Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles.
RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A.
Department of Cell Biology and Physiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA.
Melany N
MN
Lopez
Timothy J
TJ
Wilding
James E
JE
Huettner
D000200:Action Potentials; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D005227:Fatty Acids / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018092:Kainic Acid Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D000097806:GluK2 Kainate Receptor
D005227:Fatty Acids; D018092:Kainic Acid Receptors; D000097806:GluK2 Kainate Receptor