Oligomerization of a Peptide Derived from the Transmembrane Region of the Sodium Pump γ Subunit: Effect of the Pathological Mutation G41R

The Na,K-ATPase, or sodium pump, is a ubiquitously expressed membrane-bound enzyme that controls the transmembrane (TM) gradients of sodium and potassium ions in animal cells. The enzyme comprises two subunits, α and β, and in the kidney, is also associated with a small single-spanning membrane protein, the γ subunit. This 65 amino acid residues protein has been linked to a form of dominant renal hypomagnesaemia resulting from substitution of a highly conserved glycine residue (Gly41) to arginine residue. In order to characterize the quaternary structure of the γ subunit, and effects of the G41R mutation thereupon, we synthesized a series of peptides (wild-type and mutant) that span the γ subunit TM region. Using circular dichroism spectroscopy, we show that the 32-amino acid residue peptides are random coils in aqueous buffer but spontaneously adopt an α-helical conformation in the presence of detergent micelles (sodium dodecyl sulfate, SDS, and perfluorooctanoate, PFO). Furthermore, fluorescence resonance energy transfer experiments, combined with polyacrylamide gel electrophoresis, demonstrate that while γ-TM does not self-associate in SDS, it forms oligomers in PFO, a detergent that tolerates relatively weak associations between membrane proteins. Importantly, oligomerization of γ-TM is abrogated in a peptide that contains either the disease-causing mutation G41R, or the more conservative mutation G41L. On the other hand, a peptide that contains a Gly-to-Arg substitution on a different face of the helix, at position 35, retains its ability to oligomerize. Our results provide evidence for a link between renal hypomagnesaemia and γ subunit oligomerization.