[29] Vaccinia virus-T7 RNA polymerase expression system for neurotransmitter transporters

Povlock, Sue L.; Amara, Susan G.

doi:10.1016/s0076-6879(98)96031-1

Cited by 10 publications

(9 citation statements)

References 15 publications

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“…The f ull-length SA2 cDNA and SA1 cDNA (nucleotides 160 -1928) were expressed in HeLa cells using the vaccinia virus-T7 polymerase system (Povlock and Amara, 1998). Briefly, HeLa cells were plated at 1.5 ϫ 10 5 per well.…”

Section: Methodsmentioning

confidence: 99%

Glutamine Uptake by Neurons: Interaction of Protons with System A Transporters

et al. 2002

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Astrocytes provide the glutamine required by neurons to synthesize glutamate and GABA. However, the mechanisms involved in glutamine transfer from glia to neurons have remained poorly understood. Recent work has implicated the System N transporter SN1 in the efflux of glutamine from astrocytes and the very closely related System A transporters SA1 and SA2 in glutamine uptake by neurons. To understand how these closely related proteins mediate flux in different directions, we have examined their ionic coupling. In contrast to the electroneutral exchange of H ϩ for Na ϩ and neutral amino acid catalyzed by SN1, we now show that SA1 and SA2 do not couple H ϩ movement to amino acid flux. As a result, SA1 and SA2 are electrogenic and do not mediate flux reversal as readily as SN1. Differences between System N and A transporters in coupling to H ϩ thus contribute to the delivery of glutamine from glia to neurons. Nonetheless, although they are not transported, H ϩ inhibit SA1 and SA2 by competing with Na ϩ .

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Section: Methodsmentioning

confidence: 99%

Glutamine Uptake by Neurons: Interaction of Protons with System A Transporters

et al. 2002

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“…Four CNT1 variable sites were found at an allele frequency of Ն20% in the total population: CNT1 ϩ 140Val, CNT1-Val189Ile, CNT1-Gln237Lys, and CNT1-Asp521Asn. CNT1 reference and these common CNT1 variants were subcloned into the pGEMT expression vector and heterologously expressed in HeLa cells using the vaccinia virus-T7 polymerase system (Povlock and Amara, 1998;Wreden et al, 2003). The vaccinia virus and HeLa cells were generous gifts from R. H. Edwards (UCSF, San Francisco, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The vaccinia virus and HeLa cells were generous gifts from R. H. Edwards (UCSF, San Francisco, CA). Vaccinia virus stocks were prepared as described by Povlock and Amara (1998). HeLa cells were cultured using H-21 Dulbecco's modified Eagle's medium and cell culture supplies purchased from the Cell Culture Facility (UCSF, San Francisco, CA).…”

Section: Methodsmentioning

confidence: 99%

Functional and Genetic Diversity in the Concentrative Nucleoside Transporter, CNT1, in Human Populations

et al. 2004

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The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) . A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using sitedirected mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3 H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frameshift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC 50 ϭ 13.8 Ϯ 0.60 M for CNT1-reference and 23.3 Ϯ 1.5 M for CNT1-Val189Ile, p Ͻ 0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anticancer nucleoside analogs.

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“…The SA1 cDNA (nucleotides 160-1,928) was expressed by using the vaccinia virus-T7 polymerase system (29). In brief, HeLa cells were plated at 1.5 ϫ 10 5 per well in a 24-well plate, were incubated for 12 h at 37°C, and were rinsed with serum-free media, and vaccinia virus was added to each well at 5 plaque-forming units per cell.…”

Section: Methodsmentioning

confidence: 99%

Amino acid transport System A resembles System N in sequence but differs in mechanism

Reimer

Chaudhry

Gray

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

179

138

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Classical amino acid transport System A accounts for most of the Na ؉ -dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short-and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling.

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[29] Vaccinia virus-T7 RNA polymerase expression system for neurotransmitter transporters

Cited by 10 publications

References 15 publications

Glutamine Uptake by Neurons: Interaction of Protons with System A Transporters

Glutamine Uptake by Neurons: Interaction of Protons with System A Transporters

Functional and Genetic Diversity in the Concentrative Nucleoside Transporter, CNT1, in Human Populations

Amino acid transport System A resembles System N in sequence but differs in mechanism

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