ABSTRACT
Anionic lipids influence the ability of the nicotinic acetylcholine receptor to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. We show here that anionic lipids with relatively small headgroups, and thus the greatest ability to influence lipid packing/bilayer physical properties, are the most effective at stabilizing an agonist-activatable receptor. The differing abilities of anionic lipids to stabilize an activatable receptor stem from differing abilities to preferentially favor resting over both uncoupled and desensitized conformations. Anionic lipids thus modulate multiple acetylcholine receptor conformational equilibria. Our data suggest that both lipids and membrane physical properties act as classic allosteric modulators influencing function by interacting with and thus preferentially stabilizing different native acetylcholine receptor conformational states.
Subject(s)
Anions/metabolism , Lipid Bilayers/chemistry , Lipids/chemistry , Receptors, Nicotinic/chemistry , Allosteric Site , Animals , Biophysics/methods , Cell Membrane/metabolism , Chromatography, Thin Layer/methods , Electrochemistry/methods , Models, Biological , Molecular Conformation , Spectrophotometry, Infrared/methods , TorpedoABSTRACT
Annexins are a family of homologous proteins that associate with anionic phospholipid (aPL) in the presence of Ca(2+). Evidence that the function of one annexin type may be regulated by another was recently reported in studies investigating cytomegalovirus-aPL interactions, where the fusogenic function of annexin 2 (A2) was attenuated by annexin 5 (A5). This observation suggested that A2 may bind directly to A5. In the present study, we demonstrated this interaction. The A2-A5 complex was first detected utilizing (covalently linked) fluorescein-labelled A5 (F-A5) as a reporter group. The interaction required concentrations of Ca(2+) in the millimolar range, had an apparent dissociation constant [ K (d)(app)] of 1 nM at 2 mM Ca(2+) and was independent of aPL. A2 bound comparably with F-A5 pre-equilibrated with an amount of aPL that could bind just the F-A5 or to an excess amount of aPL providing sufficient binding sites for all of F-A5 and A2. A2-A5 complex formation was corroborated in an experiment, where [(125)I]A2 associated in a Ca(2+)-dependent manner with A5 coated on to polystyrene. Surface plasmon resonance was used as a third independent method to demonstrate the binding of A2 and A5 and, furthermore, supported the conclusion that the monomeric and tetrameric forms of A2 bind equivalently to A5. Together these results demonstrate an A2-A5 interaction and provide an explanation as to how A5 inhibits the previously reported A2-dependent enhancement of virus-aPL fusion.
