Determination of a Transmembrane Segment Using Cysteine-Scanning Mutants of Transposon Tn10-Encoded Metal−Tetracycline/H+Antiporter†

Biochemistry ◽  
1996 ◽  
Vol 35 (49) ◽  
pp. 15896-15899 ◽  
Author(s):  
Tomomi Kimura ◽  
Masayo Suzuki ◽  
Tetsuo Sawai ◽  
Akihito Yamaguchi
Keyword(s):  
Transmembrane Segment ◽  
Cysteine Scanning

Investigation of Na+ and K+ Transport in Halophytes: Functional Analysis of the HmHKT2;1 Transporter from Hordeum maritimum and Expression under Saline Conditions

2019 ◽  
Vol 60 (11) ◽  
pp. 2423-2435 ◽  
Author(s):  
Dorsaf Hmidi ◽  
Dorsaf Messedi ◽  
Claire Corratg�-Faillie ◽  
Th�o Marhuenda ◽  
C�cile Fizames ◽  
...  
Keyword(s):  
Absorption Efficiency ◽  
Extracellular Loop ◽  
Transmembrane Segment ◽  
Transport Pathway ◽  
Reverse Transcription Pcr ◽  
Encoding Gene ◽  
K Transport ◽  
Different Levels ◽  
Adaptation To Salinity

Abstract Control of K+ and Na+ transport plays a central role in plant adaptation to salinity. In the halophyte Hordeum maritimum, we have characterized a transporter gene, named HmHKT2;1, whose homolog HvHKT2;1 in cultivated barley, Hordeum vulgare, was known to give rise to increased salt tolerance when overexpressed. The encoded protein is strictly identical in two H. maritimum ecotypes, from two biotopes (Tunisian sebkhas) affected by different levels of salinity. These two ecotypes were found to display distinctive responses to salt stress in terms of biomass production, Na+ contents, K+ contents and K+ absorption efficiency. Electrophysiological analysis of HmHKT2;1 in Xenopus oocytes revealed distinctive properties when compared with HvHKT2;1 and other transporters from the same group, especially a much higher affinity for both Na+ and K+, and an Na+–K+ symporter behavior in a very broad range of Na+ and K+ concentrations, due to reduced K+ blockage of the transport pathway. Domain swapping experiments identified the region including the fifth transmembrane segment and the adjacent extracellular loop as playing a major role in the determination of the affinity for Na+ and the level of K+ blockage in these HKT2;1 transporters. The analysis (quantitative reverse transcription-PCR; qRT-PCR) of HmHKT2;1 expression in the two ecotypes submitted to saline conditions revealed that the levels of HmHKT2;1 transcripts were maintained constant in the most salt-tolerant ecotype whereas they decreased in the less tolerant one. Both the unique functional properties of HmHKT2;1 and the regulation of the expression of the encoding gene could contribute to H. maritimum adaptation to salinity.

scholarly journals Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H+ antiporter

FEBS Letters ◽  
1999 ◽  
Vol 461 (3) ◽  
pp. 315-318 ◽  
Author(s):  
Satoko Konishi ◽  
Shinobu Iwaki ◽  
Tomomi Kimura-Someya ◽  
Akihito Yamaguchi
Keyword(s):  
Transmembrane Segment ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO3 cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5

2007 ◽  
Vol 292 (5) ◽  
pp. C1787-C1798 ◽  
Author(s):  
Jing Lu ◽  
Walter F. Boron
Keyword(s):  
Voltage Clamp ◽  
Dose Response ◽  
Response Curve ◽  
Xenopus Oocytes ◽  
Transmembrane Segment ◽  
Initial Value ◽  
Irreversible Inhibition ◽  
Electrode Voltage

Others have shown that H2DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO3 exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (−160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK ( wt, ∼40 μM) < NKMIK ≅ NKMIN ≅ KKMIN < KNMIN ≅ KNMIK < NNMIK < NNMIN (∼400 μM) < DDMID < EEMIE (∼800 μM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80–91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.

scholarly journals Cysteine-scanning Mutagenesis around Transmembrane Segment III of Tn10-encoded Metal-Tetracycline/H+Antiporter

1998 ◽  
Vol 273 (9) ◽  
pp. 5243-5247 ◽  
Author(s):  
Tomomi Kimura ◽  
Yasuko Shiina ◽  
Tetsuo Sawai ◽  
Akihito Yamaguchi
Keyword(s):  
Transmembrane Segment ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis
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