Structure-function relationship of the fifth transmembrane domain in the Na+/H+ antiporter of Helicobacter pylori: Topology and function of the residues, including two consecutive essential aspartate residues
In: Biochemistry, Jg. 45 (2006-12-06), Heft 49
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Zugriff:
We examined the structure-function relationships of residues in the fifth transmembrane domain (TM5) of the Na + /H + antiporter A (NhaA) from Helicobacter pylori (HP NhaA) by cysteine scanning mutagenesis. TM5 contains two aspartate residues, Asp-171 and Asp-172, which are essential for antiporter activity. Thirty-five residues spanning the putative TM5 and adjacent loop regions were replaced by cysteines. Cysteines replacing Val-162, Ile-165, and Asp-172 were labeled with NEM, suggesting that these three residues are exposed to a hydrophilic cavity within the membrane. Other residues in the putative TM domain, including Asp-171, were not labeled. Inhibition of NEM labeling by the membrane impermeable reagent AMS suggests that Val-162 and Iie-165 are exposed to a water filled channel open to the cytoplasmic space, whereas Asp-172 is exposed to the periplasmic space. D171C and D172C mutants completely lost Na + /H + and Li + /H + antiporter activities, whereas other Cys replacements did not result in a significant loss of these activities. These results suggest that Asp-171 and Asp-172 and the surrounding residues of TM5 provide an essential structure for H + binding and Na + or Li + exchange. A168C and Y183C showed markedly decreased antiporter activities at acidic pH, whereas their activities were higher at alkaline pH, suggesting that the conformation of TM5 also plays a crucial role in the HP NhaA-specific acidic pH antiporter activity.
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Structure-function relationship of the fifth transmembrane domain in the Na+/H+ antiporter of Helicobacter pylori: Topology and function of the residues, including two consecutive essential aspartate residues
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Autor/in / Beteiligte Person: | Inoue, Hiroki ; Tsuboi, Yumi ; Mitsui, Keiji ; Kuwabara, Naoyuki ; Kanazawa, Hiroshi ; Matsushita, Masafumi |
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Zeitschrift: | Biochemistry, Jg. 45 (2006-12-06), Heft 49 |
Veröffentlichung: | 2006 |
Medientyp: | unknown |
ISSN: | 0006-2960 (print) |
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