Nanoscale Mobility of the Apo State and TARP Stoichiometry Dictate the Gating Behavior of Alternatively Spliced AMPA Receptors

Abstract

Neurotransmitter-gated ion channels are allosteric proteins that switch on and off in response to agonist binding. Most studies have focused on the agonist-bound, activated channel while assigning a lesser role to the apo or resting state. Here, we show that nanoscale mobility of resting a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPA receptors) predetermines responsiveness to neurotransmitter, allosteric anions and TARP auxiliary subunits. Mobility at rest is regulated by alternative splicing of the flip/flop cassette of the ligand-binding domain, which controls motions in the distant AMPA receptor N-terminal domain (NTD). Flip variants promote moderate NTD movement, which establishes slower channel desensitization and robust regulation by anions and auxiliary subunits. In contrast, greater NTD mobility imparted by the flop cassette acts as a master switch to override allosteric regulation. In AMPA receptor heteromers, TARP stoichiometry further modifies these actions of the flip/flop cassette generating two functionally distinct classes of partially and fully TARPed receptors typical of cerebellar stellate and Purkinje cells.

Genes associated with cognitive performance in the Morris water maze: an RNA-seq study

“…First of all, we should highlight the significant difference in isoform usage for genes Gria1 and Gria2 (and a marginally significant difference for genes Gria3 and Gria4) among alternative splicing events: higher production of the "flip" splice variant and lower production of the "flop" splice variant in the HP group than in the LP group. The "flip" isoform of the protein is desensitized more slowly and recovers from desensitization more rapidly, but AMPA receptor affinity for glutamate remains unchanged [47][48][49] . Besides, it is known that the ratio of these isoforms depends on the activity of cells: chronic activity deprivation by a Na + -channel blocker shifts the ratio of the alternatively spliced transcripts toward the "flop" splice variant, at least in the CA1 subfield of the hippocampus 50 .…”

Measurements of the Timescale and Conformational Space of AMPA Receptor Desensitization

“…It is likely that reformation of the interface at rest is at least as fast, otherwise the majority of receptors should simply desensitize upon binding glutamate. It is likely that the apo state is mobile and that the stability of the D1 dimer interface varies between flip and flop isoforms, impacting AMPA receptor kinetics ( 47 ). A second, less likely, effect of CTZ would be to reduce the conformational dynamics of the lower lobe of the LBD (where A665C is located) in the resting state.…”

Activation of P2X4 receptors induces an increase in the area of the extracellular region and a decrease in receptor mobility

“…For each axis ( x , y ) a Gaussian distribution through the particle reflecting the counts for brightness was assumed and the coordinates for the particle location were identified using imagej software (Rasband WS, US National Institutes of Health, Bethesda, ML, USA). The coordinates for the centre of mass of the particle for consecutive images were collected, and particle diffusion was monitored using the cumulative squared displacement (CSD; [16]), which was calculated according to the equation:where x is the distance between the centres of the particles.…”

“…Fast-scan AFM has the additional advantage of generating images with a temporal resolution of~1 s, thus enabling the direct observation of structural changes in single particles in response to manipulations such as ligand application. For example, ionotropic glutamate receptors have been shown to undergo changes in height and internal mobility in response to activation [15,16].…”