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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
15:28

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

Published on: October 1, 2010

A systematic mutagenesis study of Ile-282 in transmembrane segment M4 of the plasma membrane H+-ATPase

A Staffan Fraysse1, Anders L B Møller, Lisbeth R Poulsen

  • 1Department of Plant Biology, The Royal Danish Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

The Journal of Biological Chemistry
|April 15, 2005
PubMed

Insights

Investigating the plant plasma membrane H(+)-ATPase (AHA2), researchers found that smaller amino acid side chains at position 282 reduce proton affinity and transport. This suggests the backbone carbonyl oxygen of Ile-282 directly aids proton translocation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • P-type proton pumps, like the plasma membrane H(+)-ATPase (H+-ATPase), are crucial for cellular transport.
  • Homology models suggest specific residues in transmembrane segment M4 are vital for proton translocation.
  • The plant plasma membrane H+-ATPase AHA2 is a key target for understanding these mechanisms.

Purpose of the Study:

  • To investigate the role of residues in transmembrane segment M4 of AHA2 in proton translocation.
  • To elucidate the specific contribution of residue Isoleucine-282 (I282) to the function of the H+-ATPase.
  • To test the hypothesis that backbone carbonyl oxygen of I282 is directly involved in proton transport.

Main Methods:

  • Alanine-scanning mutagenesis was performed on 12 residues in the M4 segment of AHA2.
  • Saturation mutagenesis was used to substitute Isoleucine-282 (I282) with all other natural amino acids.
  • Mutant enzyme function was assessed by their ability to complement the yeast proton pump PMA1.

Main Results:

  • An Isoleucine-282 to Alanine (I282A) mutation resulted in reduced proton (H+) affinity.
  • Mutant enzymes' ability to substitute for yeast PMA1 correlated with the size of the side chain at position 282.
  • Smaller side chains (Gly, Ala, Ser) decreased H+ affinity and transport, while larger ones had minor effects.
  • Substitutions at I282 did not significantly alter ATP affinity or vanadate sensitivity.

Conclusions:

  • The size of the amino acid side chain at position 282 of AHA2 is critical for proton translocation efficiency.
  • Results support a model where the backbone carbonyl oxygen of Isoleucine-282 directly participates in proton translocation.
  • This finding provides insights into the molecular mechanism of proton pumping in P-type ATPases.

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