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Single-Channel Properties of Recombinant AMPA Receptors Depend on RNA Editing, Splice Variation, and Subunit Composition

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Single-Channel Properties of Recombinant AMPA Receptors Depend on RNA Editing, Splice Variation, and Subunit Composition The Journal of Neuroscience, January 1, 1997, 17(1):58–69 Single-Channel Properties of Recombinant AMPA Receptors Depend on RNA Editing, Splice Variation, and Subunit Composition Geoffrey T. Swanson,1,2,a Sunjeev K. Kamboj,1,a and Stuart G. Cull-Candy1 1Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom, and 2Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, California 92037 Non-NMDA glutamate receptor subunits of the AMPA- edited GluR2 channels had very low conductances of ;300 fS preferring subfamily combine to form ion channels with heter- (estimated from noise analysis). Additionally, the flip splice ogeneous functional properties. We have investigated the ef- variant of GluR4 conferred agonist-dependent conductance fects of RNA editing at the Q/R site, splice variation of the properties reminiscent of those found for a subset of AMPA “flip/flop” cassette, and multimeric subunit assembly on the receptors in cultured cerebellar granule cells. These results single-channel conductance and kinetic properties of the re- provide a description of the single-channel properties of certain combinant AMPA receptors formed from GluR2 and GluR4 recombinant AMPA receptors and suggest that the single- expressed in HEK 293 cells. We found that AMPA receptor channel conductance may be determined by the expression of single-channel conductance was dependent on the Q/R site edited GluR2 subunits in neurons. editing state of the subunits comprising the channel. Calcium- Key words: glutamate receptor; AMPA receptor; single- permeable (unedited) channels had resolvable single-channel channel conductance; RNA editing; alternative splicing; patch- events with main conductance states of 7–8 pS, whereas fully clamp electrophysiology Non-NMDA receptors mediate fast excitatory synaptic transmis- kainate receptor subunits at the codon for the Q/R site of the sion at glutamate synapses in the mammalian CNS. Cloning receptor involves the replacement of a glutamine by an arginine in techniques have led to the identification of nine non-NMDA the pore-lining region (M2) of GluR2, -5, and -6 (Sommer et al., receptor genes (Hollmann and Heinemann, 1994), which have 1991). This results in a reduction of the calcium permeability been separated into AMPA-preferring (GluR1-4 or GluRA–D) (Hume et al., 1991; Burnashev et al., 1992) accompanied by a loss and kainate-preferring (GluR5-7, KA-1, and KA-2) subfamilies of sensitivity to intracellular polyamines, which confer inward on the basis of structural and functional similarities. AMPA rectification on these receptors (Bowie and Mayer, 1995; Kamboj receptor subunits have two splice variants (“flip” and “flop,” et al., 1995; Koh et al., 1995). Furthermore, Q/R site editing denoted by “i” and “o”), which differ by seven residues in a 38 reduces the single-channel conductance of kainate receptors by as amino acid cassette between the third and fourth putative trans- much as 25-fold (Swanson et al., 1996). The finding that GluR2 membrane domains (Sommer et al., 1990). Furthermore, gene mRNA is completely edited at birth (Sommer et al., 1991) and splicing can give rise to alternative C-terminal sequences (Gallo et that the expression level of GluR2 subunits controls the calcium al., 1992; Ko¨hler et al., 1994). These isoforms can differ in their permeability of AMPA receptors within certain cell types (Geiger pharmacological properties as well as their desensitization kinet- et al., 1995; Jonas and Burnashev, 1995) has provided a further ics (Mosbacher et al., 1994; Partin et al., 1995). Whether the stimulus to understanding the effect of Q/R site editing on the flip/flop variation affects single-channel properties is unknown. single-channel properties of AMPA receptors. RNA editing creates further diversity among non-NMDA re- To investigate this further and to obtain clues about the subunit ceptor subunits, which show single positions occupied by one of composition of native AMPA receptor channels, we have exam- two amino acids. For AMPA subunits, these are the “Q/R” and ined the single-channel properties of combinations of GluR2 and “R/G” sites (Sommer et al., 1991; Lomeli et al., 1994; for review, GluR4 subunits. These receptors were of interest, because non- see Seeburg, 1996). Single nucleotide editing of AMPA and NMDA channels in cerebellar granule cells, which have been described in detail (Cull-Candy et al., 1988; Wyllie et al., 1993), Received July 8, 1996; revised Oct. 4, 1996; accepted Oct. 8, 1996. probably arise from these subunits (Monyer et al., 1991; Mos- This work was supported by the Wellcome Trust. G.T.S. was supported by a bacher et al., 1994). We have found that the single-channel Hitchings-Elion Fellowship from the Wellcome Trust and the Burroughs Wellcome Fund. S.K.K. was in receipt of a Wellcome Studentship. The work of S.G.C.-C. is conductance is relatively high for calcium-permeable AMPA re- supported in part by an International Scholars Award from The Howard Hughes ceptors but lower for calcium-impermeable channels containing Medical Institute. GluR2(i) and GluR4(i) plasmid cDNAs were kindly provided by edited subunits. Furthermore, heteromeric channels formed by Peter Seeburg. GluR2(o) plasmid DNA was kindly provided by Stephen Heinemann and Jim Boulter. GluR2Q(o) was generated by Andreas Sailer. We thank David coexpression of the unedited form of GluR2 [GluR2Q(o)], to- Colquhoun for generous help with software for single-channel analysis and John gether with GluR4(i), had a markedly higher conductance than Dempster for SPECTAN analysis software. We thank Mark Farrant, Zoltan Nusser, Alasdair Gibb, David Wyllie, and Mark Mayer for helpful discussions and comments GluR2R(o)/GluR4(i) channels. These results strongly suggest on this manuscript, and Stephen Heinemann for additional support for G.T.S. that one consequence of Q/R site editing is a reduction in the Correspondence should be addressed to Stuart G. Cull-Candy, Department of conductance of AMPA receptors. Finally, our results have iden- Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. aThese authors contributed equally to this work. tified at least two recombinant channels whose single-channel Copyright q 1996 Society for Neuroscience 0270-6474/96/170058-12$05.00/0 properties correspond closely to those of native non-NMDA Swanson et al. • AMPA Receptor Single-Channel Properties J. Neurosci., January 1, 1997, 17(1):58–69 59 Table 1. Conductance properties of recombinant AMPA receptors formed from GluR2 and GluR4 Unedited Edited Receptor Conductance (pS) Agonist Receptor Conductance (pS) Agonist GluR4Q(i) 7/16/27 AMPA GluR2R(i) 0.21* AMPA 8/15/24 Glutamate 0.36* Glutamate 2.5* Kainate 0.45* Kainate GluR4Q(o) 4.0* Kainate GluR2R(o) ,0.3* Kainate GluR2Q(o)/4Q(i) 7/15/24 AMPA GluR2R(o)/4Q(i) 4/9 AMPA 8/17/26 Glutamate 4/10 Glutamate – – – GluR2R(i)/4Q(i) 4/8 AMPA ;4/10 Glutamate 0.50* Kainate – – – GluR2R(i)/4Q(o) 1.0* Glutamate Single-channel conductance values were obtained either from time-course fitting or noise analysis (indicated by asterisk) when channel events were too small to be resolved directly. channels found in cerebellar granule cells (Cull-Candy et al., 1988; a critical gap length, tcrit. Bursts of openings were defined as activations Wyllie et al., 1993). This suggests that analysis of single-channel separated by shut-times shorter than tcrit (typically 1–3 msec), which was calculated from components of the shut-time distribution. The tcrit values properties of recombinant AMPA receptors can be useful in included an equal number of gaps misclassified as within bursts and gaps determining the subunits constituting native receptors and ex- misclassified as between bursts (Colquhoun and Sigworth, 1995). Higher- tends earlier work examining macroscopic properties of recombi- order activation clusters were not analyzed. nant channels (Seeburg, 1993; Hollmann and Heinemann, 1994; For spectral analysis of steady-state current responses, current records Lomeli et al., 1994; Mosbacher et al., 1994). were high-pass-filtered at 0.2 Hz, low-pass-filtered at 2 kHz (23 dB, 8-pole Butterworth), and digitized at 4 kHz (CED 1401 plus interface). MATERIALS AND METHODS Noise analysis was generally restricted to patches that gave a current response of .0.5 pA. Digitized records were split into sections 1/fres in Maintenance and transfection of HEK 293 cells. HEK 293 cells were length (with fres being 1 or 2 Hz) and edited to remove artifacts. Noise cultured in DMEM/F12 (Life Technologies, Gaithersburg, MD) with spectra were fitted with the sum of two Lorentzian components according 10% heat-inactivated fetal bovine serum, 50 mg/ml penicillin, and 50 2 2 to the relation G(f) 5 G1(0)/{1 1 (f/fc1) } 1 G2(0)/{1 1 (f/fc2) }, where mg/ml streptomycin. One day before transfection, cells were replated on G1(0) and G2(0) are the spectral densities at the zero frequency asymp- 11 mm glass coverslips coated with 100 mg/ml poly-L-lysine and 50 mg/ml tote for the two Lorentzian components, f is the frequency, and fc1 and fc2 fibronectin (Sigma, Dorset, UK). Transfections were carried out using a are the corner frequencies for the fast and slow components, respectively. standard CaPO4 protocol (Chen and Okayama, 1987) with 0.5 mg plasmid The contribution to the variance of each component was described by DNA/well for 3–6 hr at 378C. Transfected cells were allowed to grow for variance 5 G(0)fcp/2. 1–3 d before use. All cDNAs were harbored in a CMV promoter- containing vector. In addition to AMPA receptor subunits, some cells RESULTS were cotransfected with cDNA for the cell-surface marker protein CD8. Before patch-clamp recordings, cells were exposed to polystyrene beads We have examined homomeric and heteromeric AMPA receptors coated with an antibody to CD8 (Dynal, Great Neck, NY), allowing visual expressed in HEK 293 cells transfected with flip (i) and flop (o) detection of transfected cells.
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