Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Abstract

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission 11 throughout the nervous system. Conformational changes of the transmembrane domain 12 (TMD) underlying ion channel activation and desensitization remain poorly understood.13 Here, we explored the dynamics of the TMD of AMPA-type iGluRs using genetically-14 encoded unnatural amino acid (UAA) photo-crosslinkers, p-benzoyl-L-phenylalanine 15 (BzF) and p-azido-L-phenylalanine (AzF). We introduced UAAs at sites throughout the 16 TMD of the GluA2 receptor and characterized these mutants in patch-clamp recordings, 17 exposing them to glutamate and UV light. This approach revealed a range of optical 18 effects on the activity of mutant receptors. We found evidence that an interaction 19 between the Pre-M1 and the M4 TMD helix was essential for normal activation and 20 desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter, had 21 extraordinarily broad effects on gating and desensitization. This observation suggests 22 coupling to other parts of the receptor and like in other tetrameric channels, selectivity 23 filter gating.

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

“…This is particularly true for complex proteins expressed in eukaryotic cells. In fact, many groups have repeatedly observed yields of 10% or less with ncAA incorporation into transporters 37 , ion channels [38][39][40] , and G protein-coupled receptors 41,42 . Although the generally low yields observed with tPTS likely restrict the approach to applications that do not require large amounts of protein, at least some of the above limitations can be addressed by engineering more promiscuous and efficient split inteins [10][11][12]43 or by adding affinity tags to promote split intein interactions 18 .…”

Mechanism and site of action of big dynorphin on ASIC1a

“…-B)(32)(33)(34). Incorporating AzF at positions lining the ASIC1a-BigDyn interaction interface should allow covalent trapping of the complex and enable subsequent visualization using western blotting.…”

β11-12 linker isomerization governs Acid-sensing ion channel desensitization and recovery

“…The resulting free radical 270 spontaneously forms a covalent bond with a nearby atom, preferentially reacting with C-H bonds. 271 Incorporation and trapping by Bpa has been previously used to investigate conformational 272 changes in AMPA receptors (Klippenstein et al, 2014;Poulsen et al, 2019) and K + channels 273 (Murray et al, 2016;Westhoff et al, 2017). in a convincing fashion.…”