Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM
- Department of Neuroscience, Physiology and Pharmacology, University College London, United Kingdom
Figures
Figure 1
Intracellular 1-naphthylacetyl spermine (NASPM) and polyamine toxins specifically block GluA2-lacking calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) in a voltage-dependent manner.
(a) The blockers used in this study. (b) Representative responses activated by 300 µM glutamate and 50 µM cyclothiazide from outside-out patches excised from HEK293 cells expressing either GluA1 or …
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Figure 1—source data 1
RI+60/−60 values from GluA1 and GluA1/γ2 ramp current-voltage (I-V) relationships (300 µM glutamate) were recorded with intracellular spermine, NASPM, PhTx-433, or PhTx-74 (each 100 µM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig1-data1-v2.zip
Figure 2
Spermine, 1-naphthylacetyl spermine (NASPM), and polyamine toxins display different potencies of GluA1 block that are all decreased by transmembrane AMPAR regulatory proteins (TARP) γ2.
(a) Pooled, normalized conductance-voltage (G-V) relationships (from voltage ramps as in Figure 1) for GluA1 and GluA1/γ2 in the presence of spermine (left) or NASPM (right) (n=4–8). Conductance was …
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Figure 2—source data 1
Vb values for GluA1 and GluA1/γ2 obtained with intracellular spermine, NASPM, PhTx-433, or PhTx-74.
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig2-data1-v2.zip
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Figure 2—source data 2
IC50 0 mV values for GluA1 and GluA1/γ2 obtained with intracellular spermine, NASPM, PhTx-433, or PhTx-74.
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig2-data2-v2.zip
Figure 3
Block of GluA1/γ2 by 10 µM intracellular 1-naphthylacetyl spermine (NASPM) shows use-dependence and slow recovery.
(a) Representative GluA1/γ2 currents in the presence of 10 μM intracellular spermine or NASPM activated by fast applications of glutamate (10 mM, 100 ms) at positive and negative potentials. In the …
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Figure 3—source data 1
Normalized G-V data for peak and steady-state currents evoked by 10 mM glutamate from GluA1/γ2 receptors with intracellular spermine (10 μM) or NASPM (10 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig3-data1-v2.zip
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Figure 3—source data 2
τdecay values for currents evoked by 10 mM glutamate from GluA1/γ2 receptors with intracellular NASPM (10 μM) or with the polyamine-free intracellular solution at different membrane voltages.
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig3-data2-v2.zip
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Figure 3—source data 3
Recovery data (Peak 2/Peak 1) for currents evoked by 10 mM glutamate from GluA1/γ2 receptors with intracellular NASPM (10 μM) or with the polyamine-free intracellular solution at +60 and −60 mV.
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig3-data3-v2.zip
Figure 4
Calcium-permeable AMPA-type glutamate receptor (CP-AMPAR) rectification in the presence of 100 µM intracellular 1-naphthylacetyl spermine (NASPM) is unaffected by auxiliary proteins.
(a) Representative currents activated by fast applications of glutamate to outside-out patches excised from HEK293 cells transfected with GluA1/GluA2/γ2 (left) or GluA1/γ2 (right) (10 mM, 100 ms, …
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Figure 4—source data 1
Normalized current-voltage (I-V) data for peak currents evoked by 10 mM glutamate from GluA1/2/γ2, and GluA1/γ2 receptors with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig4-data1-v2.zip
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Figure 4—source data 2
RI+60/−60 values for peak currents evoked by 10 mM glutamate from GluA1, GluA1/γ2, GluA1/γ7, GluA1/γ8, GluA1/CNIH2, GluA1/CNIH3, GluA1/CKAMP59, GluA1/GSG1L, GluA1/γ8/CNIH2, and GluA1/γ8/CKAMP44 receptors with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig4-data2-v2.zip
Figure 5
Intracellular 1-naphthylacetyl spermine (NASPM) (100 μM) produces full rectification of currents carried by extrasynaptic calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) from dentate gyrus granule cells.
(a) Diagrammatic representation of the preparation. (b) Representative average glutamate-evoked currents from four outside-out patches excised from somata of wild-type (WT) and GluA2 KO dentate …
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Figure 5—source data 1
RI+60/−60 values for peak currents evoked by 10 mM glutamate from wild-type (WT) and GluA2 KO DGGCs with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig5-data1-v2.zip
Figure 6
Intracellular 1-naphthylacetyl spermine (NASPM) (100 μM) produces full rectification of calcium-permeable AMPA-type glutamate receptor (CP-AMPAR)-mediated mEPSCs.
(a) Diagrammatic representation of the preparation. (b) Representative whole-cell recording from a cultured GluA2-knockout (GluA2 KO) cerebellar stellate cell held at voltages between −80 and +60 mV …
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Figure 6—source data 1
RI+60/−60 values for cerebellar stellate cell mEPSCs were recorded with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig6-data1-v2.zip
Figure 7
Intracellular 1-naphthylacetyl spermine (NASPM) (100 μM) produces full rectification of calcium-permeable AMPA-type glutamate receptor (CP-AMPAR)-mediated MF-eEPSCs.
(a) Diagrammatic representation of the preparation. (b) Representative whole-cell recordings of mossy fiber-evoked EPSCs from wild-type (WT) cerebellar granule cells (CGCs) held at −60 and +60 mV …
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Figure 7—source data 1
MF-eEPSC peak amplitudes, 20–80% rise times and w,decay values from wild-type (WT) and GluA2-knockout (GluA2 KO) CGCs recorded with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig7-data1-v2.zip
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Figure 7—source data 2
Normalized current-voltage (I-V) data for peak MF-eEPSCs from wild-type (WT) and GluA2-knockout (GluA2 KO) CGCs recorded with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig7-data2-v2.zip
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Figure 7—source data 3
RI+60/−60 values for peak MF-eEPSCs from wild-type (WT) and GluA2-knockout (GluA2 KO) CGCs were recorded with intracellular spermine (100 μM) or NASPM (100 μM).
- https://cdn.elifesciences.org/articles/66765/elife-66765-fig7-data3-v2.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Gria2tm1Rod/J, 129 /CD1 | other | Gift from Ingo Greger, MRC LMB, Cambridge, UK | |
| Genetic reagent (Mus musculus) | oIMR6780 (Gria2) | JAX | JAX:oIMR6780 | wild-type Forward primer GGT TGG TCA CTC ACC TGC TT |
| Genetic reagent (Mus musculus) | oIMR6781 (Gria2) | JAX | JAX:oIMR6781 | Common primer TCG CCC ATT TTC CCA TAT AC |
| Genetic reagent (Mus musculus) | oIMR8444 (Gria2) | JAX | JAX:oIMR8444 | Mutant primer GCC TGA AGA ACG AGA TCA GC |
| Cell line (Homo sapiens) | HEK293 | ATCC | ATTC:CRL-1573 | |
| Transfected construct (Rattus norvegicus) | pIRES-eGFP-GluA1 | doi:10.1126/science.2166337; doi:10.1038/nn.2266 | Gria1; Flip form. Gift from Peter Seeburg (subcloned into pIRES-eGFP) | |
| Transfected construct (Rattus norvegicus) | pIRES-eGFP-GluA2 | doi:10.1126/science.2166337; doi:10.1523/JNEUROSCI.17-01-00058.1997 | Gria2; Q/R and R/G edited flip form. Gift from Peter Seeburg (subcloned into pIRES-eGFP) | |
| Transfected construct (Rattus norvegicus) | pIRES-eGFP-γ2 | doi:10.1038/1228; doi:10.1038/nn.2266 | Cacng2; Gift from Roger Nicoll, UCSF, USA | |
| Transfected construct (Homo sapiens) | pIRES-eGFP-γ7 | doi:10.1038/nn.2266 | CACNG7; OriGene Technologies pCMV6-XL4-γ7 (subcloned into pIRES-eGFP) | |
| Transfected construct (Rattus norvegicus) | pIRES-eGFP-γ8 | doi:10.1083/jcb.200212116; doi:10.1038/nn.2266 | Cacng8; gift from Roger Nicoll, UCSF, USA | |
| Transfected construct (Rattus norvegicus) | pIRES-eGFP-CNIH2 | doi:10.1126/science.1167852; doi:10.1523/JNEUROSCI.0345–12.2012 | Cnih2; gift from Bernd Fakler, University of Freiburg, Germany (subcloned into pIRES-eGFP) | |
| Transfected construct (Rattus norvegicus) | pIRES-eGFP-CNIH3 | doi:10.1016/j.neuron.2012.03.034; doi:10.1523/JNEUROSCI.0345–12.2012 | Cnih3; Gift from Bernd Fakler, University of Freiburg, Germany (subcloned into pIRES-eGFP) | |
| Transfected construct (Mus musculus) | pRK5-CKAMP44a | doi:10.1126/science.1184245 | Ckamp44; Gift from Jakob von Engelhardt, Johannes Gutenberg University, Mainz, Germany | |
| Transfected construct (Mus musculus) | pRK5-CKAMP59-short | doi:10.7554/eLife.09693.001 | Ckamp59; Gift from Jakob von Engelhardt, Johannes Gutenberg University, Mainz, Germany | |
| Transfected construct (Rattus norvegicus) | pcDNA3.1-GSG1L | doi:10.1016/j.neuron.2012.03.034; doi:10.1523/JNEUROSCI.2152–15.2015 | Gsg1l; Gift from Bernd Fakler, University of Freiburg, Germany | |
| Transfected construct (Rattus norvegicus) | pcDNA3-Homer1c-tdDsRed | doi:10.1016/j.neuron.2007.01.030 | Gift from Daniel Choquet, University of Bordeaux, France | |
| Chemical compound, drug | PhTx-433; Spermine tetrahydrochloride | Sigma-Aldrich, Merck Life Science UK Limited, Gillingham, UK | Sigma-Adrich:P207; Sigma-Adrich:85610 | |
| Chemical compound, drug | PhTx-74 | Tocris Bioscience, Bio-Techne Ltd, Abingdon, UK | Tocris:2770 | |
| Chemical compound, drug | NASPM | HelloBio, Bristol, UK | HelloBio:HB0441 | |
| Software, algorithm | IGOR Pro | Wavemetrics, Lake Oswego, Oregon, USA | RRID:SCR_000325 | version 6.35 |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | version 10 |
| Software, algorithm | WinWCP | Strathclyde Electrophysiology Software | RRID:SCR_014713 | version 5.2.7 |
| Software, algorithm | NeuroMatic | http://www.neuromatic.thinkrandom.com | RRID:SCR_004186 | version 2.8 |
| Software, algorithm | R | R Foundation for Statistical Computing | RRID:SCR_001905 | version 4.1.0 |
| Software, algorithm | RStudio Desktop | Posit Software | RRID:SCR_000432 | version 2022.12.0 |
Author response table 1
| NASPM 100 μM: | Mean −153 pA | Range −20 to −401 pA |
|---|---|---|
| Spermine 100 μM: | Mean −54 pA | Range −19 to −97 pA |
| NASPM 10 μM: | Mean −118 pA | Range −12 to −458 pA |
| Spermine 10 μM: | Mean −105 pA | Range −16 to −690 pA |
