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Mechanisms of pH activation and ion translocation in Na + /H + antiporters studied by EM. Werner Kühlbrandt Max-Planck-Institut für Biophysik Frankfurt am Main Erice 16-6-06. Na + /H + antiporters. Secondary transporters Ubiquitous in all cells (animals, plants, prokaryotes)

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mechanisms of ph activation and ion translocation in na h antiporters studied by em

Mechanisms of pH activation and ion translocation in Na+/H+ antiporters studied by EM

Werner Kühlbrandt

Max-Planck-Institut für Biophysik

Frankfurt am Main

Erice

16-6-06

na h antiporters
Na+/H+ antiporters
  • Secondary transporters
  • Ubiquitous in all cells (animals, plants, prokaryotes)
  • Maintain intracellular pH and Na+ concentration
  • pH-activated
  • E. coli Na+/H+ antiporter NhaA
  • Mammalian Na+/H+ exchanger NHE1
  • M. jannaschii Na+/H+ antiporter MjNhaP1
nhaa n a h a ntiporter a
NhaA: Na+/H+ antiporter A
  • Family prototype from E. coli
  • Well-characterized
  • 3.5 Å X-ray structure known
  • Maintains intracellular pH and Na+ concentration
  • Adaptation to high salinity, high pH
  • Inactive below pH6
  • Activated at pH7
  • Fully active at pH8
slide7

X-ray structure of NhaA monomer at 3.45 Å resolution

(Hunte et al, Nature 2005)

 hairpin

periplasmic view

cytoplasmic view

slide8

2. MjNhaP1Na+/H+ antiporter from M. jannaschiiHomologue of human NHE-1 antiporter and E. coli NhaAfrom hyperthermophilic archaeon

slide9

2D crystals of M. jannaschii NhaP1

100 nm

1 µm

Tubular crystals

slide10

NhaP1 projection data

15Å 10Å 8Å 6Å

slide15

Comparison of Na+/H+ antiporters

MjNhaP1

E. coli NhaA

pH 4

pH 8

(Williams et al, EMBO J. 1999)

(Vinothkumar et al, EMBO J. 2005)

slide16

pH induced helix movements in MjNhaP1 dimer

helix bundle

dimer interface

helix bundle

slide17

pH-dependent activity of MjNhaP1 in liposomes

MjNhaP1 liposomes, 0.3 M NaCl inside

empty liposomes (control)

pH 6

no NaCl gradient

acridine orange fluorescence (arbitrary units)

pH 7.5

add liposomes

0.6 min

NH4Cl

add NH4Cl

mjnhap1 conclusions
MjNhaP1 conclusions
  • Recombinant reconstituted Mj antiporter is active at pH6
  • Inactive at pH7.5
  • Drop in pH from 8 to acidic causes ~2Å movement of helix bundle, in addition to reorientation of helices within bundle
  • Major differences to NhaA in projection structure
  • Different mechanism of pH activation and ion translocation ?
slide20

MjNhaP1: K.R.Vinothkumar

Sander Smits

Panchali Goswami

Özkan Yildiz

NhaA: Matthias AppelCarola Hunte K.R.Vinothkumar Elena Scrapanti

Karen Williams Etana Padan

Hartmut Michel

(x-ray structure)

slide21

Max Planck Institute

of Biophysics

Frankfurt