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.

Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

“…Although infrequent and not evident in the traces in Figure 5, each record also contained openings to a larger conductance level of ϳ36 pS (Table 3). The three smallest levels seen for both GluR2-wt and T686A channels are similar to conductance levels reported previously for homomeric recombinant AMPA receptor channels (Swanson et al, 1997;Derkach et al, 1999;Banke et al, 2000;Jin et al, 2003). Although rare, the 36 pS events seen with either glutamate or quisqualate in all patches analyzed are similar to large conductance openings reported previously for both recombinant and native AMPA receptors Tomita et al, 2005).…”

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor

“…21 The electrophysiological characteristics of GluR4 flip channels expressed in HEK 293 cells are similar to those observed in a subpopulation of AMPA receptors present in cerebellar granule cells. 22 HEK-GluR4 cells were stimulated for 1 h at 371C, with 100 mM or 1 mM glutamate in sodium buffer containing 2.5 mM CaCl 2 . Glutamate did not significantly affect cell viability, assessed by the MTT test 20-22 h after stimulation, unless AMPA receptor desensitization was blocked with cyclothiazide (CTZ).…”

Molecular mechanisms controlling synaptic recruitment of GluA4 subunit-containing AMPA-receptors critical for functional maturation of CA1 glutamatergic synapses

“…The most parsimonious explanation for these results is that PKA activation increases number or conductance of GluA4-containing AMPA receptors at previously undetected release sites. Insertion of homomeric GluA4 receptors with higher single channel conductance (Swanson et al, 1997) to silent and/or labile synapses may also contribute to the observed increase in AMPA potency.…”