Differential dependence on GluR2 expression of three characteristic features of AMPA receptors

Abstract

The GluR2 subunit controls three key features of ion flux through the AMPA subtype of glutamate receptors-calcium permeability, inward rectification, and channel block by external polyamines, but whether each of these features is equally sensitive to GluR2 abundance is unknown. The relations among these properties were compared in native AMPA receptors expressed by acutely isolated hippocampal interneurons and in recombinant receptors expressed by Xenopus oocytes. The shape of current-voltage (I-V) relations between -100 and +50 mV for either recombinant or native AMPA receptors was well described by a Woodhull block model in which the affinity for internal polyamine varied over a 1000-fold range in different cells. In oocytes injected with mixtures of GluR2:non-GluR2 mRNA, the relative abundance of GluR2 required to reduce the log of internal blocker affinity by 50% was two- to fourfold higher than that needed to half-maximally reduce divalent permeability or channel block by external polyamines. Likewise, in interneurons the affinity of externally applied argiotoxin for its blocking site was a steep function of internal blocker affinity. These results indicate that the number of GluR2 subunits in AMPA receptors is variable in both oocytes and interneurons. More GluR2 subunits in an AMPA receptor are required to maximally reduce internal blocker affinity than to abolish calcium permeability or external polyamine channel block. Accordingly, single-cell RT-PCR showed that approximately one-half of the physiologically characterized interneurons exhibiting inwardly rectifying AMPA receptors expressed detectable levels of edited GluR2. The physiological effects of a moderate change in GluR2 relative abundance, such as occurs after ischemia or seizures or after chronic exposure to morphine, thus will be dependent on the ambient GluR2 level in a cell-specific manner.

Fig. 3.

Variety and stability of internal blocker affinity in hippocampal interneurons. A, I–Vcurves from two different interneurons, selected to show the extremes of inward and outward rectification, and an interneuron showing intermediate rectification. The fixed stoichiometry model (open circles) consisting of the weighted average of the two extremeI–V curves (80% outward + 20% inwardly rectifyingI–V curves) could fit the inward but not outward limb of the intermediate I–V curve. The Woodhull model [filled circles;K_D(0)/[polyamine] = 1.48,z(1 − δ) = 1.11] fit well over both inward and outward limbs of the curve. B, Stability of measured internal blocker affinity during the first 10 min after achieving whole-cell voltage clamp. Kainate currents were evoked every minute in neurons selected for initially high internal blocker affinity. Eachpoint represents the mean and SEM from three to nine cells. The hatched bar illustrates the time window for sampling neurons for the single-cell RT-PCR analysis. C, Direct comparison of rectification properties of AMPA receptors expressed by interneurons and oocytes. Each pointrepresents measurements from a different interneuron (n = 354) or oocyte (n = 170). Plots of data from the two cell types are superimposable except for the extreme GluR2-lacking recombinant receptors, which appear to be absent from the interneuron population.

Fig. 1.

Comparison of the fixed stoichiometry and Woodhull models for intermediate I–V curves.A, I–V curves of kainate-evoked currents in oocytes injected with mixtures of GluR1, 2, and 3 mRNAs in the ratios indicated to the right of eachI–V. The open circles represent a weighted average of the 1:0:1 and 1:10:1 curves, with weighting factors chosen to fit the inward limb of the intermediate (5:2:5)I–V curve. The filled circles represent the Woodhull model fit of the 5:2:5 curve [K_D(0)/[polyamine] = 0.35,z(1 − δ) = 0.85]. Each I–Vcurve is the normalized average from three to five oocytes.B, Effect of varying the relative abundance of GluR2 on the shape of kainate I–V relations in oocytes injected with mRNA encoding GluR1:R2:R3. Each of the I–Vrelations was generated from oocytes obtained from a single frog, and the Woodhull fits (symbols) are superimposed. Note that the inward limb of the 1:10:1 and 1:6:1 I–V curves are superimposed. Each curve is the normalized average of three to five oocytes. C, Relation between the rectification ratio (slope conductance ratio measured at +40 and −70 mV) and apparent affinity of cytoplasmic polyamine blocker for the channel (n = 116 oocytes injected with mixtures of the mRNA combinations indicated). GluR2:R3 mRNAs were injected in the following ratios: 0:1, 1:10, 1:3, 1:1, 3:1, 6:1, and 10:1. GluR1:R2:R3 were injected in ratios of 1:0:1, 5:1:5, 5:2:5, 5:3.33:5, 1:2:1, 1:6:1, and 1:10:1. D, A family of I–V curves in oocytes injected with the indicated ratios of GluR2:GluR3 mRNAs. Each curve is the normalized average from several oocytes (n = 76 total), and the Woodhull fits (symbols) are superimposed. Inspection of the inward and outward limbs of the I–V curves shows that the degree of rectification of the 1:6 and 1:10 I–V curves cannot be described by the fixed stoichiometry model. Note that the inward limbs of the 3:1 and 1:1 I–V curves are superimposed.

Fig. 2.

Different sensitivity of three permeation characteristics of AMPA receptors to the relative abundance of GluR2.A, Oocytes isolated from a single frog were microinjected with GluR2 and GluR3 mRNAs at different molar ratios of GluR2 and GluR3. After the oocytes were cultured for 2–3 d, the following measurements were made from each cell:K_D(0)/[polyamine] as fit by the Woodhull equations,P_Ba/P_monovalentcalculated from reversal potential measurements in high Na⁺ and high Ba²⁺ medium, and the percentage block of kainate current at −70 mV elicited by 1 mm spermine. Data are expressed as a function of the GluR2 relative abundance, and the three measurements are scaled to permit comparisons. Each point represents the mean and SEM from four to six oocytes. B, Relation among the degree of block of kainate-evoked current by external spermine at −70 mV, the barium-to-monovalent permeability ratio, and the internal polyamine blocker affinity, as determined by the Woodhull model in oocytes injected with mixtures of GluR2 and 3 mRNAs. The ratio of GluR2 to GluR3 mRNAs was varied from 1:10 to 10:1 to produce receptors with a wide range of internal blocker affinity (n = 77 oocytes). C, Mixtures of GluR3(R612) and GluR3(Q612) were coinjected and a similar analysis performed as in A(n = 64 oocytes total). The dotted line, which represents the expected binomial abundance of receptors assembled with no GluR3(R) subunits, follows the equationf(x_o) = min + (max − min) · (1 − x)⁵, wheref(x_o) is the fraction of receptors without GluR3(R) subunits, as a function ofx = relative abundance of GluR3(R) protein in a functional receptor. Max and min represent the maximum and minimum GluR3(R)-dependent effect and were the only free variables.

Fig. 5.

AMPA receptor subunit expression in physiologically characterized interneurons. A, An interneuron with outwardly rectifying kainate I–Vrelation that expressed all four AMPA receptor subunits by RT-PCR. Theinset shows an agarose gel separation of RT-PCR products from this cell. The open circles are fits of the Woodhull equation [K_D(0)/[polyamine] = 6.28,z(1 − δ) = 1.01]. B, Neuron with inwardly rectifying kainate current [K_D(0)/[polyamine] = 0.33,z(1 − δ) = 0.87] that expressed GluR1 and GluR3 mRNAs, but not GluR2 or GluR4. C, I–Vrelation from an interneuron showing strong inward rectification [K_D(0)/[polyamine] = 0.14,z(1 − δ) = 0.92]; this neuron expressed GluR2, GluR3, and GluR4 in sufficient amounts to be detected by RT-PCR.D, Cumulative distributions of the affinity of internal blocker for 38 interneurons lacking GluR2 mRNA and 44 interneurons that expressed GluR2.

Fig. 4.

Differential calcium permeability and sensitivity of kainate currents to block by polyamine toxins in isolated interneurons. A, The kainate reversal potential shifted from +13.6 to +30.4 mV in an interneuron with inwardly rectifying kainate I–V relation when the bathing solution was changed from high Na⁺ to high Ca²⁺ solution (left panel). The Woodhull fits to the two curves are superimposed [open circles for the high Na⁺ curve;K_D(0)/[polyamine] = 0.29,z(1− δ) = 0.98]. In contrast, an interneuron with weak blocker affinity [right panel;K_D(0)/[polyamine] = 16.2 withz(1 − δ) = 1.25] showed a large negative shift in kainate reversal potential (from +3.1 to −57 mV) when the bathing medium was changed from high Na⁺ to high Ca²⁺. B, Kainate-evoked current in an interneuron with strong internal block [left panel;K_D(0)/[polyamine] = 0.20,z(1 − δ) = 0.92] is inhibited in a voltage-dependent manner by 1 μm ATX-636. The open circles represent the Woodhull model fits in the absence of argiotoxin, and the open triangles represent Woodhull fits in the presence of the external polyamine toxin. Kainate currents in an interneuron with weak internal blocker affinity [right panel; K_D(0)/[polyamine] = 12.5, z(1 − δ) = 0.45] are resistant to argiotoxin block. C, The calcium-to-sodium permeability ratio decreased sharply as a function of internal blocker affinity in a sample of 39 interneurons (filled circles). Theopen symbols show a summary of data from 48 interneurons, illustrating the block of kainate-evoked currents measured at −70 mV by five polyamine spider toxins and spermine. ▵, 3 μm philanthotoxin-433; ○, 3 μm Joro spider toxin; □, 1 mm spermine; ▿, 3 μmAgel-489 toxin from the funnel web spider, Agelinopsis aperta; ⋄, 1 μm ATX-636. D, Correlation between measured affinities for internal and external channel block in interneurons. In each cell (n = 18) the kainate I–V curve was fit with the Woodhull equations to derive the affinity for internal block; then 1 μm ATX-636 was added to the external perfusing solution, and the kainate I–V curve was obtained again. The affinity of external argiotoxin for its channel-blocking site was determined by fits of the I–V curve as inB and plotted against the internal blocker affinity of the same neuron. The slope of a line fit to the initial rising phase (K_D(0)/[polyamine] between 0.18 and 3.1 μm) is 4.2.

Fig. 6.

Model accounting for the differential dependence of calcium permeability and inward rectification on GluR2 abundance. In the model two amino acid residues determine the major permeation properties: a ring of aspartates that are found in all AMPA subunits and are predicted by transmembrane topology to be located near the cytoplasmic mouth of the channel (Hollmann et al., 1994; Wo and Oswald, 1994; Bennett and Dingledine, 1995) and the Q/R site located four residues upstream (Hume et al., 1991; Burnashev et al., 1992). The essence of the model is that the ring of carbonyl oxygens in GluR2-lacking AMPA receptors (A) contributes to or forms a binding site for permeating divalent cations. Internal polyamines also interact with this ring of polar residues and, by electrostatic interactions of the positively charged amine groups, with the carboxyl groups of the ionized aspartates at D616. Incorporation of a single positively charged arginine into the Q/R site (B) disrupts the ring of carbonyl groups, which is postulated to eliminate the divalent ion binding site. This arginine also neutralizes a negative charge at the internal channel mouth by formation of a salt bridge between its guanidinium group and the carboxyl group of the aspartate, which reduces the number of anionic binding sites for internal polyamines and thus decreases their affinity for the channel. As more arginines are incorporated into the Q/R site (C), the number of negative charges at the channel mouth, and hence polyamine affinity for the internal blocking site, is progressively reduced.