Regulation Of Membrane Transport Research Articles

Role of Angiotensin II Type 1a Receptor (AT1aR) of Renal Tubules in Regulating Inwardly Rectifying Potassium Channels 4.2 (Kir4.2), Kir4.1, and Epithelial Na+ Channel (ENaC).

Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and βENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

Renal Pharmacokinetic Adaptation to Cholestasis Causes Increased Nephrotoxic Drug Accumulation by Mrp6 Downregulation in Mice

In hepatic dysfunction, renal pharmacokinetic adaptation can be observed, although information on the changes in drug exposure and the interorgan regulation of membrane transporters in kidney in liver diseases is limited. This study aimed to clarify the effects of renal exposure to nephrotoxic drugs during cholestasis induced by bile duct ligation (BDL). Among the 11 nephrotoxic drugs examined, the tissue accumulation of imatinib and cisplatin in kidney slices obtained from mice 2 weeks after BDL operation was higher than that in sham-operated mice. The uptake of imatinib in the kidney slices of BDL mice was slightly higher, whereas its efflux from the slices was largely decreased compared to that in sham-operated mice. Proteomic analysis revealed a reduction in renal expression of the efflux transporter multidrug resistance-associated protein 6 (Mrp6/Abcc6) in BDL mice, and both imatinib and cisplatin were identified as Mrp6 substrates. Survival probability after cisplatin administration was reduced in BDL mice. In conclusion, the present study demonstrated that BDL-induced cholestasis leads to the downregulation of the renal basolateral efflux transporter Mrp6, resulting in drug accumulation in renal cells and promoting drug-induced renal injury.

Effect of silica nanoparticles on cell membrane fluidity: The role of temperature and membrane composition

The increasing production and application of silica nanoparticles (SiO2 NPs) raise public concern regarding their environmental and health risks. The fluidity of the cell membrane is essential for supporting membrane proteins and regulating membrane transport. Changes in membrane fluidity inevitably influence the physiological activities of cells and even cause biological effects. In this study, the effect of SiO2 NPs on membrane fluidity was studied at 25 °C and 37 °C, and the role of membrane components in SiO2 NP-membrane interactions was investigated using giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells. SiO2 NPs cause a more serious membrane fluidity decrease at 37 °C than at 25 °C, which is revealed by the shift of Laurdan fluorescence emission and further quantified via forster resonance energy transfer (FRET) experiments. In addition, after the removal of 75 % cholesterol from the membrane, SiO2 NPs caused a greater extent of membrane gelation. These results indicate that SiO2 NPs prefer to interact with membranes that are more dynamic and less densely packed. Moreover, fluorescent experiments confirmed that the existence of phosphatidyl ethanolamine (PE) and phosphoinositide (PI) can mitigate NP-induced membrane gelation. Molecular dynamics simulation further demonstrated that SiO2 NPs form hydrogen bonds with the terminal of PE or PI but with the -PO4−- group in the middle of phosphatidylcholine (PC). The bonding that occurs in the terminal gives less restriction of phospholipid movement and a weaker effect on membrane fluidity. This research provides both evidence and mechanisms of SiO2 NP-induced membrane fluidity changes, which are helpful for understanding cell membrane damage and the biological effects of NPs.

Rab GTPases: Central Coordinators of Membrane Trafficking in Cancer.

Tumor progression involves invasion, migration, metabolism, autophagy, exosome secretion, and drug resistance. Cargos transported by membrane vesicle trafficking underlie all of these processes. Rab GTPases, which, through coordinated and dynamic intracellular membrane trafficking alongside cytoskeletal pathways, determine the maintenance of homeostasis and a series of cellular functions. The mechanism of vesicle movement regulated by Rab GTPases plays essential roles in cancers. Therefore, targeting Rab GTPases to adjust membrane trafficking has the potential to become a novel way to adjust cancer treatment. In this review, we describe the characteristics of Rab GTPases; in particular, we discuss the role of their activation in the regulation of membrane transport and provide examples of Rab GTPases regulating membrane transport in tumor progression. Finally, we discuss the clinical implications and the potential as a cancer therapeutic target of Rab GTPases.

Pharmacokinetics of Benznidazole in Experimental Chronic Chagas Disease Using the Swiss Mouse-Berenice-78 Trypanosoma cruzi Strain Model.

Chronic Chagas disease might have an impact on benznidazole pharmacokinetics with potential alterations in the therapeutic dosing regimen. This study aims to investigate the influence of chronic Trypanosoma cruzi infection on the pharmacokinetics and biodistribution of benznidazole in mice. Healthy (n = 40) and chronically T. cruzi (Berenice-78 strain)-infected (n = 40) Swiss female 10-month-old mice received a single oral dose of 100 mg/kg of body weight of benznidazole. Serial blood, heart, colon, and brain samples were collected up to 12 h after benznidazole administration. The serum and tissue samples were analyzed using a high-performance liquid chromatography instrument coupled to a diode array detector. Chronic infection by T. cruzi increased the values of the pharmacokinetic parameters absorption rate constant (Ka ) (3.92 versus 1.82 h-1), apparent volume of distribution (V/F) (0.089 versus 0.036 liters), and apparent clearance (CL/F) (0.030 versus 0.011 liters/h) and reduced the values of the time to the maximum concentration of drug in serum (Tmax) (0.67 versus 1.17 h) and absorption half-life (t1/2a ) (0.18 versus 0.38 h). Tissue exposure (area under the concentration-versus-time curve from 0 h to time t for tissue [AUC0-t,tissue]) was longer and higher in the colon (8.15 versus 21.21 μg · h/g) and heart (5.72 versus 13.58 μg · h/g) of chronically infected mice. Chronic infection also increased the benznidazole tissue penetration ratios (AUC0-t,tissue/AUC0-t,serum ratios) of brain, colon, and heart by 1.6-, 3.25-, and 3-fold, respectively. The experimental chronic Chagas disease inflammation-mediated changes in the regulation of membrane transporters probably influence the benznidazole pharmacokinetics and the extent of benznidazole exposure in tissues. These results advise for potential alterations in benznidazole pharmacokinetics in chronic Chagas disease patients with possibilities of changes in the standard dosing regimen.

Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

Prokaryotic expression, protein purification and functional verification of human homotypic fusion and vacuole protein sorting complex subunit

Homotypic fusion and vacuole protein sorting(HOPS) is a protein complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39, VPS41 and regulates membrane transport in vivo through membrane fusion mechanisms. The evidence suggests that HOPS complex as a fusion factor, facilitates autophagosome-lysosome fusion. To determine whether the HOPS complex directly interacts with the autophagic SNARE protein STX17 in vitro, the coding sequence of the six genes were amplified from the existing plasmids by PCR, and then ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA. After identification through colony PCR and DNA sequencing, 6 recombinant plasmids were constructed and transferred into Escherichia coli BL21 (DE3). The recombinant proteins were purified by glutathione sepharose 4B and nickel column. We used the tobacco etch virus protease to cut off the GST-tag or His-NusA-tag, to obtain HA-VPS11 protein of about 105 kDa, Flag-VPS16 protein of about 97 kDa, HA-VPS18 protein of about 108 kDa, Flag-VPS33 protein of about 70 kDa, HA-VPS39 protein of about 97 kDa, and Flag-VPS41 protein of about 98 kDa. The function of the purified proteins was verified by in vitro glutathione S-transferases pull-down assay, confirming that autophagic SNARE protein STX17 interacted directly with HOPS components. Our findings provide experimental basis to further study the function and mechanism of HOPS complex in the process of autophagosome-lysosome fusion.

Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

A role for Rab11 in the homeostasis of the endosome-lysosomal pathway

The small GTPases Rab11a and 11b are key regulators of membrane transport, localised to the recycling endosomes and also early endosomes. The function of Rab11 within the recycling pathway has been well defined, however, the role of Rab11 at the early endosomes remains poorly characterised. Here, we have generated HeLa cell lines devoid of either Rab11a or Rab11b using CRISPR/Cas9 to functionally dissect the roles of these two Rab11 family members in recycling and in the endosomal-lysosomal system. Both Rab11a and Rab11b contribute to the dynamics of tubulation arising from recycling endosomes whereas Rab11a has the major role in recycling of transferrin receptor. Deletion of either Rab11a or Rab11b resulted in the formation of enlarged early endosomes and perturbation of the endosomal-lysosomal pathway. Strikingly, Rab11a knock-out cells showed an increased density of functional late endosomes/lysosomes as well as lysotracker-positive organelles which were primarily concentrated in a perinuclear location, indicating that the homeostasis of the endosome/lysosome pathway had been perturbed. Moreover, in Rab11a knockout cells there was a functional defect in the intracellular recycling of the cation-independent mannose 6-phosphate receptor (CI-M6PR) between the late endosomes and the TGN, a defect associated with enhanced degradation of CI-M6PR. Expression of wild-type Rab11a in Rab11a knockout cells rescued the late endosome/lysosome phenotype. Overall, these results indicate that Rab11a and Rab11b have overlapping and distinct functions and that Rab11a, unexpectedly, plays a central role in the homeostasis of endosomal-lysosomal biogenesis.

Multiple Roles of Rab GTPases at the Golgi.

The Golgi apparatus is a central sorting station in the cell. It receives newly synthesized molecules from the endoplasmic reticulum and directs them to different subcellular destinations, such as the plasma membrane or the endocytic pathway. Importantly, in the last few years, it has emerged that the maintenance of Golgi structure is connected to the proper regulation of membrane trafficking. Rab proteins are small GTPases that are considered to be the master regulators of the intracellular membrane trafficking. Several of the over 60 human Rabs are involved in the regulation of transport pathways at the Golgi as well as in the maintenance of its architecture. This chapter will summarize the different roles of Rab GTPases at the Golgi, both as regulators of membrane transport, scaffold, and tethering proteins and in preserving the structure and function of this organelle.

AMP-Activated Protein Kinase (AMPK)-Dependent Regulation of Renal Transport.

AMP-activated kinase (AMPK) is a serine/threonine kinase that is expressed in most cells and activated by a high cellular AMP/ATP ratio (indicating energy deficiency) or by Ca2+. In general, AMPK turns on energy-generating pathways (e.g., glucose uptake, glycolysis, fatty acid oxidation) and stops energy-consuming processes (e.g., lipogenesis, glycogenesis), thereby helping cells survive low energy states. The functional element of the kidney, the nephron, consists of the glomerulus, where the primary urine is filtered, and the proximal tubule, Henle’s loop, the distal tubule, and the collecting duct. In the tubular system of the kidney, the composition of primary urine is modified by the reabsorption and secretion of ions and molecules to yield final excreted urine. The underlying membrane transport processes are mainly energy-consuming (active transport) and in some cases passive. Since active transport accounts for a large part of the cell’s ATP demands, it is an important target for AMPK. Here, we review the AMPK-dependent regulation of membrane transport along nephron segments and discuss physiological and pathophysiological implications.

Vitamin E intestinal absorption: Regulation of membrane transport across the enterocyte.

Vitamin E is an essential molecule for our development and health. It has long been thought that it was absorbed and transported through cellular membranes by a passive diffusion process. However, data obtained during the past 15 years showed that its absorption is actually mediated, at least in part, by cholesterol membrane transporters including the scavenger receptor class B type I (SR-BI), CD36 molecule (CD36), NPC1-like transporter 1 (NPC1L1), and ATP-binding cassettes A1 and G1 (ABCA1 and ABCG1). This review focuses on the absorption process of vitamin E across the enterocyte. A special attention is given to the regulation of this process, including the possible competition with other fat-soluble micronutrients, and the modulation of transporter expressions. Overall, recent results noticeably increased the comprehension of vitamin E intestinal transport, but additional investigations are still required to fully appreciate the mechanisms governing vitamin E bioavailability. © 2018 IUBMB Life, 71(4):416-423, 2019.

Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution

The scaffolding protein PDZK1 has been associated with the regulation of membrane transporters. It contains four conserved PDZ domains, which typically recognize a 3-5-residue long motif at the C terminus of the binding partner. The atomic structures of the individual domains are available but their spatial arrangement in the full-length context influencing the binding properties remained elusive. Here we report a systematic study of full-length PDZK1 and deletion constructs using small-angle X-ray scattering, complemented with biochemical and functional studies on PDZK1 binding to known membrane protein partners. A hybrid modeling approach utilizing multiple scattering datasets yielded a well-defined, extended, asymmetric L-shaped domain organization of PDZK1 in contrast to a flexible "beads-on-string" model predicted by bioinformatics analysis. The linker regions of PDZK1 appear to play a central role in the arrangement of the four domains underlying the importance of studying scaffolding proteins in their full-length context.

Modeling, dynamics and phosphoinositide binding of the pleckstrin homology domain of two novel PLCs: η1 and η2

PH domains mediate interactions involved in cell signaling, intracellular membrane transport regulation and cytoskeleton organization. Some PH domains bind phosphoinositides with different affinity and specificity. The two novel PLCη (1 and 2) possess an N-terminal PH domain (PHη1 and PHη2 respectively) that has been implicated in membrane association and induction of PLC activity. Understanding of the structure and dynamics is crucial for future modulation of lipid-protein interactions in PHη1, PHη2 and other PH domains. Therefore, the three-dimensional structure of PHη1 and PHη2 was modeled using ITASSER and phosphoinositides (IP3 and IP4) were docked in the inferred binding site using HADDOCK server. Molecular Dynamics simulations of unliganded and phosphoinositide bound PHη1 and PHη2 were performed using AMBER14 to study the mechanism of interaction, and conformational dynamics in response to phosphoinositide binding. The binding affinity was predicted using Kdeep server. The models of PHη1 and PHη2 had a conserved structural core consisting of seven β-strands and a C-terminal α-helix as seen in other PH domains. Sequence/structure analysis showed that phosphoinositide ligands bind PHη1 and PHη2 at the canonical binding site. Phosphoinositide binding induced movement of positively charged side chains towards the ligand, changes in the secondary structure especially at the β5-β6 loop and allosteric changes at the interface of β1-β2 and β5-β6 loops. Dynamics studies showed that the size of the binding site and differential affinity for IP3/IP4 binding is coordinated by the number, length, flexibility, secondary structure and allosteric interactions of the loops surrounding the phosphoinositide binding site.

Farnesoid X receptor (FXR) activation induces up‐regulation of multidrug and toxin extrusion proteins (MATEs) in human renal proximal tubular cells

Farnesoid X receptor (FXR) is a nuclear transcriptional factor which highly expressed in the liver, intestine, and kidney. Its plays an essential role in regulation of membrane transporters to eliminate endogenous and xenobiotic compounds. The previous study has shown that endogenous ligand, chenodeoxycholic acid (CDCA) induced by cholestasis down‐regulated hepatic organic cation transporter 1 (OCT1) which plays a critical role in the hepatic secretion of organic cations. It is interesting whether FXR activation regulates multidrug and toxin extrusion protein1 (MATE1) and kidney specific MATE2‐K that play an essential role in the renal cationic drugs efflux into the lumen. The present study showed that treatment with 20 μM CDCA, an endogenous ligand of FXR, for 24 hours stimulated MATE1‐ and MATE2‐K‐mediated 3H‐MPP+ transport in human renal proximal tubular cell line (RPTEC/TERT1 cells). The effect of CDCA was abolished by co‐treatment with a FXR antagonist, Z‐guggulsterone. These data indicated that the stimulatory effect of CDCA on MATEs required FXR‐dependent mechanism. Furthermore, treatment RPTEC/TERT1 cells with CDCA at both physiological level (20 mM) and pathophysiological level (cholestasis) (80 μM) increased mRNA expression of MATE1 and MATE2‐K. Taken together, FXR activation up‐regulates mRNA expression of MATE1 and MATE2‐K resulting in increased cation transports in the proximal tubular cells in both physiological and pathological conditions.Support or Funding InformationThe study was supported by the grant from Mahidol UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Stress-Induced O-GlcNAc Protein Modification in the Regulation of Membrane Transport.

O-linked N-acetylglucosamine (O-GlcNAc) is a posttranslational modification that is increasingly recognized as a signal transduction mechanism. Unlike other glycans, O-GlcNAc is a highly dynamic and reversible process that involves the addition and removal of a single N-acetylglucosamine molecule to Ser/Thr residues of proteins. UDP-GlcNAc—the direct substrate for O-GlcNAc modification—is controlled by the rate of cellular metabolism, and thus O-GlcNAc is dependent on substrate availability. Serving as a feedback mechanism, O-GlcNAc influences the regulation of insulin signaling and glucose transport. Besides nutrient sensing, O-GlcNAc was also implicated in the regulation of various physiological and pathophysiological processes. Due to improvements of mass spectrometry techniques, more than one thousand proteins were detected to carry the O-GlcNAc moiety; many of them are known to participate in the regulation of metabolites, ions, or protein transport across biological membranes. Recent studies also indicated that O-GlcNAc is involved in stress adaptation; overwhelming evidences suggest that O-GlcNAc levels increase upon stress. O-GlcNAc elevation is generally considered to be beneficial during stress, although the exact nature of its protective effect is not understood. In this review, we summarize the current data regarding the oxidative stress-related changes of O-GlcNAc levels and discuss the implications related to membrane trafficking.

376 An RNAi Screen for Factors that Regulate Membrane Transport in Polarized Epithelia

376 An RNAi Screen for Factors that Regulate Membrane Transport in Polarized Epithelia

Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment.

Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL-CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses.

Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic.

Glycosphingolipids (GSLs) are predominantly found in the outer leaflet of the plasma membrane, where they play a role in important processes such as cell adhesion, migration and signaling. However, by which mechanisms GSLs regulate these processes remains elusive. In this study, we therefore took advantage of the fact that some GSLs also serve as receptors for certain protein toxins, which rely on receptor binding for internalization and intoxication. Here, we demonstrate that Shiga and cholera toxins, which both possess multivalent GSL-binding capacity, induce dissociation of the cytosolic cPLA2α-AnxA1 complex in HeLa and HMEC-1 cells. The dissociation is mediated through an increase in cytosolic calcium levels and activation of the tyrosine kinase Syk. Ricin, a protein toxin that does not cross-link surface molecules, has no effect on the same complex. Importantly, we find that antibody-mediated cross-linking of Gb3 and GM1, the GSL receptors for Shiga and cholera toxin, respectively, also induces dissociation. These data demonstrate that cross-linking of GSLs at the plasma membrane mediates the intracellular signaling events resulting in dissociation of the complex. After dissociation, cPLA2α and AnxA1 are translocated to intracellular membranes where they are known to function in regulating membrane transport processes. In conclusion, we have characterized a novel mechanism for cell surface-induced initiation of intracellular signaling and transport events.

Abstract 4415: Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs

Abstract Cisplatin and oxaliplatin are among the most widely used anticancer drugs in both children and adults. The clinical use of these agents is associated with dose-limiting damage to kidneys (nephrotoxicity) and peripheral nerves (neurotoxicity). Mechanistic understanding of the underlying etiology remains incomplete and preventive strategies are currently unavailable. We previously reported that cisplatin nephrotoxicity (Filipski et al, CPT 2009) and oxaliplatin neurotoxicity (Sprowl et al, PNAS 2013) are dependent on organic cation transporter-mediated uptake mechanisms and can be reduced by genetic or pharmacological inhibition of OCT2. With a goal to identify small molecule OCT2 inhibitors, that could be used to prevent these toxicities, we carried out a high-throughput chemical library screen. Multiple tyrosine kinase inhibitors (TKIs), including dasatinib and vandetanib, were identified as potent OCT2 inhibitors. Further studies showed that TKIs inhibit OCT2 through a non-competitive mechanism raising the possibility that tyrosine phosphorylation by an unknown TKI-sensitive protein kinase(s) may be essential for OCT2 function. Using in vitro cell culture models and proteomics, site-directed mutagenesis and functional assays we found that OCT2 is indeed tyrosine phosphorylated and this phosphorylation is essential for OCT2 function and sensitive to inhibition by TKIs. A subsequent RNAi kinase library screen identified the Src-family member YES1 as the OCT2-phosphorylating kinase. This was further supported by experiments where a TKI-resistant YES1 mutant was able to rescue OCT2 inhibition by dasatinib. Non-TKI inhibitors of YES1 were also found to suppress OCT2 function. Importantly, tyrosine phosphorylation of OCT2 was observed in vivo, in murine kidneys and dorsal root ganglions, major sites of OCT2 expression and toxicities of platinum-based drugs. siRNA mediated in vivo knockdown of YES1 reduced OCT2 phosphorylation and suppressed its function in the kidneys. Treatment with the YES1 targeting TKI, Dasatinib, suppressed OCT2 phosphorylation in dorsal root ganglions, reduced oxaliplatin uptake and significantly prevented oxaliplatin induced neurotoxicity in mice models. These findings have not only provided new mechanistic insights into the regulation of membrane transporters; but have also provided a rationale for the translational development of new targeted interventions using TKI pulse-exposure to mitigate the debilitating side effects associated with platinum-based chemotherapeutics. Note: This abstract was not presented at the meeting. Citation Format: Navjotsingh Pabla, Jason A. Sprowl, Su Sien Ong, Alice A. Gibson, Guoqing Du, Wenwei Lin, Shuiying Hu, Lie Li, Taosheng Chen, Alex Sparreboom. Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4415. doi:10.1158/1538-7445.AM2015-4415