Gonzalo L. Vilas

Metabolon disruption: a mechanism that regulates bicarbonate transport

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3−. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl−/HCO3− exchanger with a suggested role in pancreatic HCO3− secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl−/HCO3− exchange in cells coexpressing SLC26A6 and AT1a‐AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog

Myristoylation is the attachment of the 14‐carbon fatty acid myristate to the N‐terminal glycine residue of proteins. Typically a co‐translational modification, myristoylation of proapoptotic cysteinyl‐aspartyl proteases (caspase)‐cleaved Bid and PAK2 was also shown to occur post‐translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1–3 months). Consequently, we developed a nonradioactive detection methodology in which a bio‐orthogonal azidomyristate analog is specifically incorporated co‐ or post‐translationally into proteins at N‐terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million‐fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase‐cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post‐translationally myristoylatable proteins (PKCε, CD‐IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post‐translational myristoyl‐ation in apoptotic Jurkat T cells. This suggests that post‐translational myristoylation of caspase‐cleaved proteins represents a novel mechanism widely used to regulate cell death.—Martin, D. D. O., Vilas, G. L., Prescher, J. A., Rajaiah, G., Falck, J. R., Bertozzi, C. R., Berthiaume, L. G. Rapid detection, discovery, and identification of post‐translationally myristoylated proteins during apoptosis using a bio‐orthogonal azidomyr‐istate analog. FASEB J. 22, 797–806 (2008)

Palmitoylation of ATP-Binding Cassette Transporter A1 Is Essential for Its Trafficking and Function

ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.

Transmembrane water-flux through SLC4A11: a route defective in genetic corneal diseases

Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11−/− mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.

A Biochemical Framework for SLC4A11, the Plasma Membrane Protein Defective in Corneal Dystrophies

Mutations in the SLC4A11 protein, reported as a sodium-coup-led borate transporter of the human plasma membrane, are responsible for three corneal dystrophies (CD): congenital hereditary endothelial dystrophy type 2, Harboyan syndrome, and late-onset Fuchs CD. To develop a rational basis to understand these diseases, whose point mutations are found throughout the SLC4A11 sequence, we analyzed the protein biochemically. Hydropathy analysis and an existing topology model for SLC4A1 (AE1), a bicarbonate transporter with the lowest evolutionary sequence divergence from SLC4A11, formed the basis to propose an SLC4A11 topology model. Immunofluorescence studies revealed the cytosolic orientation of N- and C-termini of SLC4A11. Limited trypsinolysis of SLC4A11 partially mapped the folding of the membrane and cytoplasmic domains of the protein. The binding of SLC4A11 to a stilbenedisulfonate inhibitor resin (SITS-Affi-Gel) was prevented by preincubation with H(2)DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A11 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H(2)DIDS] required for resin displacement and the fraction of protein binding H(2)DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of HEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 °C.

Oligomerization of SLC4A11 protein and the severity of FECD and CHED2 corneal dystrophies caused by SLC4A11 mutations

Mutations in the SLC4A11 gene, which encodes a plasma membrane borate transporter, cause recessive congenital hereditary endothelial corneal dystrophy type 2 (CHED2), corneal dystrophy and perceptive deafness (Harboyan syndrome), and dominant late‐onset Fuchs endothelial corneal dystrophy (FECD). We analyzed missense SLC4A11 mutations identified in FECD and CHED2 patients and expressed in transfected HEK 293 cells. Chemical cross‐linking and migration in nondenaturing gels showed that SLC4A11 exists as a dimer. Furthermore, co‐immunoprecipitation of epitope‐tagged proteins revealed heteromeric interactions between wild‐type (WT) and mutant SLC4A11 proteins. When expressed alone, FECD‐ and CHED2‐causing mutant SLC4A11 proteins are primarily retained intracellularly. Co‐expression with WT SLC4A11 partially rescued the cell surface trafficking of CHED2 mutants, but not FECD mutants. CHED2 alleles of SLC4A11 did not affect cell surface processing of WT SLC4A11. In contrast, FECD mutants reduced WT cell surface processing efficiency, consistent with dominant inheritance of FECD. The reduction in movement of WT protein to the cell surface caused by FECD SLC4A11 helps to explain the dominant inheritance of this disorder. Similarly, the failure of CHED2 mutant SLC4A11 to affect the processing of WT protein, explains the lack of symptoms found in CHED2 carriers and the recessive inheritance of the disorder. Hum Mutat 33:419–428, 2012.

Characterization of an epilepsy-associated variant of the human Cl−/HCO3− exchanger AE3

Anion exchanger 3 (AE3), expressed in the brain, heart, and retina, extrudes intracellular HCO(3)(-) in exchange for extracellular Cl(-). The SLC4A3 gene encodes two variants of AE3, brain or full-length AE3 (AE3(fl)) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. The AE3-A867D allele in humans has been associated with the development of IGE (IGE), which accounts for approximately 30% of all epilepsies. To examine the molecular basis for the association of the A867D allele with IGE, we characterized wild-type (WT) and AE3(fl)-A867D in transfected human embryonic kidney (HEK)-293 cells. AE3(fl)-A867D had significantly reduced transport activity relative to WT (54 +/- 4%, P < 0.01). Differences in expression levels or the degree of protein trafficking to the plasma membrane did not account for the defect of AE3(fl)-A867D. Treatment with 8-bromo-cAMP (8-Br-cAMP) increased Cl(-)/HCO(3)(-) exchange activity of WT and AE3(fl)-A867D to a similar degree, which was abolished by preincubation with the protein kinase A (PKA)-specific inhibitor H89. This indicates that PKA regulates WT and AE3(fl)-A867D Cl(-)/HCO(3)(-) exchange activity. No difference in Cl(-)/HCO(3)(-) exchange activity was found between cultures of mixed populations of neonatal hippocampal cells from WT and slc4a3(-/-) mice. We conclude that the A867D allele is a functional (catalytic) mutant of AE3 and that the decreased activity of AE3(fl)-A867D may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexcitability and the generation of seizures.

Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1.

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney α-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in α-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.

Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.

Adenosine A1 receptor activation modulates human equilibrative nucleoside transporter 1 (hENT1) activity via PKC-mediated phosphorylation of serine-281

Equilibrative nucleoside transporter subtype 1 (ENT1) is critical for the regulation of the biological activities of endogenous nucleosides such as adenosine, and for the cellular uptake of chemotherapeutic nucleoside analogs. Previous studies have implicated protein kinase C (PKC) in the regulation of ENT1 expression/function. It was hypothesized that hENT1 activity at the plasma membrane is regulated by PKC-mediated phosphorylation of Ser281. WT (wild-type)-hENT1 or S281A-hENT1 was stably transfected into a PK15 cell variant that is deficient in nucleoside transport. Using [(3)H]nitrobenzylthioinosine (NBMPR) binding and [(3)H]2-chloroadenosine uptake analyses, it was determined that S281A-hENT1 exhibited functional characteristics similar to WT-hENT1. Direct activation of PKC with PMA or indirect activation with the adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) led to significant increases in [(3)H]NBMPR binding and [(3)H]2-chloroadenosine uptake in WT-hENT1 transfected cells. The PKC inhibitor Gö6983 blocked these effects of both PMA and CCPA, and the CCPA-mediated increase was also blocked by the A1 adenosine receptor antagonist DPCPX. In contrast, neither PMA nor CCPA affected [(3)H]NBMPR binding or [(3)H]2-chloroadenosine uptake in cells transfected with S281A-hENT1. shRNAi silencing studies implicated PKCδ in this regulation of hENT1 activity. Immunocytochemical analysis and cell surface biotinylation assays showed that activation of PKC with PMA, but not CCPA, led to a significant increase in the plasma membrane localization of hENT1. These data suggest that phosphorylation of hENT1 by PKC has effects on both the function and subcellular trafficking of hENT1. This signaling pathway represents a feedback loop whereby adenosine receptor signaling can lead to increased adenosine reuptake into cells via hENT1.