Crosslinking glutamate receptor ion channels
Publikation: Bidrag til bog/antologi/rapport › Bidrag til bog/antologi › Forskning › fagfællebedømt
Combining crosslinking strategies with electrophysiology, biochemistry, and structural in silico analysis is a powerful tool to study transient movements of ion channels during gating. This chapter describes crosslinking in living cells using cysteine and photoactive unnatural amino acids (UAAs) that we have used on glutamate receptor ion channels. Here, we share the protocol for building a perfusion tool to enable rapid chemical modification of glutamate-gated AMPA receptors, optimized for their fast activation. This system can be used to perform state-dependent crosslinking in receptors modified by cysteines or UAA incorporation on the millisecond timescale. Introducing UAAs results in receptors with lower expression levels relative to the introduction of cysteine residues. Reduced expression is principally a challenge for biochemical studies, and we share here our approach to capture the light driven oligomerization of AMPA receptors containing UAA crosslinkers. Finally, we describe strategies for computational analysis to make sense of the crosslinking results in terms of structure and function.
| Originalsprog | Engelsk |
|---|---|
| Titel | Methods in Enzymology |
| Antal sider | 30 |
| Forlag | Elsevier |
| Publikationsdato | 2021 |
| Sider | 161-192 |
| DOI | |
| Status | Udgivet - 2021 |
| Navn | Methods in Enzymology |
|---|---|
| Vol/bind | 652 |
| ISSN | 0076-6879 |
Bibliografisk note
Funding Information:
We thank Jelena Baranovic, Hector Salazar, Valentina Ghisi, Viktoria Klippenstein and Anahita Poshtiban for their refinements to the protocols. We thank Thomas Sakmar (Rockefeller) for the gift of optimised tRNAs and RNA-synthetase constructs. We thank Hector Salazar and Eva Meyer-Keller for some of the photographs of the glass tools in Figs. 4 and 6. This work was funded by DFG (PL 619/7-1, Heisenberg Professor and Cluster of Excellence NeuroCure EXC2049) and ERC (CoG ?GluActive? 647895). We thank Sam Goodchild and Chris Ahern for their advice on UV crosslinking and Mark Mayer for comments on the perfusion tool protocol.
Funding Information:
We thank Jelena Baranovic, Hector Salazar, Valentina Ghisi, Viktoria Klippenstein and Anahita Poshtiban for their refinements to the protocols. We thank Thomas Sakmar (Rockefeller) for the gift of optimised tRNAs and RNA-synthetase constructs. We thank Hector Salazar and Eva Meyer-Keller for some of the photographs of the glass tools in Figs. 4 and 6 . This work was funded by DFG (PL 619/7-1, Heisenberg Professor and Cluster of Excellence NeuroCure EXC2049) and ERC (CoG “GluActive” 647895). We thank Sam Goodchild and Chris Ahern for their advice on UV crosslinking and Mark Mayer for comments on the perfusion tool protocol.
Publisher Copyright:
© 2021 Elsevier Inc.
ID: 273635608