Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM

  1. Ian Coombs
  2. Cécile Bats
  3. Craig A Sexton
  4. Dorota Studniarczyk
  5. Stuart G Cull-Candy
  6. Mark Farrant

  • Curated by eLife

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    Evaluation Summary:

    Here the authors identify that inclusion of intracellular NASPM can fully block Ca-permeable AMPA receptors regardless of association with auxiliary subunits. The distinction between Ca-permeable and Ca-impermeable AMPA receptors is critical to synaptic physiology, and thus these results will be of interest within the field of excitatory synaptic transmission. The data is of high quality and the experimental analysis is rigorous, but the key claim that the approach provides an unambiguous functional measure of CP-AMPAR prevalence has not been fully supported.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits – including transmembrane AMPAR regulatory proteins, cornichons, and GSG1L – which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here, we show that the inclusion of the spider toxin analog 1-naphthylacetyl spermine (NASPM) in the intracellular solution results in a complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content.

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  1. Version published to 10.7554/elife.66765 on eLife
  2. eLife

    Reviewer #4 (Public Review):

    Coombs et al. aimed to establish a pharmacological tool to distinguish calcium-permeable (CP) AMPA receptors (AMPAR) from calcium impermeable AMPA receptors unambiguously. Towards this end, the authors examined the effects of intracellularly applied NASPM, PhTx-433, PhTx-74, and spermine. The authors showed that NASPM completely blocked outward glutamate-evoked currents with a desensitization blocker, cyclothiazide, from outside-out patch membranes from HEK cells expressing GluA1. In contrast, spermine and PhTx-433/74 partially blocked the outward currents in a voltage-dependent manner (Figure 1). TARPg-2 co-expression reduced potencies of spermine and NASPM, and altered shapes of their conductance-voltage relationship (Figure 2) as well as various kinetics of GluA1, including decay kinetics and recovery kinetics (Figure 3). Further, the authors showed that NASPM blocked GluA1 co-expressed with one of the AMPAR auxiliary subunits, TARPg-2, g-7, CNIH2 GSG1L (Figure 4). Finally, the authors showed that NASPM blocked AMPAR-mediated mEPSC events at +60 mV, but not -70mV, in cultured cerebellar stellate neurons from GluA2 knockout mice. Overall, this manuscript provides high-quality data and critical information about TARPg-2, GluA1, and GluA2 knockout mice.

    This provides a solid analysis of GluA1, TARPg-2, 7, CNIH2, GSG1L, and GluA2 knockout neurons. However, it remains unclear whether intracellular NASPM allows an unambiguous functional measure of CP-AMPAR, especially considering many combinations of AMPARs and auxiliary subunits, e.g., GluA1-4 with splicing isoforms, six TARPs, four CNIHs, GSG1L and CKAMP44, etc.

    Strengths:

    The experimental design to evaluate drugs and receptors with outside-out patch membranes and a piezoelectric device provides the highest-resolution analysis and meaningful information.

    Both experiments and analyses are rigorous and of high quality. However, it remains unclear if intracellular NASPM allows an unambiguous functional measure of CP-AMPAR.

    Weaknesses:

    Because the authors tested a limited combination of receptors and auxiliary subunits, it is difficult to conclude whether NASPM blocks all CP-AMPAR unambiguously.

    Slopes of the conductance-voltage relationships are changed upon TARPg-2 co-expression or different concentrations of NASPM.

  3. eLife

    Reviewer #3 (Public Review):

    Calcium-permeable AMPA receptors (CP-AMPARs) have been shown to have important roles in modulating many aspects of neuronal function. They are distinguished from calcium-impermeable AMPARs (CI-AMPARs) by a property known as inward rectification and block by relatively selective polyamine compounds; this relative lack of selectivity has led to caveats in the interpretations of the roles of CP-AMPARs. The authors here demonstrate that complete block of CP-AMPARs, with no apparent effect on CI-AMPARs, can be achieved by intracellular application of the polyamine NASPM. Importantly, the authors provide evidence that this block is apparently not affected by the presence of auxiliary subunits, one of the key caveats regarding prior interpretations of the effects of polyamines and the roles of CP-AMPARs. The authors hypothesize that this new approach, use of intracellular NASPM, can provide greater clarity regarding the role of CP-AMPARs in future.

    The approach is sound, the experiments are performed appropriately, the data provided is robust, the presentation is clear, the analyses including statistics are appropriate, the immediate interpretations are therefore fully supported, and the overall manuscript outstanding. The authors appropriately used both a heterologous expression system as well as in vitro neuronal preparation to address their hypotheses. The use of intracellular NASPM to unambiguously distinguish CP-AMPARs from CI-AMPARs has the potential to be transformative in future interpretations about the role of CP-AMPARs, so these findings are very relevant and highly impactful to the field.

  4. eLife

    Reviewer #2 (Public Review):

    This study compares the pharmacology of intracellular polyamine blockers for Ca-permeable (CP-AMPAR) and Ca-impermeable (CI-AMPAR) AMPA receptors in the absence/presence of auxiliary subunits. Spermine is a widely used polyamine blocker to identify CP-AMPARs in native tissue, but the blocking action of spermine varies depending on which auxiliary subunits are associated with the CP-AMPARs. Hence, spermine has limitations. The goal of the present work was to identify if other polyamine blockers would be more efficient than spermine in identifying CP-AMPARs.

    The authors studied CP- and CI-AMPARs in heterologous cells (HEK293T) and in primary cerebellar stellate interneurons from mice lacking the GluA2 subunit. They primarily used electrophysiology to assay channel block by various polyamines. While the technology is standard, the experiments are carried out in a rigorous manner and encompass numerous controls and variations on appropriate constructs (GluA2-containing and GluA2-lacking AMPARs and various prominent auxiliary subunits - TARPs, cornichons, and GSG1L).

    The main conclusion of the work is that 100 uM NASPM fully blocks CP-AMPAR regardless of the associated auxiliary subunit. This conclusion is strongly supported by experiments including testing various auxiliary subunits in the defined conditions of HEK293T cells as well as recording and demonstrating that NASPM fully blocks AMPAR-mediated currents in stellate cells lacking GluA2 subunits.

    I have no major criticisms of the work.

  5. eLife

    Reviewer #1 (Public Review):

    Using various voltage and concentration protocols in a heterologous expression system, the authors provide compelling evidence for strong block of GluR1 AMPA receptors by intracellular NASPM, and unlike spermine, the block is independent of auxiliary subunit expression. The authors also show that intracellular NASPM provides a more complete block than spermine of synaptic currents in GluR2-KO neurons.

    Overall the manuscript contains high quality data that is clearly presented. It seems likely that this approach will be useful for assessing the contribution of CP-AMPARs in various scenarios. However, currently the authors have fallen short of providing a comprehensive analysis of the use of NASPM to differentiate between CP and CI AMPARs in intact systems containing multiple AMPAR subunits and auxiliary proteins.

  6. eLife

    Evaluation Summary:

    Here the authors identify that inclusion of intracellular NASPM can fully block Ca-permeable AMPA receptors regardless of association with auxiliary subunits. The distinction between Ca-permeable and Ca-impermeable AMPA receptors is critical to synaptic physiology, and thus these results will be of interest within the field of excitatory synaptic transmission. The data is of high quality and the experimental analysis is rigorous, but the key claim that the approach provides an unambiguous functional measure of CP-AMPAR prevalence has not been fully supported.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  7. Version published to 10.1101/2021.02.18.431828v1 on bioRxiv