Lys38, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys38 or Lys290 of MCT1 and Lys160 or Lys164 of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys38 in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys38 accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp302− and Asp306+, an ion pair already identified as important for transport and located adjacent to Phe360, which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data

Distribution properties of lentiviral vectors administered into the striatum by convection-enhanced delivery

Before the successful use of lentiviral vectors in clinical trials it is essential that strategies for direct vector delivery into the brain be evaluated in vivo, particularly as these vectors are significantly larger than the brain extracellular space. To date no such studies have been undertaken. In this study, convection-enhanced delivery (CED) was employed in an attempt to achieve widespread lentiviral delivery in the striatum. Infusions of equine infectious anemia virus (EIAV) and HIV vector constructs expressing the reporter gene β-galactosidase (β-Gal) were undertaken into the striatum at a range of flow rates and viral titers. In rats, all EIAV and HIV infusions led to the extensive transduction of cells in perivascular spaces throughout the brain. Although infusions were performed under standardized conditions, the number and volume of distribution of transduced cells were highly variable, with approximately one-third of EIAV infusions leading to no concentrated cell transduction in the striatum. Heparin coinfusion had no effect on EIAV distribution, although coinfusion of nimodipine resulted in a significant reduction in the number and volume of distribution of transduced cells. Intrastriatal EIAV delivery in pigs led to extensive transduction of mainly neurons, which could be effectively visualized in real time by T(2)-weighted magnetic resonance imaging. No infusions were associated with a significant inflammatory response. Therefore, despite its large size, lentiviral vectors can be administered by CED to the striatum in both small and large animal models. However, the variability in vector distribution under standardized conditions and widespread vector distribution through the perivascular spaces raise serious concerns regarding the practicality of lentivirus-mediated gene therapy in the brain in clinical practice.</p