Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition

Abstract

Non-NMDA glutamate receptor subunits of the AMPA-preferring subfamily combine to form ion channels with heterogeneous functional properties. We have investigated the effects of RNA editing at the Q/R site, splice variation of the "flip/flop" cassette, and multimeric subunit assembly on the single-channel conductance and kinetic properties of the recombinant AMPA receptors formed from GluR2 and GluR4 expressed in HEK 293 cells. We found that AMPA receptor single-channel conductance was dependent on the Q/R site editing state of the subunits comprising the channel. Calcium-permeable (unedited) channels had resolvable single-channel events with main conductance states of 7-8 pS, whereas fully edited GluR2 channels had very low conductances of approximately 300 fS (estimated from noise analysis). Additionally, the flip splice variant of GluR4 conferred agonist-dependent conductance properties reminiscent of those found for a subset of AMPA receptors in cultured cerebellar granule cells. These results provide a description of the single-channel properties of certain recombinant AMPA receptors and suggest that the single-channel conductance may be determined by the expression of edited GluR2 subunits in neurons.

Fig. 1.

Agonist-dependent conductances of the homomeric GluR4(i) receptor. A, Current noise in control solution and in the presence of kainate, glutamate, and AMPA from outside-out patches of HEK 293 cells expressing GluR4(i). Mean inward currents activated by the three agonists are similar in amplitude and illustrate the difference in noise variance. The calibration is the same for all the responses (0.5 pA, 500 msec). B, Noise spectrum for currents activated by 100 μm kainate. The spectrum was fit to two Lorentzian components (dashed lines), as detailed in Materials and Methods. The conductance (γ) obtained from this spectrum was 2.0 pS. The corner frequencies were 30 and 395 Hz.C, Noise spectrum for currents activated by 10 μm AMPA. The γ obtained from this spectrum was 5.8 pS, and corner frequencies were 26 and 280 Hz. The holding potential was −80 mV.

Fig. 2.

GluR4(i) single channels and amplitude histograms.A, Baseline noise and single-channel responses elicited by kainate, AMPA, and glutamate in GluR4(i)-containing patches. A channel-like event superimposed on the kainate-induced noise can be seen in the second trace. The dashed lines indicate the approximate size of the conductance levels activated by AMPA and glutamate as determined from time-course fitting (see B, C). The holding potential was −80 mV in each case. B, Amplitude histogram generated by time-course fitting of AMPA-activated events. The distribution was fitted to three Gaussian components, which gave mean conductance levels of 6, 16, and 27 pS. C, Amplitude histogram generated by time-course fitting of glutamate-activated events. The distribution was fitted to three Gaussian components, which gave mean conductances of 8, 15, and 24 pS.

Fig. 3.

Kinetics of GluR4(i) channel events when activated by AMPA and glutamate. A, Open-time histogram for AMPA-activated events (5 μm). The distribution was fitted to a single exponential with a time constant of 0.20 msec.B, Shut-time histogram from the same patch as shown inA. The distribution is fitted with four exponentials with time constants as shown. C, Burst-length histogram for AMPA-activated events from the same patch as A. The distribution is fitted to two exponential components. The burst-length time constants were 0.18 and 1.8 msec. D, Open-time histogram for glutamate-activated events (100 μm). The distribution was fitted to a single exponential with a time constant of 0.17 msec. E, Shut-time histogram from the same patch as shown in A. The distribution is fitted with four exponentials with time constants as shown. F, Burst-length histogram for glutamate-activated events from the same patch as A. The distribution is fitted with two exponential components. The burst-length time constants were 0.14 and 3.3 msec.

Fig. 4.

Homomeric GluR2(i) channels have a very low conductance. A, Whole-cell response activated by 300 μm glutamate in the presence of 30 μm CZD from a cell expressing GluR2(i) (arrow indicates application). B, A current-variance plot (fitted with a linear regression) for a whole-cell response from a cell expressing GluR2(i). The slope gave a mean conductance of 313 fS for this cell. The holding potential was −80 mV.

Fig. 5.

GluR2(i)/4(i) and GluR2(o)/4(i) channels and amplitude histograms. A, GluR2(i)/4(i) single-channel events activated by 100 μm glutamate (top two traces) and 20 μm AMPA (bottom two traces) in an outside-out patch. Channel openings in response to glutamate were very fast and poorly resolved and did not yield sufficient data to construct amplitude histograms. In contrast, time-course fitting revealed two AMPA-activated conductance levels of ∼4 and ∼8 pS, which are marked by dashed lines. The calibration is 0.5 pA and 25 msec. B, An amplitude histogram for AMPA-activated events fitted to two Gaussian components. The AMPA-activated conductances for this patch were 3.8 and 7.7 pS.C, GluR2(o)/4(i) single-channel events activated by 100 μm glutamate (top two traces) and 20 μm AMPA (bottom two traces) in an outside-out patch. Time-course fitting revealed two conductance levels of ∼4 and ∼8 pS. D, An amplitude histogram for AMPA-activated events fitted to two Gaussian components with means of 4.0 and 8.1 pS. The holding potential was −80 mV.

Fig. 6.

Open-time distributions of GluR2(i)/4(i) and GluR2(o)/4(i) channel events. A, Open-time distributions for AMPA-activated events in a patch from a cell expressing GluR2(i)/GluR4(i) receptors. In this record the resolution was set to 0.3 msec. The distribution was fitted with two exponential components with time constants of 0.7 and 1.8 msec. B, Open-time distribution for AMPA-activated events in a patch containing GluR2(o)/4(i) receptors. In this record the resolution was set to 0.4 msec. The distribution was fitted with two exponential components with time constants of 0.4 and 1.2 msec.

Fig. 7.

GluR2Q(o)/4(i) single channels and amplitude histogram. A, GluR2Q(o)/4(i) single-channel events activated by 30 μm glutamate (top two traces) and 20 μm AMPA (bottom two traces) in an outside-out patch. Time-course fitting of channel openings gave conductance levels of 7, 12, and 25, which are marked by dashed lines. The calibration is 1.0 pA and 10 msec. B, An amplitude histogram for AMPA-activated events from a different patch than that inA fitted to three Gaussian components. The holding potential was −80 mV.

Fig. 8.

Kinetics of GluR2Q(o)/4(i) channel events when activated by AMPA. A, Open-time histogram for AMPA-activated events (5 μm). The distribution was fitted to a single exponential with a time constant of 0.26 msec.B, Shut-time histogram from the same patch as shown inA. The distribution is fitted with the sum of four exponentials with time constants as shown. C, Burst-length histogram for AMPA-activated events from the same patch asA. The distribution is fitted with two exponential components. The burst-length time constants were 0.31 and 2.3 msec. These distributions were compiled from time-course fitted events.