Role of proline residues in the expression and function of the human noradrenaline transporter

Paczkowski, F. A.; Bryan-Lluka, Lesley J.

doi:10.1111/j.1471-4159.2004.02149.x

Cited by 9 publications

(9 citation statements)

References 15 publications

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“…As V max of NE transport is dependent on the number of functional NET transporters, we estimated the number of NET transporters inserted in the plasma membrane of transfected cells by determining NE-sensitive binding of [ 3 H]NIS to intact cells at 4°C. This method has also been used in previous studies and by other groups Paczkowski et al 2002;Sucic et al 2002;Paczkowski and Bryan-Lluka 2004), and Blakely and coworkers have confirmed its utility by parallel biotinylation analysis of surface transporters in hNET transfected HEK293 cells (Apparsundaram et al 1998b). This method is based on the assumption that NE competes with nisoxetine binding exclusively at the cell surface since at 4°C NE should not enter the cells by transport and diffusion should be very small.…”

Section: Discussionmentioning

confidence: 84%

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Functional importance of the C‐terminus of the human norepinephrine transporter

Distelmaier

Wiedemann

Brüss

et al. 2004

Journal of Neurochemistry

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Three C-terminal variants of the human norepinephrine transporter (hNET) are known: the wild-type hNET in which exon 14 encodes the last seven amino acids and two variants with either three or 18 amino acids encoded by an alternatively spliced exon 15. type hNET with those of the more abundant long splice variant containing exon 15 (hNET-Ex15L) and of two artificial hNET mutants lacking either the last three (hNET-Ex14-4) or all seven (hNET-Ex14-0) C-terminal amino acids of exon 14. No differences among the NET isoforms were observed concerning the K m for uptake of NE and the K D for binding of NIS.However, compared with the wild-type hNET, the three isoforms (hNET-Ex15L, hNET-Ex14-4 and hNET-Ex14-0) showed a pronounced decrease in V max of [ 3 H]NE uptake and B max of [ 3 H]NIS binding which correlated with strongly reduced surface expression of the transporter isoforms. The decrease in surface expression of the hNET isoforms is probably a consequence of the lack of the three amino acids leucine, alanine and isoleucine at the C-terminal end which may represent a motif facilitating cell surface expression of the hNET. Expression of hNET-Ex15L exerted a dominant negative effect on plasma membrane expression of the wild-type hNET and thus may represent a novel mechanism for regulation of noradrenergic neurotransmission. Keywords: alternative splicing, C-terminus, norepinephrine transporter, surface expression, trafficking. The human norepinephrine transporter (hNET) plays a crucial role in the rapid removal of neuronally released norepinephrine (NE). This Na + and Cl --dependent reuptake into noradrenergic neurons is essential for a fine-tuned control of sympathetic activity . The hNET gene has been assigned to the long arm of chromosome 16 (Brüss et al. 1993). Its coding region consists of 14 exons spanning 45 kb (Pörzgen et al. 1995(Pörzgen et al. , 1998. The sequence predicts a protein of 617 amino acids with 12 hydrophobic transmembrane domains (TMDs) (Amara and Kuhar 1993;Brüss et al. 1995), intracellular N-and C-termini, and a large extracellular loop between TMD 3 and TMD 4 with consensus sites for N-linked glycosylation. hNET shows significant homology to other members of the monoamine neurotransmitter transporter family (Borowsky and Hoffman 1995;Masson et al. 1999).By influencing sympathetic activity the NET is implicated in cardiac function and vascular tone and an impaired NET function has been shown to be involved in the genesis of hypernoradrenergic hypertension, orthostatic dysregulation and congestive heart failure (Shannon et al. 2000;Eisenhofer 2001). The NET is the primary target for clinically important antidepressants (e.g. desipramine and reboxetine) and drugs of abuse such as cocaine and amphetamine. Disturbances in catecholamine reuptake have been proposed to be involved in the etiology and pathophysiology of depression, anxiety and alcohol dependence (Crabbe et al. 1994;Hadley et al. 1995).Expression of the NET and related Na + /Cl --dependent monoamine transporters have been show...

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Section: Discussionmentioning

confidence: 84%

“…2002; Sucic et al . 2002; Paczkowski and Bryan‐Lluka 2004), and Blakely and coworkers have confirmed its utility by parallel biotinylation analysis of surface transporters in hNET transfected HEK293 cells (Apparsundaram et al . 1998b).…”

Section: Discussionmentioning

confidence: 94%

Functional importance of the C‐terminus of the human norepinephrine transporter

Distelmaier

Wiedemann

Brüss

et al. 2004

Journal of Neurochemistry

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“…Proline residues play important structural and functional roles in transporters. They can form conformational switches in TM ␣-helices that enable the changes critical for substrate translocation (Sansom, 1992;Paczkowski and Bryan-Lluka, 2004). Because initial mutation of Pro290 did not significantly decrease transport activity, we can conclude that the presence of proline at this position is not strictly required.…”

Section: Discussionmentioning

confidence: 93%

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Hussainzada¹,

Banerjee²,

Swaan³

2006

Mol Pharmacol

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Recent evidence implicating transmembrane (TM) segment 7 of the apical sodium-dependent bile acid transporter (ASBT) in substrate interaction warranted examination of its aqueous accessibility. Therefore, cysteine substitution of 22 consecutive amino acids was performed against a methanethiosulfonate (MTS)-resistant background (C270A). Activity and susceptibility to polar MTS derivatives [(2-aminoethyl)-methanethiosulfonate (MTSEA), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and methanethiosulfonate ethylsulfonate (MTSES)] of mutants were evaluated in COS-1 cells. Thr289, Tyr293, Gln297, Ala301, Phe307, and Tyr308 represented loss-of-function mutants; furthermore, the measurable residual activities for T289C, Y293C, and A301C (Յ20% control) proved insensitive to MTS treatment. MTSES and MTSET inhibition was confined to residues lining the extracellular half of TM7; amino acids situated deeper within the membrane were unaffected. In contrast, the entire length of TM7 was susceptible to the relatively smaller MTSEA; moreover, MTSEA sensitivity was significantly amended by coapplication with substrates. This selective pattern of modification suggests that the highly conserved lower half of TM7 lies within a water-filled cavity easily accessible from the extracellular milieu, whereas residues approaching the cytosolic/membrane interface reside in pores for which accessibility is modulated by molecular volume. Functionally inactive and MTS-inaccessible residues (T289C, Y293C, Q297C, and A301C) within TM7 may play a structural role critical to transporter function; conversely, MTS-sensitive residues are spatially distinct and may demarcate a face of the TM involved in substrate translocation. In addition, computational analysis of solvent-accessible domains identified five key solvent pockets that predominantly line the hydrophilic face of TM7. Combined, our data suggest that TM7 plays a dominant role in the hASBT translocation process.

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“…Recent studies with mammalian dopamine transporters suggested structural as well as functional importance of TM prolines in plasma membrane targeting, assembly, membrane insertion and contribution to dopamine recognition (29). Prolines in the human noradrenaline transporter are involved in assembly of the transporter, inhibitor binding as well as in conformational changes associated with substrate translocation (30). Prolines are also required for cell surface expression and targeting in various membrane proteins (31).…”

Section: Discussionmentioning

confidence: 99%

Role of Conserved Prolines in the Structure and Function of the Na⁺/Dicarboxylate Cotransporter 1, NaDC1

Joshi

Pajor

2006

Biochemistry

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The sodium dicarboxylate cotransporter, NaDC1, is a low affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TM) 7 and 10. These transmembrane domains are of particular importance as they may be involved in determining substrate or cation binding affinity in NaDC1. Four conserved proline residues in TM 7 and 10 of rabbit NaDC1 were replaced with alanine to promote ideal α-helix or glycine to promote free conformation and the mutant transporters were expressed in the HRPE cell line. Mutations of prolines in TM 10 produced decreased protein expression and activity, whereas mutations of prolines in TM 7 completely abolished protein expression and activity. The chemical chaperone glycerol was found to improve expression of the Pro-351 mutants in TM 7, suggesting that these mutants had defects in trafficking. The inactive mutant transporters at position 351 could also be rescued by addition of a proline at a second site. For example, the P351A-F347P mutant had restored activity, although its substrate specificity was altered. We conclude that in TM 7, Pro-327 may be of particular importance in the function of the transporter whereas Pro-351 may affect protein targeting. The prolines in TM 10, at positions 523 and 524, may not be directly involved in transporter function but may be necessary for maintaining structure.The absorption of tricarboxylic acid cycle intermediates, such as succinate and citrate, across the apical membrane of the kidney proximal tubule and the small intestine is mediated by the Na + /dicarboxylate cotransporter, NaDC1 (1). NaDC1 belongs to the SLC13 gene family that includes transporters for di-and tricarboxylates as well as sulfate (2;3). NaDC1 seems to play a crucial role in the regulation of urinary citrate concentrations and low urinary citrate concentrations or hypocitraturia are usually associated with an increased risk of kidney stone formation (4). NaDC1 may also affect longevity or metabolic status since decreased expression of a related protein, the Indy transporter from Drosophila, produces a doubling of lifespan (5). There is little information about the protein structure of NaDC1 or any member of the SLC13 family. There is experimental evidence that the amino-terminus is located intracellularly and the carboxy-terminus, containing the N-glycosylation site at Asn-578, is located extracellularly (6;7). Furthermore, the carboxy-terminal half of NaDC1 contains determinants of substrate recognition and cation selectivity (8).Prolines play important structural as well as functional roles in membrane proteins. Normally, hydrogen bonds between backbone amino acids contribute to the structure and stability of transmembrane α-helices. Proline lacks an amide hydrogen which prevents it from forming

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Role of proline residues in the expression and function of the human noradrenaline transporter

Cited by 9 publications

References 15 publications

Functional importance of the C‐terminus of the human norepinephrine transporter

Functional importance of the C‐terminus of the human norepinephrine transporter

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Role of Conserved Prolines in the Structure and Function of the Na⁺/Dicarboxylate Cotransporter 1, NaDC1

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Role of proline residues in the expression and function of the human noradrenaline transporter

Cited by 9 publications

References 15 publications

Functional importance of the C‐terminus of the human norepinephrine transporter

Functional importance of the C‐terminus of the human norepinephrine transporter

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Role of Conserved Prolines in the Structure and Function of the Na+/Dicarboxylate Cotransporter 1, NaDC1

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Role of Conserved Prolines in the Structure and Function of the Na⁺/Dicarboxylate Cotransporter 1, NaDC1