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Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts
- Source :
- JOURNAL OF BIOLOGICAL CHEMISTRY. 283(4):1911-1920
- Publication Year :
- 2008
- Publisher :
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2008.
-
Abstract
- The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.
- Subjects :
- Patch-Clamp Techniques
Potassium Channels
Osmotic shock
Nicotiana tabacum
Molecular Sequence Data
Saccharomyces cerevisiae
Biochemistry
Cell membrane
Gene Expression Regulation, Plant
Osmotic Pressure
Tobacco
Escherichia coli
medicine
Homeostasis
Osmotic pressure
Amino Acid Sequence
Patch clamp
Promoter Regions, Genetic
Molecular Biology
Ion transporter
Plant Proteins
Ion Transport
biology
Cell Membrane
Sodium
Cell Biology
Hydrogen-Ion Concentration
Membrane transport
biology.organism_classification
Recombinant Proteins
Potassium channel
medicine.anatomical_structure
Vacuoles
Potassium
Biophysics
Calcium
Subjects
Details
- Language :
- English
- ISSN :
- 00219258
- Volume :
- 283
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- JOURNAL OF BIOLOGICAL CHEMISTRY
- Accession number :
- edsair.doi.dedup.....9471333066d8a0f07321d84f97cc5f3a