Molecular determinants responsible for differences in desensitization kinetics of AMPA receptor splice variants

Abstract

Flip (i) and flop (o) alternatively spliced variants of the four glutamate AMPA receptor subunits (GluR1-4) are differentially expressed in the CNS and can display distinct rates of desensitization that contribute to the heterogeneity of native AMPA receptor-dependent synaptic responses. In the present study, we initially compared the kinetics of desensitization in response to fast application of glutamate (1 mm) for the eight different homomeric recombinant human AMPA receptors (hGluR1-4i and o) heterologously expressed in mammalian cells. Consistent with previous reports on recombinant rat AMPA receptors, the time constants of desensitization between human GluR1i and GluR1o receptors were the same, whereas the flip isoforms for GluR2-4 receptors exhibited significantly slower rates of desensitization compared with the flop isoforms. To identify the molecular determinants responsible for these functional differences, the effects of exchanging amino acid residues in the flip-flop cassette of GluR2i and GluR2o were investigated. Three amino acid residues in the flip-flop region (Thr765, Pro766, and Ser775 in flip and Asn765, Ala766, and Asn775 in flop) were identified that contribute to splice-variant differences in the rate of desensitization. Recent structural data show that these three residues are located on helix J, which forms part of the intradimer interface of AMPA receptor ligand-binding cores, and that the stability of this interface may regulate desensitization. The present results suggest that these three residues may confer differences in flip and flop receptor desensitization rates by directly and/or indirectly influencing the stability of the interface between adjacent subunits.

Figure 1.

Desensitization kinetics for human homomeric AMPA receptors. A, Bar graph of the time constants of desensitization (mean ± SEM). Open bars represent flip isoform, and closed bars represent flop isoform. Asterisks indicate that for GluR2-4, the τ_DES for the flop isoform is significantly faster than for the flip isoform (p < 0.001). B, Representative traces of GluR2i and GluR2o. Calibration bar, 10 msec.

Figure 2.

Desensitization kinetics are sensitive to the exchange of three residues in GluR2i and GluR2o. A, Sequence alignment of the flip and flop domains for GluR1-4. Numbering is based on GluR2 (accession number NP_000817), showing four regions of heterogeneity (regions 1, 2, 3, and Val/Leu779 residue). B, Representative traces from mutant GluR2i with exchanges of region 1 (GluR2i_o,i,i), region 2 (GluR2i_i,o,i), V/L residue (GluR2i_V779L), region 3 (GluR2i_i,i,o), and region 1 and 2 (GluR2i_o,o,i). Traces from GluR2i and GluR2o are superimposed in gray for comparison. Above each trace, open rectangles indicate GluR2i residues, and filled rectangles indicate GluR2o residues. C, Representative traces from GluR2i_T765N and GluR2i_P765A. Trace from GluR2i_o,i,i is superimposed in gray for comparison. D, Bar graph of the τ_DES (mean ± SEM) for mutant GluR2i. Asterisks indicate mutant receptors that have faster τ_DES than GluR2i (p < 0.001). The mean τ_DES for GluR2i (R2i) and GluR2o (R2o) wild-type receptors are indicated for comparison. E, Representative traces from GluR2o_i,o,o, GluR2o_o,i,o, or GluR2o_i,i,o. Traces from GluR2i and GluR2o are superimposed in gray for comparison. Calibration bar, 10 msec. F, Bar graph of the τ_DES (mean ± SEM) for mutant GluR2o. Asterisks indicate mutant receptors having slower τ_DES than GluR2o (^*p < 0.05; ^**p < 0.001). The mean τ_DES for GluR2I and GluR2o wild-type receptors are indicated for comparison.

Figure 3.

Recovery from desensitization kinetics are sensitive to the exchange of three residues in GluR2i. A-C, Recovery from desensitization was evaluated by delivering pairs of 10 msec pulses of 1 mm glutamate separated by interstimulus intervals ranging from 20 to 500 msec. Traces represent superimposed responses from cells expressing GluR2i, GluR2o, and mutant GluR2i with exchanges of region 1 and 2 (GluR2i_o,o,i). Arrows denote on set of glutamate pulses. Calibration bar, 50 msec. D, The time course of recovery from desensitization was determined for each cell by measuring the amplitude of the second response (I₂) relative to the first response (I₁) of a pair of pulses and plotting these values as a function of interstimulus interval. The τ_REC for each cell was determined by fitting these recovery plots with a single exponential function. Statistically significant differences were found between τ_REC for GluR2i, GluR2o, and GluR2i_o,o,i (F_(2,27) = 7.2; p < 0.005). Post hoc comparisons showed that although the τ_REC for GluR2i_o,o,i was not different from the τ_REC for GluR2o (p > 0.05), both differed from the τ_REC for GluR2i (p < 0.05).