Hemanta K. Sarkar

Molecular characterization and in situ localization of a mouse retinal taurine transporter

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high‐affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na+‐ and Cl−‐dependent [3H]taurine transport activity in Xenopus laevis oocytes that saturated with an average Km of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl− was varied, a single saturation process with an average apparent equilibrium constant (KCl−) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na+ was varied (apparent equilibrium constant, KNa+∼54.8 mM). Analyses of the Na+‐ and Cl−‐concentration dependence data suggest that at least two Na+ and one Cl− are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β‐alanine > l‐diaminopropionic acid > guanidinoethane sulfonate > β‐guanidinopropionic acid > chloroquine > γ‐aminobutyric acid > 3‐amino‐1‐propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α‐phorbol 12,13‐didecanoate but was inhibited significantly by the active phorbol ester phorbol 12‐myristate 13‐acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.

The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain.

To identify interacting proteins of survival motor neuron (SMN) in neurons, a fetal human brain cDNA library was screened using the yeast two‐hybrid system. One identified group of SMN interacting clones encoded the DNA transactivator FUSE binding protein (FBP). FBP overexpressed in HEK293 cells or endogenously expressed in fetal and adult mouse brain bound specifically in vitro to recombinant SMN protein. Furthermore, an anti‐FBP antibody specifically co‐immunoprecipitated SMN when both proteins were overexpressed in HEK293 cells. These results demonstrate that FBP is a novel interacting partner of SMN and suggests a possible role for SMN in neuronal gene expression.

Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes

The goal was to investigate the role of protein kinases in modulating taurine transporter activity in Xenopus laevis oocytes expressing the mouse retinal Na+/C−/taurine transporter. The currents generated by the taurine transporter were studied with a two‐electrode voltage clamp and we recorded the maximal current (I max ), presteady‐state charge transfer Q, and membrane capacitance C m . 8‐BR‐cAMP, a membrane‐permeable activator of the cAMP‐dependent protein kinase (PKA), decreased I max (41%), Q (41%) and C m (10%). Similarly, 1 μM sn‐1,2‐dioctanoylglycerol (DOG), an activator of the Ca2+ I diacylglycerol‐dependent protein kinase (PKC), decreased I max (56%), Q (37%), and C m (9%). Calyculin A, a specific inhibitor of protein phosphatases 1 and 2A, also produced effects similar to those of 8‐Br‐cAMP and DOG, and decreased I max (64%), Q (38%), and C m (10%). We conclude that the taurine transporter is regulated by activators of PKA and PKC, and regulation occurs largely by changes in the number of transporters in the plasma membrane.

Cyclosporin A inhibits creatine uptake by altering surface expression of the creatine transporter.

The immunosuppressive drug cyclosporin A (CsA) inhibited the hCRT-1 cDNA-induced creatine uptake inXenopus oocytes and the endogenous creatine uptake in cultured C2C12 muscle cells in a dose- and time-dependent manner. FK506, another potent immunosuppressant, was unable to mimic the effect of CsA suggesting that the inhibitory effect of CsA was specific. To delineate the mechanism underlying, we investigated the effect of CsA on theK m and V max of creatine transport and also on the cell surface distribution of the creatine transporter. Although CsA treatment did not affect theK m (20–24 μm) for creatine, it significantly decreased the V max of creatine uptake in both oocytes and muscle cells. CsA treatment reduced the cell surface expression level of the creatine transporter in the muscle cells by ∼60% without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in creatine uptake. Taken together, our results suggest that CsA inhibited creatine uptake by altering the surface abundance of the creatine transporter. We propose that CsA impairs the targeting of the creatine transporter by inhibiting the function of an associated cyclophilin, resulting in an apparent loss in surface expression of the creatine transporter. Our results also suggest that prolonged exposure to CsA may result in chronically creatine-depleted muscle, which may be a cause for the development of CsA-associated clinical myopathies in organ transplant patients.

Molecular characterization of taurine transport in bovine aortic endothelial cells.

Cultured bovine aortic endothelial (BAE) cells expressed a Na(+)/Cl(-)-dependent taurine uptake activity that saturated with an apparent K(0.5) of approximately 4.9 microM for taurine and was inhibited by beta-alanine, guanidinoethane sulfonate, and homotaurine. We isolated a taurine transporter clone from a BAE cell cDNA library that revealed >91% sequence identity at the amino acid level to the previously cloned high-affinity mammalian taurine transporters. The biochemical and pharmacological properties of the bovine taurine transporter cDNA expressed in Xenopus oocyte was similar to those of the high-affinity taurine transporter. Surprisingly, F(-) blocked taurine uptake in BAE cells with an IC(50) of approximately 17.5 mM. The endogenous taurine uptake was also inhibited by the protein kinase C activator phorbol 12-myristate 13-acetate, but not by its inactive analog, 4 alpha-phorbol 12,13-didecanoate. The endogenous uptake was stimulated, however, by hypertonic stress and the increase was due to an increase in the V(max) of taurine uptake. Our results provide the first description of a molecular mechanism that may be responsible for maintaining the intracellular taurine content in the endothelial cells.

Orientation of d-Tubocurarine in the Muscle Nicotinic Acetylcholine Receptor-binding Site

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist,d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR γ-subunit. Because data for mutations at residue γTyr117 were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of γTyr117 with both the 2-N and the 13′-positions of dTC. Examination of a dTC analog with a negative charge at the 13′-position failed to reveal electrostatic interaction with charged side-chain substitutions at γ117, but the effects of side-chain substitutions remained consistent with proximity of Tyr117 to the cationic 2-N of dTC. The apparent interaction of γTyr117with the 13′-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-π electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to γTyr117. This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.

Inhibition of taurine transport by cyclosporin A is due to altered surface abundance of the taurine transporter and is reversible.

We have investigated the underlying mechanism of the CsA-induced inhibition of taurine transport using a cell line permanently expressing the mouse taurine transporter (mTauT) tagged with the green-fluorescence protein (GFP). CsA inhibited the uptake activity of the expressed mTauT.GFP fusion protein in both dose and time dependent manner. Surface biotinylation assay revealed that the CsA-treatment reduced the relative surface abundance of the taurine transporter without affecting its total expression level. CsA treatment reduced both the taurine uptake and the relative surface abundance of the transporter by similar magnitudes. Conversely, when the CsA was washed off, both the uptake and the relative surface abundance of the transporter recovered fully to the control level. Remarkably, the recovery process was insensitive to the protein synthesis inhibitor cycloheximide. These results suggested that the CsA inhibited taurine transport by altering the surface abundance, possibly by internalization of the expressed taurine transporters.

Taurine and Guanidinoethanesulfonic Acid (GES) Differentially Affects the Expression and Phosphorylation of Cellular Proteins in C6 Glial Cells

Effects of taurine and guanidinoethanesulfonic acid (GES), a taurine transport inhibitor, on the expression and phosphorylation of cellular proteins in C6 glial cells were examined using two-dimensional (2-D) gel electrophoresis and 2-D immunoblots. 2-D gels stained with Coomassie Blue or SYPRO Ruby showed differential distribution patterns of cellular proteins in the control, taurine-supplemented and GES-supplemented cells. 2-D immunoblot analysis using the anti-phosphotyrosine antibody recognized only few immuno-reactive proteins in all three samples, and their distribution patterns were different. On the other hand, 2-D immunoblot analysis using the anti-phosphothreonine antibody recognized many immuno-reactive proteins with distinctly different distribution patterns in the control, taurine-supplemented and GES-supplemented cells. In GES-supplemented cells, the relative number of the anti-phosphotyrosine immuno-reactive protein spots increased modestly, whereas the relative number of the anti-phosphothreonine immuno-reactive protein spots decreased markedly than those in the control and taurine-supplemented cells.

Taurine Uptake and Release by the Pancreatic β-Cells

The β-amino acid taurine is abundantly present in its free form in various mammalian cells and tissues1, including the islet of Langerhans2. Although the exact physiological role of taurine in the pancreas is not understood, a number of studies suggest a possible role of taurine in glucose metabolism. Taurine has been shown to attenuate streptozotocin-induced diabetes in animals3, to increase the stimulatory effects of conventional secretagogues in cultured fetal β-cells4, and to protect against β-cells injury by NO and IL-1 beta5. Most interestingly, hyposmotic challenge that induces taurine release in many cells1 has been shown to increase secretion of insulin6,7.