Regulation of Kinetic Properties of GluR2 AMPA Receptor Channels by Alternative Splicing

Abstract

The four subunits of the AMPA-type glutamate receptor (GluR1-GluR4 or GluR-A-GluR-D) exist in two distinct forms, flip and flop, generated by alternative splicing of a 115 bp region. The GluR2 subunit plays a key role in determining the functional properties of the AMPA receptor channel. In this study, we examined the differences in kinetic properties between the flip and flop splice variants of the GluR2 subunit expressed in Xenopus oocytes using fast agonist application techniques. Glutamate was applied to outside-out patches from oocytes with piezo-driven double-barreled application pipettes. Because homomeric receptor channels composed of the edited form of GluR2 (GluR2R) produce no appreciable current responses, we expressed the unedited form of GluR2 (GluR2Q) in oocytes, which produced large current responses sufficient for analysis of the kinetic properties. The time constant for desensitization during application of 1 mM glutamate was 5.89 +/- 0. 17 msec (n = 50) in flip and 1.18 +/- 0.05 msec (n = 37) in flop. The deactivation time constant was 0.62 +/- 0.06 msec (n = 10) in flip and 0.54 +/- 0.05 msec (n = 10) in flop. The steady-state nondesensitizing current was 6.8 +/- 0.4% (n = 53) of the peak current in flip, whereas it was almost negligible in flop, being only 1.1 +/- 0.1% (n = 36). The slower desensitization kinetics and larger steady-state current responses in the flip variant were also observed in heteromeric receptors assembled from GluR2Q/GluR2R. Thus, desensitization occurred much more prominently in the flop variant in the recombinant GluR2 receptor channels.

Mechanisms Underlying Developmental Speeding in AMPA-EPSC Decay Time at the Calyx of Held

“…Recombinant AMPARs composed of GluR2 flop or GluR4 flop have a faster desensitization time than those composed of the flip form, whereas deactivation time is similar between flip-and flop-form GluRs (Mosbacher et al, 1994;Koike et al, 2000). Consistently, in neocortical neurons, the abundance of GluR flop is associated with the fast desensitization time of AMPA patch currents, whereas it is unrelated with the deactivation time (Lambolez et al, 1996).…”

“…Double-barreled application pipettes were fabricated from theta glass tubes (outer diameter, 2.0 mm; TGC200-10; Harvard Apparatus) pulled for a tip diameter of 200 -300 m (Koike et al, 2000). Control and test solutions were continuously passed through each barrel of the application pipette using static pressure.…”

“…Recombinant AMPARs composed of GluR4 flop (Mosbacher et al, 1994) or GluR2 flop (Koike et al, 2000) show the fastest desensitization among those composed of different GluR subunits. In the cerebral cortex, AMPAR subunit mRNAs in pyramidal cells are mostly in the flip form, and those in nonpyramidal cells are in the flop form (Lambolez et al, 1996).…”

“…The GluR4 flop abundance was correlated with the qEPSC decay time only at immature synapses. Compared with GluR flip, GluR flop AMPARs have a faster desensitization time but similar deactivation time (Mosbacher et al, 1994;Koike et al, 2000). Also, the abundance of GluR flop is associated with fast desensitization but not with deactivation of the AMPA patch currents in neocortical neurons (Lambolez et al, 1996).…”

Block of AMPA Receptor Desensitization by a Point Mutation outside the Ligand-Binding Domain

“…In terms of the %des, this effect was strongest in the GluR-B i background, but the fold increase for plateau/peak ratios was approximately the same in all backgrounds (Table 1). Indeed, the %des for wild-type GluR-6, GluR-A i , and GluR-B i (Table 1) decreased in parallel with the ratio of the rate of exit from (␥) and entry into (␦) desensitization [␥/␦ Ӎ 10 Ϫ5 -10 Ϫ4 for GluR-6 (Bowie et al, 1998), 10 Ϫ3 for GluR-A i (Partin et al, 1996;Robert and Howe, 2003), and 10 Ϫ2 -10 Ϫ1 for GluR-B i (Koike et al, 2000)]. In addition, the subunit-specific effect of the R 3 E substitution can be simulated by the same modification of the wildtype models, specifically increasing the rate of exit from the desensitized state by ϳ10 2 times as well as decreasing the rate of agonist dissociation (k off ) by ϳ10 times (data not shown).…”