Clathrin-mediated endocytosis is required for compensatory regulation of GLR-1 glutamate receptors after activity blockade.

Chronic changes in neural activity trigger a variety of compensatory homeostatic mechanisms by which neurons maintain a normal level of synaptic input. Here we show that chronic activity blockade triggers a compensatory change in the abundance of GLR-1, a Caenorhabditis elegans glutamate receptor. In mutants lacking a voltage-dependent calcium channel (unc-2) or a vesicular glutamate transporter (VGLUT; eat-4), the abundance of GLR-1 in the ventral nerve cord was increased. Similarly, the amplitude of glutamate-evoked currents in ventral cord interneurons was increased in eat-4 VGLUT mutants compared with wild-type controls. The effects of eat-4 VGLUT mutations on GLR-1 abundance in the ventral cord were eliminated in double mutants lacking both the clathrin adaptin protein unc-11 AP180 and eat-4 VGLUT. In contrast, mutations that decreased ubiquitination of GLR-1 did not prevent increased ventral cord abundance of GLR-1 in eat-4 VGLUT mutants. Taken together, our results suggest that GLR-1 is regulated in a homeostatic manner and that this effect depends on clathrin-mediated endocytosis but does not require ubiquitination of GLR-1.

Molecular determinants of KCNQ1 channel block by a benzodiazepine.

KCNQ1 channels underlie the slow delayed rectifier K+ current, mediate repolarization of cardiac action potentials, and are a potential therapeutic target for treatment of arrhythmia. (E)-(+)-N-[(3R)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3-(2,4-dichlorophenyl)-2-propenamide [L-735821 (L-7)] is a potent blocker of KCNQ1 channels. Here we describe the structural determinants of KCNQ1 that are critical for high-affinity block by L-7 using site-directed mutagenesis to alter specific residues and voltage clamp to record channel currents in Xenopus laevis oocytes. Chimeric channels were constructed by combination of regions from L-7-sensitive KCNQ1 and L-7-insensitive KCNQ2 channel subunits. This approach localized the drug interaction site to the pore and S6 domains of KCNQ1. Substitution of single amino acids identified Thr-312 of the pore domain and Ile-337, Phe-339, Phe-340, and Ala-344 of the S6 domain as the most important molecular determinants of channel block. Some mutations also altered the inactivation properties of KCNQ1, but there was no correlation between extent of inactivation and sensitivity to block by L-7. Modeling was used to simulate the docking of L-7 to the KCNQ1 channel pore. The docking was consistent with our experimental data and predicts that L-7 blocks K+ conductance by physically precluding the occupancy of a K+ ion to a pore helix-coordinated site within the central hydrated cavity, a crucial step in ion permeation.

Glutamate receptor subunit 3 is modified by site-specific limited proteolysis including cleavage by gamma-secretase.

Ionotropic glutamate receptor (GluR) expression and function is regulated through multiple pre- and post-translational mechanisms. We find that limited proteolytic cleavage of GluR3 at two distinct sites generates stable GluR3 short forms that are glycosylated and found in association with other full-length GluRs in the mouse brain and cultured primary neurons. A combination of mutagenesis and transfection into HEK293 cells revealed cleavage by a gamma-secretase-like activity within the membrane-localized re-entry loop at or near the leucine-glycine pair (amino acids 585-586, GluR3sbeta) and a second site within a proline-rich PEST-like sequence in the first cytoplasmic loop (Asp570-Pro571, GluR3salpha). Generation of the prominent GluR3salpha form was effectively abolished in the mutant, GluR3D570A, but inhibitors of lysosomes, the proteasome, caspases, or calpains had no effect. The possible impact of cleavage on receptor function was suggested when the co-expression of the GluR3P571Stop mutant (creating GluR3salpha) co-assembled with other GluR subunits and decreased receptor function in Xenopus oocytes. In transiently transfected HEK293 cells, co-expression of GluR3salpha alters the relative association between GluR1 and GluR3 during assembly, and the presence of the novel C-terminal proline-rich domain of GluR3salpha imparts lateral membrane mobility to GluR complexes. These results suggest that limited proteolysis is another post-translational mechanism through which functional diversity and specialization between closely related GluR subunits is accomplished.

Kainate-binding proteins are rendered functional ion channels upon transplantation of two short pore-flanking domains from a kainate receptor.

Kainate-binding proteins belong to an elusive class of putative ionotropic glutamate receptors that to date have not been shown to form functional ion channels in heterologous expression systems, despite binding glutamatergic agonists with high affinity. To test the hypothesis that inefficient or interrupted signal transduction from the ligand-binding site via linker domains to the ion pore (gating) might be responsible for this apparent lack of function, we transplanted the short homologous linker sequences from the fully functional rat kainate receptor GluR6 into frog kainate-binding protein. We were able to generate chimeric receptors that are functional in the Xenopus oocyte expression system and in human embryonic kidney 293 cells. The linker domains A and B in particular appear to be crucial for gating, because a functional kainate-binding protein was observed when at least parts of both linkers were derived from GluR6. We speculate that to enable signal transduction from the ligand-binding site to the ion pore of the frog kainate-binding protein, the linker structure of the protein has to undergo an essential conformational alteration, possibly mediated by an as yet unknown subunit or modulatory protein.