Assembly of Taurine Transporter (Slc6a6) with Na<sup>+</sup>–H<sup>+</sup> Exchanger Regulatory Factor 1 (Slc9a3r1) Improves GABA Transport Activity by Increasing the Maximum Transport Velocity

A cDNA clone highly related to the rat brain taurine transporter has been isolated from a human placental cDNA library. Transfection of this cDNA into HeLa cells results in a marked elevation of taurine transport activity. The activity of the cDNA-induced transporter is dependent on the presence of Na+ as well as Cl-. The Na+/Cl-/taurine stoichiometry for the cloned transporter is 2:1:1. The transporter is specific for taurine and other beta-amino acids, including beta-alanine, and exhibits high affinity for taurine (Michaelis-Menten constant approximately 6 microM). The clone consists of a coding region 1863 bp long (including the termination codon), flanked by a 376 bp-long 5′ non-coding region and a 625 bp-long 3′ non-coding region. The nucleotide sequence of the coding region predicts a 620-amino acid protein with a calculated M(r) of 69,853. Northern-blot analysis of poly(A)+ RNA from several human tissues indicates a complex expression pattern differing across tissues. The principal transcript, 6.9 kb in size, is expressed abundantly in placenta and skeletal muscle, at intermediate levels in heart, brain, lung, kidney and pancreas and at low levels in liver. Cultured human cell lines derived from placenta (JAR and BeWo), intestine (HT-29), cervix (HeLa) and retinal pigment epithelium (HRPE), which are known to possess Na(+)- and Cl(-)-coupled taurine transport activity, also contain the 6.9 kb transcript. Somatic cell hybrid and in situ hybridization studies indicate that the cloned taurine transporter is localized to human chromosome 3 p24-->p26.

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Cloning and Characterization of a pH-Sensing Regulatory Factor That Modulates Transport Activity of the Human H+/Peptide Cotransporter, PEPT1

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1997.7129 ◽

1997 ◽

Vol 237 (3) ◽

pp. 577-582 ◽

Cited By ~ 23

Author(s):

Hideyuki Saito ◽

Hideyuki Motohashi ◽

Mayumi Mukai ◽

Ken-ichi Inui

Keyword(s):

Ph Sensing ◽

Transport Activity ◽

Regulatory Factor

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Kinetics of 2-deoxyglucose transport in skeletal muscle: effects of insulin and contractions

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.1.c30 ◽

1995 ◽

Vol 268 (1) ◽

pp. C30-C35 ◽

Cited By ~ 53

Author(s):

P. Hansen ◽

E. Gulve ◽

J. Gao ◽

J. Schluter ◽

M. Mueckler ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucose Transport ◽

Skeletal Muscles ◽

Contractile Activity ◽

Transport Activity ◽

Muscle Type ◽

Glut 1 ◽

Transport Velocity ◽

Kinetics Of

There is some controversy regarding whether insulin or contractile activity alters the affinity of skeletal muscle glucose transporters for glucose and its analogues. The effects of insulin and contractions on the kinetics of glucose transport were therefore reexamined in isolated rat skeletal muscles. Concentration-dependent rates of 2-deoxyglucose (2-DG) transport were measured in the absence or presence of insulin (2 mU/ml) in the epitrochlearis and split soleus muscles. The apparent half-maximal saturating substrate concentration (Km) for basal 2-DG transport (approximately 12 mM) was similar for the split soleus and epitrochlearis, and the apparent Km was not changed by insulin in either muscle type. The presence of 2 mU/ml insulin increased the maximal transport velocity (Vmax) approximately fourfold in the epitrochlearis and approximately eightfold in the split soleus. In the epitrochlearis, in vitro muscle contractions also resulted in an approximately fourfold increases in Vmax with no change in apparent Km. The combined effects of insulin and contractions on Vmax were completely additive, but the apparent Km was not different from insulin alone. The apparent Km values for basal and insulin-stimulated glucose transport were further characterized in the epitrochlearis isolated from transgenic mice overexpressing the GLUT-1 isoform in the sarcolemma and their nontransgenic littermates. The apparent Km for basal 2-DG transport in the transgenic muscle (9 mM) was not significantly different from the apparent Km for insulin-stimulated transport in the control muscle (10 mM). The present study provides evidence that insulin and contractions, either alone or in combination, increase glucose transport activity in skeletal muscle by increasing Vmax, with no significant change in Km. Our results also suggest that, in intact skeletal muscle, the Km for basal glucose transport (a process mediated primarily by GLUT-1) is similar to the Km values for stimulated transport, mediated predominantly by GLUT-4.

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Ser-322 Is a Critical Site for PKC Regulation of the MDCKCell Taurine Transporter (pNCT)

Journal of the American Society of Nephrology ◽

10.1681/asn.v1091874 ◽

1999 ◽

Vol 10 (9) ◽

pp. 1874-1879

Author(s):

XIAOBIN HAN ◽

ANDREA M. BUDREAU ◽

RUSSELL W. CHESNEY

Keyword(s):

Expression System ◽

Site Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

Taurine Transporter ◽

Taurine Transport ◽

Transporter Protein ◽

Oocyte Expression System ◽

Pkc Activation ◽

Critical Site

Abstract. Previous studies have shown that the Madin—Darby canine kidney cell taurine transporter (pNCT) is downregulated by protein kinase C (PKC) activation. In this study, it is hypothesized that the highly conserved serine-322 (Ser-322) located in the fourth intracellular segment (S4) may play an important role in the function of taurine transporter, which is modulated by PKC phosphorylation. It is demonstrated that Ser-322 is the critical site of PKC phosphorylation, as determined by site-directed mutagenesis. When Ser-322 of pNCT was changed to alanine (S322A) and this mutant was evaluated in an oocyte expression system, taurine transport activity increased threefold compared with control (wild-type pNCT). Activation of PKC by the active phorbol ester 12-myristate 13-acetate did not influence taurine transport by mutant S322A. Kinetic analysis showed that the mutation of Ser-322 essentially changed the Vmax, rather than the Km, of the transporter. Mutation of all other PKC consensus sites did not affect transporter activity when expressed in the oocyte system. Western blot analysis showed that expression of taurine transporter protein was similar in oocytes injected with either wild-type or mutant pNCT cRNA, indicating that the enhanced taurine transport activity by mutant S322A was not caused by a greater amount of transporter expressed in the oocyte. Furthermore, this study demonstrated that the taurine transporter was phosphorylated after PKC activation, and this effect was not observed in mutant S322A. In conclusion, Ser-322 is critical in PKC regulation of taurine transporter activity. The steady-state taurine transporter activity is tightly controlled by endogenous PKC phosphorylation of Ser-322, which is located in the fourth intracellular segment of the taurine transporter.

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Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8-9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals

Biochemical Journal ◽

10.1042/bj3300771 ◽

1998 ◽

Vol 330 (2) ◽

pp. 771-776 ◽

Cited By ~ 13

Author(s):

A. Liaoyuan HU ◽

C. Steven KING

Keyword(s):

Escherichia Coli ◽

Nervous System ◽

Deleterious Effect ◽

Transmembrane Helix ◽

Transport Activity ◽

Gaba Transport ◽

Gaba Transporters ◽

Gabaergic Synapses ◽

Gaba Analogues ◽

The Many

The gab permease (GabP) catalyses transport of GABA (4-aminobutyrate) into Escherichia coli. Although GabP can recognize and transport many GABA analogues that exhibit activity at GABAergic synapses in the nervous system, the protein domains responsible for these transport and ligand recognition properties have not been studied. Here we report that an amphipathic domain extending through putative transmembrane helix 8 and into the adjoining cytoplasmic region (loop 8-9) contains a critical 20 residue zone within which mutagenesis of polar amino acids has a deleterious effect on [3H]GABA transport activity. This functionally important amphipathic domain is found to be highly conserved in the many APC family transporters that are homologous to GabP. And even though members of the GAT family of GABA transporters from the animal nervous system are not homologous to GabP, an analogous amphipathic structure is found in their loop 8-9 region. These results and observations suggest: (1) that the consensus amphipathic region (CAR) in the putative helix 8 and loop 8-9 region of GabP has functional significance, and (2) that nature has repeatedly used this CAR in transporters from bacteria to mammals.

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Cardiac antisecretory peptide inhibits intestinal chloride secretion

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.4.c775 ◽

1989 ◽

Vol 257 (4) ◽

pp. C775-C780 ◽

Cited By ~ 2

Author(s):

S. M. O'Grady ◽

P. J. Wolters

Keyword(s):

Atrial Natriuretic Factor ◽

Chloride Secretion ◽

Low Molecular Weight ◽

Transport Activity ◽

Regulatory Factor ◽

Cl Transport ◽

Heat Stable ◽

Antisecretory Effect ◽

The Right ◽

Intestinal Chloride Secretion

A heat-stable low-molecular-weight peptide isolated from porcine heart inhibited vasoactive intestinal polypeptide and ionomycin (a Ca ionophore) -stimulated Cl secretion across the rat ileum. The antisecretory effects of this peptide were resistant to the neuronal conduction blocker tetrodotoxin, suggesting that submucosal nerves were not involved in mediating its effects on Cl transport. Activity was found in extracts of the right and left atria and in the ventricles of the heart. The antisecretory effect was not mimicked by atrial natriuretic factor, brain natriuretic peptide, or by putative antisecretory factors (neuropeptide Y, somatostatin, enkephalin, and norepinephrine), suggesting that cardiac antisecretory peptide is a unique regulatory factor that may regulate Cl transport in the intestinal mucosa and other epithelia.

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