Localization and functional characterization of the human NKCC2 isoforms

I Carota, F Theilig, M Oppermann, P Kongsuphol, A Rosenauer, R Schreiber, B L Jensen, S Walter, K Kunzelmann, H Castrop

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl(-) affinity as determined by (86)Rb(+) uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. CONCLUSION: The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.
OriginalsprogEngelsk
TidsskriftActa Physiologica (Print)
Vol/bind199
Udgave nummer3
Sider (fra-til)327-38
Antal sider12
ISSN1748-1708
DOI
StatusUdgivet - 1. jul. 2010

Fingeraftryk

Protein Isoforms
Oocytes
Member 1 Solute Carrier Family 12
Rodentia
Protein Sequence Analysis
Furosemide
Salts
Cell Membrane
Rabbits
Kidney
Polymerase Chain Reaction
Proteins

Citer dette

Carota, I., Theilig, F., Oppermann, M., Kongsuphol, P., Rosenauer, A., Schreiber, R., ... Castrop, H. (2010). Localization and functional characterization of the human NKCC2 isoforms. Acta Physiologica (Print), 199(3), 327-38. https://doi.org/10.1111/j.1748-1716.2010.02099.x
Carota, I ; Theilig, F ; Oppermann, M ; Kongsuphol, P ; Rosenauer, A ; Schreiber, R ; Jensen, B L ; Walter, S ; Kunzelmann, K ; Castrop, H. / Localization and functional characterization of the human NKCC2 isoforms. I: Acta Physiologica (Print). 2010 ; Bind 199, Nr. 3. s. 327-38.
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abstract = "AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl(-) affinity as determined by (86)Rb(+) uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. CONCLUSION: The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.",
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Carota, I, Theilig, F, Oppermann, M, Kongsuphol, P, Rosenauer, A, Schreiber, R, Jensen, BL, Walter, S, Kunzelmann, K & Castrop, H 2010, 'Localization and functional characterization of the human NKCC2 isoforms', Acta Physiologica (Print), bind 199, nr. 3, s. 327-38. https://doi.org/10.1111/j.1748-1716.2010.02099.x

Localization and functional characterization of the human NKCC2 isoforms. / Carota, I; Theilig, F; Oppermann, M; Kongsuphol, P; Rosenauer, A; Schreiber, R; Jensen, B L; Walter, S; Kunzelmann, K; Castrop, H.

I: Acta Physiologica (Print), Bind 199, Nr. 3, 01.07.2010, s. 327-38.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Localization and functional characterization of the human NKCC2 isoforms

AU - Carota, I

AU - Theilig, F

AU - Oppermann, M

AU - Kongsuphol, P

AU - Rosenauer, A

AU - Schreiber, R

AU - Jensen, B L

AU - Walter, S

AU - Kunzelmann, K

AU - Castrop, H

PY - 2010/7/1

Y1 - 2010/7/1

N2 - AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl(-) affinity as determined by (86)Rb(+) uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. CONCLUSION: The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.

AB - AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl(-) affinity as determined by (86)Rb(+) uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. CONCLUSION: The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.

KW - Amino Acid Sequence

KW - Animals

KW - Biological Transport, Active

KW - Chlorine

KW - Diuretics

KW - Exons

KW - Humans

KW - Immunohistochemistry

KW - In Situ Hybridization

KW - Isomerism

KW - Isotopes

KW - Kidney

KW - Mice

KW - Molecular Sequence Data

KW - Oocytes

KW - Rabbits

KW - Rats

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Rubidium Radioisotopes

KW - Sodium-Potassium-Chloride Symporters

KW - Transcription, Genetic

KW - Xenopus laevis

U2 - 10.1111/j.1748-1716.2010.02099.x

DO - 10.1111/j.1748-1716.2010.02099.x

M3 - Journal article

VL - 199

SP - 327

EP - 338

JO - Acta Physiologica (Print)

JF - Acta Physiologica (Print)

SN - 1748-1708

IS - 3

ER -

Carota I, Theilig F, Oppermann M, Kongsuphol P, Rosenauer A, Schreiber R et al. Localization and functional characterization of the human NKCC2 isoforms. Acta Physiologica (Print). 2010 jul 1;199(3):327-38. https://doi.org/10.1111/j.1748-1716.2010.02099.x