Crosslinking glutamate receptor ion channels
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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Crosslinking glutamate receptor ion channels. / Plested, Andrew J.R.; Poulsen, Mette H.
Methods in Enzymology. Elsevier, 2021. p. 161-192 (Methods in Enzymology, Vol. 652).Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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TY - CHAP
T1 - Crosslinking glutamate receptor ion channels
AU - Plested, Andrew J.R.
AU - Poulsen, Mette H.
N1 - Publisher Copyright: © 2021 Elsevier Inc.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - Electrophysiology
KW - Fast perfusion
KW - Piezo
KW - Unnatural amino acids
KW - UV light
U2 - 10.1016/bs.mie.2021.03.005
DO - 10.1016/bs.mie.2021.03.005
M3 - Book chapter
C2 - 34059281
AN - SCOPUS:85103727538
T3 - Methods in Enzymology
SP - 161
EP - 192
BT - Methods in Enzymology
PB - Elsevier
ER -
ID: 273635608