Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

Hager, Karl; Hazama, Akihiro; Kwon, H. Moo; Loo, Donald D. F.; Handler, Jerome S.; Wright, Ernest M.

doi:10.1007/bf00234656

Cited by 106 publications

(97 citation statements)

References 16 publications

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“…4). The current showed no evidence of having attained a maximal level at the most negative potential used (Ϫ175 mV), which is similar to the I-V curve seen with SMIT1 expression but unlike the shape of the I-V curve for SGLT1 (28). The currents for the phlorizin-sensitive sodium leak associated with SMIT2 were also examined at different membrane potentials (Fig.…”

Section: Fig 1 Presteady-state Current Recordingsmentioning

confidence: 73%

Identification of a Novel Na+/myo-Inositol Cotransporter

Coady

Wallendorff

Gagnon

et al. 2002

Journal of Biological Chemistry

146

141

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rkST1, an orphan cDNA of the SLC5 family (43% identical in sequence to the sodium myo-inositol cotransporter SMIT), was expressed in Xenopus laevis oocytes that were subsequently voltage-clamped and exposed to likely substrates. Whereas superfusion with glucose and other sugars produced a small inward current, the largest current was observed with myo-inositol. The expressed protein, which we have named SMIT2, cotransports myo-inositol with a K m of 120 M and displays a current-voltage relationship similar to that seen with SMIT (now called SMIT1). The transport is Na ؉ -dependent, with a K m of 13 mM. SMIT2 exhibits phlorizin-inhibitable presteady-state currents and substrate-independent "Na ؉ leak" currents similar to those of related cotransporters. The steady-state cotransport current is also phlorizin-inhibitable with a K i of 76 M. SMIT2 exhibits stereospecific cotransport of both D-glucose and D-xylose but does not transport fucose. In addition, SMIT2 (but not SMIT1) transports D-chiro-inositol. Based on previous publications, the tissue distribution of SMIT2 is different from that of SMIT1, and the existence of this second cotransporter may explain much of the heterogeneity that has been reported for inositol transport.The first members of the vertebrate cotransporter protein family SLC5, which includes the high affinity Na ϩ /glucose cotransporter (SGLT1) and the Na ϩ /myo-inositol cotransporter (SMIT), were isolated over a decade ago based on expression of the proteins in Xenopus laevis oocytes (1, 2). Although substrates as diverse as proline, iodide, and vitamins (3) are transported by this family of proteins, the best characterized transporters remain SGLT1 and SMIT. There are also several "orphan" transporters whose cDNA has been cloned either by using labeled cDNA from members of the SLC5 family as biochemical probes or by comparing SLC5 sequence information in silico to data stored in DNA data bases (3); the newly discovered sequences are orphans in that they have no known function. Some of the orphan protein sequences are particularly similar to the protein sequences for SGLT1 and SMIT (4, 5) and presumably transport substrates similar or identical to either glucose or its isomer myo-inositol. The SLC5 proteins with known functions have generally been studied by voltageclamp experiments because these proteins are electrogenic. Also, presteady-state currents are associated with expression of these proteins at the cell surface, and some (but not all, e.g.

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Section: Fig 1 Presteady-state Current Recordingsmentioning

confidence: 73%

Identification of a Novel Na+/myo-Inositol Cotransporter

Coady

Wallendorff

Gagnon

et al. 2002

Journal of Biological Chemistry

146

141

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“…Na ϩ -dependent leakage currents have also been reported in nonexcitable tissues (e.g. a thyroid Na ϩ /I Ϫ uptake system (15), myo-inositol (16), and Na ϩ /glucose (SGLT1) transporters (3,17) and ASCT-x Na ϩ , Cl Ϫ dependent transporters broadly selective for both neutral and acid amino acids (2,5)). The mammalian amine and amino acid transporters are strictly dependent on Cl Ϫ , unlike CAATCH1 (Fig.…”

Section: Fig 3 Changes In Amino Acid-dependent Current-voltage Relamentioning

confidence: 99%

“…This dual role permits direct modulation of electrochemical events in the central nervous system by illicit drugs, such as cocaine on DAT or antidepressants on SERT (8, 10 -14). Rare examples of dual solute-transporter/leakage-channel activity have been reported in membrane proteins cloned from sources other than excitable tissues (2,(15)(16)(17).…”

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confidence: 99%

A Novel Electrogenic Amino Acid Transporter Is Activated by K+ or Na+, Is Alkaline pH-dependent, and Is Cl−-independent

Feldman

Harvey

Stevens

2000

Journal of Biological Chemistry

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A new eukaryotic nutrient amino acid transporter has been cloned from an epithelium that is exposed to high voltages and alkaline pH. The full-length cDNA encoding this novel CAATCH1 (cation-anion-activated Amino acid transporter/channel) was isolated using a polymerase chain reaction-based strategy, and its expression product in Xenopus oocytes displayed a combination of several unique, unanticipated functional properties. CAATCH1 electrophysiological properties resembled those of Na ؉ ,Cl ؊ -coupled neurotransmitter amine transporters, although CAATCH1 was cloned from a gut absorptive epithelium rather than from an excitable tissue. Amino acids such as L-proline, L-threonine, and L-methionine elicited complex current-voltage relationships in alkaline pH-dependent CAATCH1 that were reminiscent of the behavior of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, NET) in the presence of their substrates and pharmacological inhibitors such as cocaine or antidepressants. These I-V relationships indicated a combination of substrate-associated carrier current plus an independent CAATCH1-associated leakage current that could be blocked by certain amino acids. However, unlike all structurally related proteins, CAATCH1 activity is absolutely independent of Cl ؊ . Unlike related KAAT1, CAATCH1 possesses a methionine-inhibitable constitutive leakage current and is able to switch its narrow substrate selectivity, preferring threonine in the presence of K ؉ but preferring proline in the presence of Na ؉ .The distinction between ion-activated transporters and channels is becoming blurred. For example, the Na ϩ and Cl Ϫ -dependent neurotransmitter transporters, which serve substrates such as dopamine (DAT), norepinephrine (NET), serotonin (SERT), GABA (GATx), and selected amino acids (via EAATx, GLAST, GLT, GlyTx, PROT, or ASCTx) act as both solute carriers and ion channels (1-6). The presence of a ligand-modulated leakage current (7) (i.e. a small background conductance of undetermined ionic basis) was identified in DAT by an inverted U-shaped current-voltage relationship (8 -10). This dual role permits direct modulation of electrochemical events in the central nervous system by illicit drugs, such as cocaine on DAT or antidepressants on SERT (8,[10][11][12][13][14]. Rare examples of dual solute-transporter/leakage-channel activity have been reported in membrane proteins cloned from sources other than excitable tissues (2, 15-17).Here we report the cloning and characterization of a cDNA that encodes a new membrane protein, CAATCH1 (cation-anion-activated amino acid transporter/channel), from a caterpillar absorptive epithelium. When expressed in Xenopus oocytes, CAATCH1 acts as a Cl Ϫ -independent, and Na ϩ -or K ϩ -activated nutrient amino acid transporter that also yields currentvoltage relationships like those ascribed to ligand-modulated leakage behavior in DAT (8 -10). These unusual characteristics of CAATCH1, which are different from those of the structurally related KAAT1 (18), suggest that CAATCH1 ...

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“…The deduced 718-amino acid polypeptide was predicted to have multiple potential protein kinase A/C (PKA/PKC) phosphorylation sites (16) and multiple hydrophobic ''membrane-spanning'' domains, homologous with the mammalian Na ϩ -dependent glucose transporter (SGLT) family (12). More significantly, voltage-clamp studies of Na ϩ -myo-inositol cotransport in Xenopus oocytes expressing the recombinant canine SMIT cDNA confirm the functional identity of the recombinant transporter through its substrate specificity and kinetic parameters (11). With the use of this cDNA as a probe, SMIT transcripts ranging from ϳ1.0 to Ͼ13.5 kb have been identified following hypertonic stress in Northern blots of RNA from kidney (16), lens (33), neural (21), and vascular (28) endothelial cells.…”

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confidence: 99%

Human Na⁺-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts

Porcellati¹,

Hlaing²,

Togawa³

et al. 1998

American Journal of Physiology-Cell Physiology

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splicing generates diverse transcripts. Am. J. Physiol. 274 (Cell Physiol. 43): C1215-C1225, 1998.-Na ϩ -myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myoinositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na ϩ -myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na ϩ -dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and ϳ1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress. human retinal pigment epithelial cells; hypertonic stress; exon splicing; osmoregulation; diabetes mellitus THE WATER-SOLUBLE CYCLIC hexitol myo-inositol is a constituent of virtually every living cell and an essential nutrient for most mammalian cells in culture. Intracellular concentrations 50-to 1,000-fold greater than that of extracellular fluid are with few exceptions (25) ascribed to the action of membrane-associated, Na ϩ -dependent myo-inositol cotransporters (SMITs) (2). At the cellular level, myo-inositol is an obligate and sometimes rate-limiting (19) substrate for phosphoinositide synthesis. Along with sorbitol, taurine, and betaine, myo-inositol belongs to a family of alternative nonionic organic intracellular osmolytes whose regulated accumulation and efflux in response to hypertonic stress preserve intracellular volume and tonicity without perturbing the ionic milieu (2, 5). Organic osmolyte accumulation is coordinated by the regulated expression and/or activity of osmotically responsive, osmolytespecific genes or gene products: distinct Na ϩ -cotransporters for myo-inositol, taurine, and betaine and aldose reductase (AR) for the synthesis of sorbitol from glucose. Gradient-dependent organic osmolyte efflux is thought to be regulated primarily but not exclusively by a nonselective, ATP-dependent, volume-sensitive organic anion channel (2,14,27). Thus, at any given tonicity, the relative abundance of each osmolyte depends in part on substrate availability, the abundance and activity of their transporter or biosynthetic enzyme, and their efflux or degradation. T...

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Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

Cited by 106 publications

References 16 publications

Identification of a Novel Na+/myo-Inositol Cotransporter

Identification of a Novel Na+/myo-Inositol Cotransporter

A Novel Electrogenic Amino Acid Transporter Is Activated by K+ or Na+, Is Alkaline pH-dependent, and Is Cl−-independent

Human Na⁺-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts

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Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

Cited by 106 publications

References 16 publications

Identification of a Novel Na+/myo-Inositol Cotransporter

Identification of a Novel Na+/myo-Inositol Cotransporter

A Novel Electrogenic Amino Acid Transporter Is Activated by K+ or Na+, Is Alkaline pH-dependent, and Is Cl−-independent

Human Na+-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts

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Human Na⁺-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts