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Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.

ABSTRACT: RNA editing of kainate receptor subunits at the Q/R site determines their susceptibility to inhibition by cis-unsaturated fatty acids as well as block by cytoplasmic polyamines. Channels comprised of unedited (Q) subunits are strongly blocked by polyamines, but insensitive to fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), whereas homomeric edited (R) channels resist polyamine block but are inhibited by AA and DHA. In the present study, we have analyzed fatty acid modulation of whole-cell currents mediated by homomeric recombinant GluK2 (formerly GluR6) channels with individual residues in the pore-loop, M1 and M3 transmembrane helices replaced by scanning mutagenesis. Our results define three abutting surfaces along the M1, M2, and M3 helices where gain-of-function substitutions render GluK2(Q) channels susceptible to fatty acid inhibition. In addition, we identify four locations in the M3 helix (F611, L614, S618, and T621) at the level of the central cavity where Arg substitution increases relative permeability to chloride and eliminates polyamine block. Remarkably, for two of these positions, L614R and S618R, exposure to fatty acids reduces the apparent chloride permeability and potentiates whole-cell currents approximately 5 and 2.5-fold, respectively. Together, our results suggest that AA and DHA alter the orientation of M3 in the open state, depending on contacts at the interface between M1, M2, and M3. Moreover, our results demonstrate the importance of side chains within the central cavity in determining ionic selectivity and block by cytoplasmic polyamines despite the inverted orientation of GluK2 as compared with potassium channels and other pore-loop family members.

SUBMITTER: Wilding TJ

PROVIDER: S-EPMC2931155 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.

Wilding Timothy J TJ Chen Kevin K Huettner James E JE

The Journal of general physiology 20100901 3

RNA editing of kainate receptor subunits at the Q/R site determines their susceptibility to inhibition by cis-unsaturated fatty acids as well as block by cytoplasmic polyamines. Channels comprised of unedited (Q) subunits are strongly blocked by polyamines, but insensitive to fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), whereas homomeric edited (R) channels resist polyamine block but are inhibited by AA and DHA. In the present study, we have analyzed fatty acid modu ...[more]

PMID: 20805577

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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.

Dataset Information

Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.

Publications

Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.

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