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.

Fig. 1.

Current responses of homomeric GluR2Q AMPA receptor channels in outside-out patches to fast application of glutamate and kainate. A, Schematic drawing of fast application of agonist to excised outside-out patch with a piezo-driven double-barreled application pipette. Inset shows an open tip response caused by a 1 msec solution change between normal Ringer's solution and 10% normal Ringer's solution.B, C, Currents evoked by 100 msec pulses of 1 mm glutamate and 1 mm kainate in outside-out membrane patches excised from oocytes injected with cRNAs of flip (B) and flop (C) variants of the unedited form of GluR2 (GluR2Q). Glutamate and kainate responses were obtained from the same patch. Duration of agonist application is indicated by the rectangular pulse at thetop of the current traces. Membrane potential was held at −100 mV.

Fig. 2.

Dose–response relationships for responses of GluR2Q flip (A) and flop (B) AMPA receptor channels to glutamate. The relative peak amplitudes of the current activated by each glutamate concentration, with the amplitude to 1 mm glutamate as a reference, are plotted against the concentration of glutamate. Eachcircle and bar represent the mean ± SEM of the relative amplitude of glutamate response obtained from five to 10 patches. Data were fitted to the equation I =I_max/[1 + (EC₅₀/C)ⁿ], whereI is the peak amplitude of glutamate response,I_max is the maximal response, andC is the concentration of glutamate. EC₅₀ is the concentration producing a half maximal response, andn is the Hill coefficient. The Hill coefficients obtained by the best nonlinear least-squares fit were 1.08 (flip) and 1.10 (flop), and EC₅₀ values were 1.39 (flip) and 1.38 (flop) mm. Insets show current responses induced by 100 (flip) and 50 (flop) msec pulses of 0.1, 1, and 10 mm glutamate. Membrane potential was held at −100 mV.

Fig. 3.

Dependence of desensitization time constant on agonist concentrations. A, B, Current responses to 0.1 (a), 1 (b), and 10 (c) mm glutamate in outside-out patches expressing GluR2Q flip (A) and flop (B) AMPA receptor channels. Each trace is the average of 10 responses (thin lines). The time constant for desensitization (τ) was obtained by fitting the decay of the current response with a single exponential function (thick lines). In the graphs in d, the means ± SEM of the desensitization time constant were plotted against the concentration of glutamate. Data were pooled from four to 10 patches.

Fig. 4.

Kinetics of desensitization and deactivation in GluR2Q receptor channels. A, B, Superimposed current traces evoked by 1, 10, and 100 msec pulses of 1 mm glutamate in flip (A) and flop (B) variants. All traces are averages of 10 responses. The decay time constants of current responses after cessation of 1 msec glutamate pulse and during 100 msec pulse were defined as deactivation and desensitization time constants, respectively. Rectangular steps above traces indicate the duration of glutamate application. C, Comparison of desensitization time constants between flip (5.89 ± 0.17 msec,n = 50) and flop (1.18 ± 0.05 msec,n = 37) variants. D, Comparison of deactivation time constants between flip (0.62 ± 0.06 msec,n = 10) and flop (0.54 ± 0.05 msec,n = 10) variants. There was a significant difference in the desensitization time constant between flip and flop variants (p < 0.001, Student'st test), whereas no differences were observed in the deactivation time constant (p > 0.3).

Fig. 5.

Glutamate-activated currents simulated by the kinetic model. A, Model used to describe kinetic reactions of GluR2Q flip AMPA receptor channels. The model assumes two binding sites for glutamate, three closed states, three desensitized states, and one open state. Thus, it has a total of seven states in which C is the unliganded closed state,CA is the single-liganded closed state,CA2 is the double-liganded closed state,OA2 is the double-liganded open state, D1is the single-liganded desensitized state, and D2 andD3 are double-liganded desensitized states.c denotes glutamate concentration. The rate constants of the respective transitions were obtained by fitting to the actual current responses using a combination of trial and error with optimization. The kinetic responses were described satisfactorily with the following rate constant values: k1 = 1.8 × 10⁶m⁻¹sec⁻¹; k2 = 2.4 × 10³ sec⁻¹;k3 = 1.0 × 10⁷m⁻¹ sec⁻¹;k4 = 1.0 × 10⁴sec⁻¹; k5 = 1.6 × 10⁴ sec⁻¹;k6= 5.0 × 10³ sec⁻¹;k7 = 7.0 × 10²sec⁻¹; k8 = 1.5 × 10² sec⁻¹;k9 = 1.0 × 10²sec⁻¹; k10 = 2.1 sec⁻¹; k11 = 3.0 × 10² sec⁻¹;k12 = 1.5 × 10 sec⁻¹; k13 = 1.0 × 10⁷m⁻¹sec⁻¹; k14 = 1.0 × 10³ sec⁻¹;k15 = 1.6 × 10⁴sec⁻¹; and k16 = 1.2 × 10⁴ sec⁻¹.B, Simulated responses calculated using the kinetic model with the values of transition rate constants described inA (broken lines) were superimposed on actual current responses of GluR2Q flip AMPA receptor channels to 1, 5, 10, and 20 msec pulses of 1 mm glutamate (thin continuous lines). The peak amplitude of 120 pA obtained experimentally was adjusted to the maximum open probability of 0.32 in the simulated responses.

Fig. 6.

Recovery of GluR2Q flip (A) and flop (B) AMPA receptor channels from desensitization produced by a brief glutamate pulse. a, Superimposed current traces evoked by two 1 msec pulses of 1 mm glutamate separated by different intervals. Each response to the second pulse is the average of three responses.b, Time course of recovery from desensitization. The amplitude of the second response relative to the first is plotted against the interval. Each circle and barrepresent the mean ± SEM of the relative amplitude of the second response obtained from six experiments in both flip and flop variants. The recovery time course was fitted with a single exponential function. The time constants for recovery were 11.7 msec in flip and 31.3 msec in flop. Membrane potential was held at −100 mV.

Fig. 7.

Effects of CTZ on responses to glutamate in GluR2Q flip (A) and flop (B) AMPA receptor channels. Current responses to 500 msec pulses of 1 mm glutamate in the presence and absence of 100 μm CTZ are superimposed.Insets show the initial portion of the response with a faster sweep speed. When CTZ was applied, the control saline also contained 100 μm CTZ. Membrane potential was held at −100 mV.

Fig. 8.

Recovery from desensitization of GluR2Q flip (A) and flop (B) AMPA receptor channels produced by a brief glutamate pulse in the presence of CTZ. a, Superimposed current traces evoked by two 1 msec pulses of 1 mm glutamate separated by different intervals in the presence of 100 μm CTZ. Each response to the second pulse is the average of three responses. b, Time course of recovery from desensitization. The amplitude of the second response relative to the first is plotted against the interval. Each circle and bar represent the mean ± SEM of the relative amplitude of the second response obtained from four to six experiments. Broken linesindicate the time courses of recovery in the absence of CTZ, which are shown in Figure 6. Note that CTZ almost completely abolished the reduction of the second response in both flip and flop variants. Membrane potential was held at −100 mV.

Fig. 9.

Current responses of heteromeric GluR2Q/GluR2R receptor channels. A, I–V relationships of responses to glutamate obtained from outside-out patches excised from oocytes injected with the unedited (Q) and edited (R) forms of GluR2 cRNA at a ratio of 1:4. Both GluR2Q and GluR2R were of flip form in a and of flop form in b.I–V relationships were obtained by plotting the peak amplitudes of glutamate-induced currents against the membrane potential between −100 and +80 mV in 20 mV steps. The current traces (average of 3 responses to 1 mm glutamate) at the different membrane potentials are superimposed in the insets.B, Kinetics of desensitization and deactivation in heteromeric GluR2Q/GluR2R (1:4) receptor channels. Current responses to brief (1 msec) and long (100 msec) glutamate pulses (average of 10 responses) are superimposed for flip in a and for flop in b. Membrane potential was held at −100 mV.c, Comparison of desensitization time constants between flip (6.40 ± 0.50 msec, n = 6) and flop (1.00 ± 0.07 msec, n = 6) variants.d, Comparison of deactivation time constants between flip (0.63 ± 0.14 msec, n = 5) and flop (0.58. ± 0.06 msec, n = 6) variants. There was a significant difference in the desensitization time constants between flip and flop variants (p < 0.001, Student's t test), whereas no differences were observed in the deactivation time constants (p > 0.3).