Elsevier

Neuropharmacology

Volume 45, Issue 6, November 2003, Pages 768-776
Neuropharmacology

Mutations in the ligand-binding and pore domains control exit of glutamate receptors from the endoplasmic reticulum in C. elegans

https://doi.org/10.1016/S0028-3908(03)00274-0Get rights and content

Abstract

The abundance of ion channels and neurotransmitter receptors in the plasma membrane is limited by the efficiency of protein folding and subunit assembly in the endoplasmic reticulum (ER). The ER has a quality-control system for monitoring nascent proteins, which prevents incompletely folded and assembled proteins from being transported from the ER. Chaperone proteins identify unfolded and misassembled proteins in the ER via retention motifs that are normally buried at intersubunit contacts or via carbohydrate residues that are attached to misfolded domains. Here, we examined the trafficking of a C. elegans non-NMDA glutamate receptor (GLR-1). We show that mutations in the pore domain (predicted to block ion permeation) and mutations in the ligand-binding domain (predicted to block glutamate binding) both caused a dramatic reduction in the synaptic abundance of GLR-1 and increased retention of GLR-1 in the ER. These results suggest that the structural integrity of the ligand-binding site and the pore domain of GLR-1 are monitored in the ER during the process of quality control.

Introduction

A variety of cellular functions, among these neuronal signaling, depends on the proper functioning of ion channels. A key event in the early trafficking of ion channels is efficient export from the endoplasmic reticulum (ER). Newly synthesized proteins are subject to quality control mechanisms that restrict forward transport to correctly folded and assembled proteins (Ellgaard and Helenius, 2001, Ellgaard and Helenius, 2003). Misfolded and incompletely assembled proteins are retained in the ER and are often degraded. ER-resident chaperones promote protein folding and assembly and assist in the retention of incorrectly folded proteins. Deficiencies in protein folding and subunit assembly in the ER decrease total expression at the cell surface (Kopito, 1999, Ma and Jan, 2002) and may underlie the pathology of neurological and other diseases (Kim and Arvan, 1998, Aridor and Balch, 1999, Dobson, 1999, Soto, 2003).

Prior work on quality control mechanisms has focused on identifying signals or covalent modifications that lead to retention of ion channels and other membrane proteins in the ER. Misfolded domains are marked by the attachment of N-linked carbohydrates and subsequently recognized by a lectin-based chaperone system in the ER (Trombetta and Helenius, 1998). Specific examples of retention motifs include dibasic residues in cytosolic domains (Zerangue et al., 1999, Margeta-Mitrovic et al., 2000) and polar residues in membrane spanning domains (Bonifacino et al., 1991, Cosson et al., 1991). Masking of these retention motifs is thought to be coupled to folding and assembly of membrane proteins; consequently, these motifs are selectively exposed in misfolded or misassembled proteins, which are subsequently retained in the ER or subjected to ER-associated degradation. Since single subunits can dictate the permeability and agonist sensitivity of ion channels (Dingledine et al., 1992, Jonas and Burnashev, 1995, Schwarz et al., 2001), the functional properties are dependent on the proper stoichiometric assembly of subunits. Many studies have focused on defining these properties on a molecular level. Ion permeability is determined by specific residues of the channel-lining pore (Dingledine et al., 1999, Kuner et al., 2003), and agonist sensitivity depends on the molecular identity of the ligand-binding domain (Kuusinen et al., 1995, Lampinen et al., 1998, Kuusinen et al., 1999, Abele et al., 2000). Since neuronal excitability and other cellular functions are critically dependent on specific ion channel properties, it may be advantageous to survey these properties early in the secretory pathway. Could they be subject to quality control in the ER?

Here, we test this idea by examining the trafficking of the C. elegans glutamate receptor GLR-1 upon limiting ion permeation and ligand binding. Glutamate receptors (GluRs) constitute the major neurotransmitter receptor at excitatory synapses and their channel properties help to shape the strength of a synapse. GluRs are heteromeric glutamate-activated cation channels (Dingledine et al., 1999) and mutations in pore and ligand-binding domains have been shown to change their functional properties (Dingledine et al., 1992, Lampinen et al., 1998, Kuusinen et al., 1999, Abele et al., 2000). Here, we examine the effects of these mutations on the trafficking of GLR-1.

Section snippets

Strains

Standard methods were used to culture the following alleles and transgenes: eat-4(n2474),unc-11(e47) glr-1(n2461), nuIs114 GLR-1::YFP, nuIs96 GLR-1(E770A)::YFP, nuIs116 GLR-1(AAA)::YFP, nuIs105 GLR-1(Q651D)::YFP.

Transgenes and germline transformation

Plasmids were constructed by standard techniques, and sequences were verified where appropriate; full details are available on request. Many plasmids contain fragments derived from vectors kindly provided by A. Fire. Transgenic strains were isolated by microinjecting various plasmids

Glutamate-binding site and pore mutations generate inactive GLR-1 receptors

To test the effects of ligand binding on GLR-1 trafficking, we introduced mutations into the ligand-binding domain of GLR-1. The ligand-binding site of GluRs consists of sequences in the first and second ectodomains, termed S1 and S2. A soluble fusion protein in which the S1 and S2 domains are connected by a short linker sequence has ligand-binding characteristics that are similar to intact GluRs (Armstrong et al., 1998, Kuusinen et al., 1999, Armstrong and Gouaux, 2000, Paas et al., 2000).

Discussion

We examined how mutations in the ligand-binding site and pore domain affected trafficking of GLR-1 receptors. Our work leads to three conclusions: First, mutant GLR-1 receptors that cannot bind glutamate or that cannot permeate ions are less abundant at synapses. Second, these mutant receptors are retained in the ER. Third, inactive receptors do not participate in membrane recycling at synapses. These results, together with prior studies on rodent GluRs, suggest that the ER quality control

Acknowledgements

This work was supported by a research grant from the National Institutes of Health to J.K. (NS32196). M.G. was supported by a fellowship from the American Heart Association (California Division). We thank the following for strains and reagents: A. Fire, O. Hobert, M. Nonet, The C. elegans Genetic Stock Center, and A. Coulson. We also thank Lars Dreier and Jeremy Dittman for help, advice and reagents, and members of the Kaplan lab for comments on the manuscript.

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