Reconstitution of invertebrate glutamate receptor function depends on stargazin-like proteins

Abstract

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are a major subtype of ionotropic glutamate receptors (iGluRs) that mediate rapid excitatory synaptic transmission in the vertebrate brain. Putative AMPARs are also expressed in the nervous system of invertebrates. In Caenorhabditis elegans, the GLR-1 receptor subunit is expressed in neural circuits that mediate avoidance behaviors and is required for glutamate-gated current in the AVA and AVD interneurons. Glutamate-gated currents can be recorded from heterologous cells that express vertebrate AMPARs; however, when C. elegans GLR-1 is expressed in heterologous cells, little or no glutamate-gated current is detected. This finding suggests that other receptor subunits or auxiliary proteins are required for function. Here, we identify Ce STG-1, a C. elegans stargazin-like protein, and show that expression of Ce STG-1 together with GLR-1 and the CUB-domain protein SOL-1 reconstitutes glutamate-gated currents in Xenopus oocytes. Ce STG-1 and homologues cloned from Drosophila (Dro STG1) and Apis mellifera (Apis STG1) have evolutionarily conserved functions and can partially substitute for one another to reconstitute glutamate-gated currents from rat, Drosophila, and C. elegans. Furthermore, we show that Ce STG-1 and Apis STG1 are primarily required for function independent of possible roles in promoting the surface expression of invertebrate AMPARs.

Fig. 1.

C. elegans STG-1, Drosophila STG1, and Apis STG1 are related to members of the TARP family. (A) The predicted amino acid sequences encoded by C. elegans stg-1, Drosophila stg1, vertebrate stg, and A. mellifera stg1. Amino acids are numbered beginning with the first predicted methionine. For Ce STG-1, bars and asterisks indicate predicted transmembrane domains and putative N-linked glycosylation sites, respectively. Underlined in gray are the potential type I PDZ-domain-binding sites for vertebrate stargazin and Apis STG1. (B) Phylogenetic tree of the amino acid sequences for stargazin-like proteins (figure modified from ref. 16).

Fig. 2.

C. elegans STG-1 is expressed in the nervous system. Confocal images of transgenic worms that expressed Pstg-1::GFP (A) or both Pglr-1::CFP and Pstg-1::YFP (B) are shown. Expression is limited to neuronal cell bodies and processes.

Fig. 3.

GLR-1-mediated glutamate-gated currents in Xenopus oocytes are dependent on Ce STG-1 and SOL-1. (A) Currents recorded in response to 1 mM glutamate application in Xenopus oocytes injected with combinations of SOL-1, GLR-1, and C. elegans STG-1, vertebrate stargazin, Apis STG1, or Dro STG1 cRNA. Oocytes were voltage-clamped at a holding potential of −70 mV. (B) Average peak glutamate-gated current amplitude in oocytes coinjected with GLR-1, SOL-1, and Ce STG-1 cRNA as a function of Ce STG-1 (black). Values are also indicated for vertebrate stargazin, Apis STG1, and Dro STG1 (gray). GLR-1 plus SOL-1 plus 0.1 ng of Ce STG-1, n = 21; plus 0.5 ng of Ce STG-1, n = 19; plus 2.5 ng of Ce STG-1, n = 16; plus 12.5 ng of Ce STG-1, n = 18; plus vert. stargazin, n = 9; plus Apis STG1, n = 6; plus Dro STG1, n = 5. In A and B, oocytes were injected with 8.3 ng of GLR-1 and 8.3 ng of SOL-1 cRNA. (C) Glutamate-gated currents in Xenopus oocytes injected with rat GluR1 alone or coinjected with Ce STG-1, Apis STG1, Dro STG1, or vertebrate stargazin cRNAs. (D) Average peak glutamate-gated current amplitude as a function of stargazin cRNA. GluR1, n = 12; plus vert. stargazin, n = 5; plus Ce STG-1, n = 12; plus Apis STG1, n = 5; plus Dro STG1, n = 4. In C and D, oocytes were injected with 0.1 ng of rat GluR1 and 8.3 ng of the indicated stargazin cRNA. (E) Western blot showing the relative surface expression of C. elegans HA::GLR-1 in the presence or absence of Ce STG-1. (F) Coimmunoprecipitation of HA::GLR-1 and STG-1::MYC from HEK 293 cells. IB, immunoblot.

Fig. 4.

Glutamate-gated current in the absence of Ce STG-1 depends on modifying the gating properties of GLR-1. (A–C) Currents measured in response to 1 mM glutamate application in Xenopus oocytes coinjected with cRNAs for SOL-1 and GLR-1(Q552Y) (A), GLR-1(A687T) (B), or GLR-1(Q552Y; A687T) (C) both with (Upper) and without (Lower) Ce STG-1. Oocytes were voltage-clamped at −70 mV. (D) The ratio of the current amplitude 1 s after the start of glutamate application (steady state) to the peak current both with (black) and without (gray) Ce STG-1. GLR-1(Q/Y) plus SOL-1 plus Ce STG-1, n = 3; GLR-1(Q/Y) plus SOL-1, n = 3; GLR-1(A/T) plus SOL-1 plus Ce STG-1, n = 4; GLR-1(A/T) plus SOL-1, n = 4; GLR-1(Q/Y;A/T) plus SOL-1 plus Ce STG-1, n = 4; GLR-1(Q/Y;A/T) plus SOL-1, n = 6.

Fig. 5.

Apis and Drosophila STG1 differentially affect Dro GluRIA. (A) Currents measured in response to 1 mM glutamate application in Xenopus oocytes that expressed Dro GluRIA and various combinations of vertebrate stargazin, Ce STG-1, Dro STG1, Apis STG1, and Ce SOL-1. Oocytes were voltage-clamped at −70 mV. (B) Average peak current (black) and current amplitude 1 s after glutamate application (gray) for currents mediated by Dro GluRIA coexpressed with Apis STG1, n = 10. (C) Western blot showing the relative surface expression of HA::Dro GluRIA in the presence or absence of Apis STG1.