Dataset Information

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles.

ABSTRACT: 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.

SUBMITTER: Lopez MN

PROVIDER: S-EPMC3753602 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles.

Lopez Melany N MN Wilding Timothy J TJ Huettner James E JE

The Journal of general physiology 20130812 3

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 ...[more]

PMID: 23940260

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Project description:Kainate receptors are ligand-gated ion channels that have two major roles in the central nervous system: they mediate a postsynaptic component of excitatory neurotransmission at some glutamatergic synapses and modulate transmitter release at both excitatory and inhibitory synapses. Accumulating evidence implicates kainate receptors in a variety of neuropathologies, including epilepsy, psychiatric disorders, developmental delay, and cognitive impairment. Here, to gain a deeper understanding of the conformational changes associated with agonist binding and channel opening, we generate a series of Cys substitutions in the GluK2 kainate receptor subunit, focusing on the M3 helices that line the ion pore and form the bundle-crossing gate at the extracellular mouth of the channel. Exposure to 50 µM Cd produces direct activation of homomeric mutant channels bearing Cys substitutions in (A657C), or adjacent to (L659C), the conserved SYTANLAAF motif. Activation by Cd is occluded by modification with 2-aminoethyl MTS (MTSEA), indicating that Cd binds directly and specifically to the substituted cysteines. Cd potency for the A657C mutation (EC50 = 10 µM) suggests that binding involves at least two coordinating residues, whereas weaker Cd potency for L659C (EC50 = 2 mM) implies that activation does not require tight coordination by multiple side chains for this substitution. Experiments with heteromeric and chimeric channels indicate that activation by Cd requires Cys substitution at only two of the four subunits within a tetrameric receptor and that activation is similar for substitution within subunits in either the A/C or B/D conformations. We develop simple kinetic models for the A657C substitution that reproduce several features of Cd activation as well as the low-affinity inhibition observed at higher Cd concentrations (5-20 mM). Together, these results demonstrate rapid and reversible channel activation, independent of agonist site occupancy, upon Cd binding to Cys side chains at two specific locations along the GluK2 inner helix.

Project description:GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the kinetic data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant and thus a channel-opening probability of 0.85 vs 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K(1) for hGluK2, like the EC(50) value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms share 99% sequence homology.

Dataset Information

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles.

Publications

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles.

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