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

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation AMPA-type glutamate receptors that lack the GluA2 subunit are calcium-permeable and contribute to calcium influx in plasticity and disease. The authors of this Tools and Resources manuscript describe a method for evaluating the presence of GluA2-lacking receptors using intracellular NASPM that avoids complications related to auxiliary subunits that affect biophysical properties. The compelling results provide a valuable new approach for unambiguously differentiating the presence of Ca-permeable and -impermeable AMPA receptors. https://doi.org/10.7554/eLife.66765.sa0 Decision letter Reviews on Sciety eLife's review process Introduction AMPA-type glutamate receptors (AMPARs) mediate the fast component of excitatory postsynaptic currents (EPSCs) throughout the mammalian brain (Baranovic and Plested, 2016; Greger et al., 2017; Hansen et al., 2021) and their regulation allows lasting changes in synaptic strength that are essential for normal brain function (Huganir and Nicoll, 2013). AMPARs are cation-permeable channels that exist as homo- or heterotetrameric assemblies of the homologous pore-forming subunits GluA1-4, encoded by the genes Gria1-4. While a majority of central AMPARs are GluA2-containing di- or tri-heteromeric assemblies (Lu et al., 2009; Wenthold et al., 1996; Zhao et al., 2019), those lacking GluA2 constitute an important functionally distinct and widely occurring subtype. Editing of Gria2 pre-mRNA at codon 607 results in the substitution of a genetically encoded glutamine (Q) with an arginine (R). This switch from a neutral to a positively charged residue in the pore-forming loop causes receptors containing Q/R edited GluA2 to have a greatly reduced Ca2+ permeability compared to those lacking GluA2 (Burnashev et al., 1992; Kuner et al., 2001; Sommer et al., 1991). As this mRNA editing is essentially complete, AMPARs are commonly divided into GluA2-containing Ca2+-impermeable (CI-) and GluA2-lacking Ca2+-permeable (CP-) forms (Bowie, 2012; Burnashev et al., 1992; Cull-Candy et al., 2006). Aside from allowing Ca2+ flux, CP-AMPARs differ from CI-AMPARs in having greater single-channel conductance (Benke and Traynelis, 2018, Swanson et al., 1997) and susceptibility to voltage-dependent block by the endogenous intracellular polyamines spermine and spermidine (Bowie and Mayer, 1995; Donevan and Rogawski, 1995; Kamboj et al., 1995; Koh et al., 1995a). They can also be blocked in a use-dependent manner by extracellular application of the same polyamines (Washburn and Dingledine, 1996) or various exogenous organic cations, such as the spermine analog N-(4-hydroxyphenylpropanoyl)-spermine (HPP-SP; Washburn and Dingledine, 1996), the polyamine-amide wasp toxin philanthotoxin-4,3,3 (PhTx-433; Washburn and Dingledine, 1996), the spider toxin analog 1-naphthylacetyl spermine (NASPM; Tsubokawa et al., 1995), and the dicationic adamantane derivative IEM-1460 (Magazanik et al., 1997). Block of CP-AMPARs by intracellular polyamines results in inwardly- or bi-rectifying current-voltage relationships. This characteristic rectification – seen during whole-cell patch-clamp recordings in the presence of residual endogenous polyamines or added exogenous spermine – has been utilized extensively to identify the presence of CP-AMPARs in neurons. Importantly, this approach has enabled the identification of cell- and synapse-specific CP-AMPAR expression (Koh et al., 1995b, Tóth and McBain, 1998), changes in CP-AMPAR prevalence during development (Brill and Huguenard, 2008; Kumar et al., 2002; Soto et al., 2007; Yang et al., 2010), and roles for CP-AMPARs in multiple synaptic plasticities, including long-term potentiation (LTP) and depression (LTD) (Fortin et al., 2010; Lamsa et al., 2000; Liu and Cull-Candy, 2000; Mahanty and Sah, 1998; Manz et al., 2020; Plant et al., 2006; Sanderson et al., 2016). In addition, its use has suggested CP-AMPAR changes associated with various conditions, including ischemia (Dixon et al., 2009; Liu et al., 2004; Peng et al., 2006), brain trauma (Korgaonkar et al., 2020), schizophrenia (Druart et al., 2021), stress (Kuniishi et al., 2020), and glaucoma (Sladek and Nawy, 2020), and in circuit remodeling associated with fear-related behaviors (Clem and Huganir, 2010; Liu et al., 2010), drug addiction (Bellone and Lüscher, 2006; Conrad et al., 2008; Lee et al., 2013; Parrilla-Carrero et al., 2021; Scheyer et al., 2014; Van den Oever et al., 2008), and neuropathic or inflammatory pain (Goffer et al., 2013; Katano et al., 2008; Park et al., 2009; Sullivan et al., 2017; Vikman et al., 2008). Native AMPARs co-assemble with various transmembrane auxiliary subunits that influence receptor biogenesis, synaptic targeting, and function (Greger et al., 2017; Jackson and Nicoll, 2011b, Schwenk et al., 2019). Importantly, several of these, including transmembrane AMPAR regulatory proteins (TARPs), cornichons, and germ cell-specific gene 1-like protein (GSG1L), have been shown to modify the block of CP-AMPARs by intracellular spermine. In the case of TARPs and CNIHs inward rectification is reduced (Brown et al., 2018; Cho et al., 2007; Coombs et al., 2012; Soto et al., 2007; Soto et al., 2009), whereas with GSG1L the rectification is increased (McGee et al., 2015). This can complicate the interpretation of measures of spermine-dependent rectification. Moreover, any change in a rectification that might be attributed to changes in the prevalence of CP-AMPARs could instead reflect a change in auxiliary subunit content. Here, we show that intracellular NASPM, PhTx-433, and PhTx-74 can be used to specifically and voltage-dependently block recombinant CP-AMPARs. Unlike spermine, which is permeant, these blockers allow negligible outward current at positive potentials. At +60 mV, 10 µM intracellular NASPM produces a use-dependent block, while at 100 µM it fully blocks outward currents in a use-independent manner. Critically, this block is unaffected by the presence of auxiliary subunits. Furthermore, in three neuronal populations from GluA2-knockout (GluA2 KO) mice (Jia et al., 1996), known to express different auxiliary proteins (Fukaya et al., 2006; Khodosevich et al., 2014; Tomita et al., 2003; Yamazaki et al., 2010), we show that 100 µM intracellular NASPM causes full rectification of glutamate-evoked currents from extrasynaptic and synaptic CP-AMPARs, something that cannot be achieved with spermine. Together, our results reveal that the use of intracellular NASPM provides a simple and effective method for determining the relative contribution of CP-AMPARs to AMPAR-mediated currents, regardless of their associated auxiliary subunits. Results Intracellular polyamine toxins block CP-AMPARs To determine whether intracellularly applied NASPM or polyamine toxins might offer advantages over spermine for the identification of native CP-AMPARs we examined three compounds: NASPM, which contains the same polyamine tail as spermine, PhTx-433, which has a different distribution of amines, and PhTx-74, which lacks one amine group (Figure 1a). Initially, we recorded currents in outside-out patches from HEK cells transiently transfected with GluA1 alone or with GluA1 and GluA2, to produce homomeric CP- and heteromeric CI-AMPARs, respectively. The receptors were activated by glutamate (300 μM) in the presence of cyclothiazide (50 μM) to minimize AMPAR desensitization and we applied voltage ramps (100 mV/s) to generate current-voltage (I-V) relationships. Figure 1 Download asset Open asset 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 GluA1/2. The voltage was ramped linearly from −80 to +60 mV (100 mV/s). GluA1 displayed outward rectification with a polyamine-free pipette solution. In the presence of 100 μM spermine GluA1 displayed a doubly rectifying relationship, and with 100 μM NASPM GluA1 displayed full inward rectification. GluA1/2 did not rectify in the presence of NASPM. (c) Normalized and pooled current-voltage (I-V) relationships for GluA1 and GluA1/2 in the presence of 100 μM intracellular polyamines (n=3–8). Colored traces denote the mean and gray shading ± standard error of the mean. For all blockers the RI+60/−60 value with GluA1 was less than that with GluA1/2; the unpaired mean differences and 95% confidence intervals were −0.85 [−1.09,–0.63] with spermine (n=4 GluA1/2 patches and 5 GluA1 patches), −1.07 [−1.7,–0.79] with NASPM (n=8 and 4),–0.82 [−0.96,–0.60] with PhTx-433 (n=3 and 4), and −0.78 [−0.91,–0.70] with PhTx-74 (n=3 and 4). 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 Download elife-66765-fig1-data1-v2.zip As expected (McGee et al., 2015; Soto et al., 2007), in the absence of intracellular polyamines homomeric GluA1 receptors generated outward currents at positive potentials, showing clear outward rectification, while in the presence of 100 μM spermine, they displayed doubly rectifying responses (Figure 1b). By contrast, when the intracellular solution contained 100 μM NASPM, GluA1 receptors displayed inwardly rectifying responses with negligible current passed at positive potentials (Figure 1b). Unlike responses from GluA1 alone, currents from GluA1/2 receptors in the presence of NASPM were non-rectifying (Figure 1b). I-V plots showed that, when added to the intracellular solution at 100 μM, each blocker conferred marked inward rectification on the currents mediated by GluA1 (rectification index, RI+60/−60 0.02–0.26), but not on those mediated by GluA1/2 (RI+60/−60 0.84–1.30) (Figure 1c). Thus, although differing in structure, they all produced selective voltage-dependent block of the GluA2-lacking CP-AMPARs. Of note, the block by intracellular PhTx-74 was restricted to CP-AMPARs, despite the fact that it produces low-affinity block of CI-AMPARs when applied extracellularly (Jackson et al., 2011b; Nilsen and England, 2007). We next investigated the concentration- and auxiliary subunit-dependence of the block. Specifically, we generated conductance-voltage (G-V) relationships and fit those from inwardly rectifying responses with a single Boltzmann function and those from doubly rectifying responses with a double Boltzmann function (Panchenko et al., 1999). This revealed that as the concentration of added blocker was increased (from 0.1 or 1 μM to 500 μM) there was a progressive negative shift in Vb (the potential at which 50% block occurs) (Figure 2a and b). Plotting Vb against polyamine concentration (Figure 2b) allowed us to determine the IC50, 0 mV (the concentration expected to result in a half maximal block at 0 mV) and thus estimate the potency of each polyamine. This showed that, for steady-state conditions, the order of potency for the GluA1 block was spermine >NASPM >PhTx-433 >PhTx-74 (Figure 2b and c). The same analysis demonstrated that the potency of each blocker was reduced when GluA1 was co-expressed with TARP γ2 (between 7- and 18-fold reduction; Figure 2b and c). Figure 2 Download asset Open asset 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 corrected for the outwardly rectifying response seen in polyamine-free conditions and fitted with single or double Boltzmann relationships (solid lines). (b) Vb values from fitted G-V relationships of GluA1 and GluA1/γ2 in the presence of varying concentrations of blockers. When Vb was plotted against log [blocker] a linear relationship could be fitted in all cases, the x-intercept of which gave IC50, 0 mV. (c) The IC50, 0 mV values for each polyamine demonstrate relative potency in the order spermine >NASPM >PhTx-433 >PhTx-74, with γ2 co-expression reducing the potency of the blockers by 7–18-fold. 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 Download elife-66765-fig2-data1-v2.zip 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 Download elife-66765-fig2-data2-v2.zip Onset and recovery of the block by NASPM Next, we examined GluA1/γ2 currents elicited by rapid application of 10 mM glutamate (in the absence of cyclothiazide) and compared the effect of NASPM – the second most potent of the blockers – with that of spermine. We first tested the blockers at a concentration of 10 μM, as this allowed us to examine the onset of the block. At both positive and negative voltages, glutamate application produced currents that showed a clear peak and rapid desensitization to a steady-state level. However, with intracellular NASPM the steady state-currents at positive voltages were much reduced compared to the corresponding currents at negative voltages (Figure 3a). For both peak and steady-state currents the RI+60/−60 values obtained with NASPM were less than those obtained with spermine. For peak current, RI+60/−60 was 0.37 ± 0.04 for NASPM and 0.56 ± 0.07 for spermine (n=11 and 10, respectively; unpaired mean difference −0.19 [−0.34,–0.045], p=0.031, two-sided Welch two-sample t-test). For steady-state current, RI+60/−60 was 0.04 ± 0.02 for NASPM and 0.90 ± 0.14 for spermine (n=8 and 7), respectively (unpaired mean difference −0.86 [−1.12,–0.62], p=0.00075). This difference was also evident in the G-V relationships; those determined from peak responses in the presence of spermine or NASPM were largely overlapping, while those from steady-state responses were markedly different at potentials positive to +40 mV, with a large outward conductance seen in the presence of spermine but not NASPM (Figure 3b). Figure 3 Download asset Open asset 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 presence of NASPM, relative peak outward currents are smaller than those seen with spermine, while steady-state currents are negligible. (b) Pooled, averaged conductance-voltage (G-V) relationships for peak and steady-state currents obtained with spermine (n=9 and 6, respectively) and NASPM (n=11 and 8, respectively). Symbols and error bars indicate the mean ± standard error of the mean. Note the stronger inhibition by NASPM of the steady-state compared with peak currents. (c) Representative GluA1/γ2 currents with 10 μM intracellular NASPM at the indicated voltages (left) and normalized currents from the same patch at +60 and −60 mV (right). Applications of glutamate at each voltage were preceded by an application at −60 mV to relieve the NASPM block. The dashed lines are single exponential fits. (d) Pooled data showing weighted time constants of decay with polyamine-free (open symbols, n=8) and 10 μM NASPM (n=8) intracellular solutions measured between −110 mV and +80 mV. Symbols and error bars indicate the mean ± standard error of the mean. The kinetics of the negative limb (−110 mV to −10 mV) were voltage and NASPM independent (fitted lines indicate τdecay values of 5.7 ms for polyamine free and 5.4 ms for NASPM). The kinetics of the positive limb (+10 to +80 mV) was markedly accelerated in the presence of intracellular NASPM. (e) Superimposed GluA1/γ2 currents in the presence or absence of 10 μM NASPM elicited by pairs of glutamate applications at 4, 2, 1, 0.5, 0.25, and 0.125 Hz (10 mM, 100 ms,+/−60 mV). The first responses with NASPM were scaled to those in the polyamine-free condition. With NASPM at −60 mV and in the absence of polyamine at both voltages, the second currents broadly recovered to the initial levels. With NASPM at +60 mV, however, peak currents recovered much more slowly. (f) Pooled recovery data recorded in the polyamine-free condition (open symbols, n=9) and with added 10 μM NASPM (filled symbols, n=9). Symbols and error bars indicate the mean ± standard error of the mean. 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 Download elife-66765-fig3-data1-v2.zip 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 Download elife-66765-fig3-data2-v2.zip 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 Download elife-66765-fig3-data3-v2.zip Intracellular spermine is a weakly permeable open-channel blocker of both CP-AMPARs and kainate receptors (KARs) (Bowie et al., 1998; Brown et al., 2016). However, for GluK2(Q) KARs, permeation of intracellular polyamines has been shown to vary with molecular size, with two molecules of greater width than spermine – N-(4-hydroxyphenylpropanoyl)-spermine (HPP-SP) and PhTx-433 – showing little or no detectable relief from the block with depolarization (Bähring et al., 1997). We reasoned that NASPM, with its naphthyl headgroup, might also be expected to display limited permeability of CP-AMPAR channels. This could account for the shape of the ramp I-V (Figure 1c) and G-V plots with NASPM (Figure 2a). Indeed, the pronounced effect of 10 μM intracellular NASPM on steady-state GluA1/γ2 currents at positive potentials (Figure 3a) is also consistent with limited permeation, leading to the accumulation of channel block. In line with this, we found that the decay of GluA1/γ2 currents recorded in the presence of NASPM was strongly voltage-dependent (Figure 3a and c). At negative potentials, τdecay values were similar in the presence and absence of NASPM. However, at positive potentials (from +10 to +80 mV) the kinetics in the two conditions differed markedly. In the absence of NASPM current decay was slowed at positive potentials, while in the presence of NASPM, the decay was progressively accelerated (Figure 3d). In the absence of NASPM, τdecay was slower at +60 mV than at −60 mV (7.3 ± 0.6 ms versus 5.7 ± 0.3 ms, n=8; paired mean difference 1.55 ms [0.86, 2.62], p=0.019 two-sided paired Welch t-test). By contrast, in the presence of NASPM τdecay was markedly faster at +60 mV than at −60 mV (1.7 ± 0.2 ms versus 5.6 ± 0.6 ms, n=10; paired mean difference −4.14 ms [−5.44,–3.07], p=0.00012, two-sided paired Welch t-test). Of note, at +60 mV, along with the accelerated decay in the presence of NASPM, we also observed a dramatic slowing of the recovery of peak responses following the removal of glutamate (Figure 3e). Currents were elicited by pairs of 100 ms glutamate applications at frequencies from 0.125 to 4 Hz. The peak amplitude of successive responses is normally shaped solely by the kinetics of recovery from desensitization. In the absence of polyamines, a small degree of residual desensitization was apparent at 4 Hz (150 ms interval), but full recovery was seen at all other intervals, at both +60 and −60 mV (Figure 3f). However, in the presence of NASPM, although responses at −60 mV were indistinguishable from those in the absence of polyamines, at +60 mV an additional slow component of recovery (τrec slow 4.9 s; Figure 3f) was present. The biphasic recovery is suggestive of two populations of receptors, those that desensitized before being blocked (fast component of recovery), and those that were blocked by NASPM before they desensitized (slow component of recovery). Taken together, our data suggest that 10 μM intracellular NASPM produces a pronounced, rapid and long-lasting inhibition of CP-AMPARs at positive potentials. NASPM can induce complete rectification that is unaffected by auxiliary subunits We found that with 10 µM NASPM, the block of CP-AMPARs was incomplete (Figure 3a and b) and depended on the recent history of the channel (Figure 3f). However, as intracellular polyamines produce both closed- and open-channel blocks of CP-AMPARs (Bowie et al., 1998; Rozov et al., 1998), we reasoned that a higher concentration of NASPM would block more effectively and cause pronounced rectification, regardless of recent activity. With a higher concentration of NASPM not only would closed-channel block be more favored, rendering a higher proportion of receptors silent prior to activation, but unblocked closed receptors would more rapidly enter a state of open-channel block once activated at positive potentials. For example, an open channel block with 100 µM would be approximately 10 times faster than with 10 µM NASPM and would be expected to rapidly curtail charge transfer. Importantly, the block of CP-AMPARs by intracellular spermine is known to be affected by AMPAR auxiliary proteins. Different TARP and CNIH family members reduce inward rectification to varying extents (Brown et al., 2018; Cho et al., 2007; Coombs et al., 2012; Soto et al., 2007; Soto et al., 2009), while GSG1L increases rectification (McGee et al., 2015). Thus, an experimentally observed change in spermine-induced rectification may not arise solely from a change in CP-AMPAR prevalence. Accordingly, we next sought to determine whether intracellular NASPM was able to produce complete rectification, and whether its action was affected by the presence of different auxiliary subunits. We first examined the effect of NASPM and spermine on CI-AMPARs. When 100 μM spermine or NASPM was added to the internal solution (Figure 4a) the RI+60/−60 values were 0.90 ± 0.03 and 0.93 ± 0.06 (both n=6), while with 400 μM the corresponding RI+60/−60 values were 0.85 ± 0.11 and 0.77 ± 0.09 (n=5 and 6). This suggests either, that both spermine and NASPM have a small effect on CI-AMPARs, or that there may have been a minor complement of CP-AMPARs (GluA1 homomers) in some patches. Given that the RI+60/−60 values were ≥0.9 with 100 μM spermine or NASPM this concentration was chosen for all subsequent experiments. Figure 4 Download asset Open asset 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, −110 to +80 mV). The intracellular solution was supplemented with 100 µM spermine (blue) or NASPM (purple). (b) Mean normalized peak current-voltage (I-V) relationships for GluA1/GluA2/γ2 and GluA1/γ2 with 100 μM spermine (blue) or NASPM (purple). Symbols and error bars indicate the mean ± standard error of the mean. (c) Pooled rectification data (RI+60/−60) for GluA1 and GluA1/auxiliary subunit combinations with 100 μM spermine (Spm) or NASPM. Box-and-whisker plots indicate the median (black line), the first and third quartiles (Q1 and Q3; 25–75th percentiles) (box), and the range min(x[x>Q1+1.5 * inter-quartile range]) to max(x[x<Q3+1.5 * inter-quartile range]) (whiskers). Indicated p-values are from two-sided Welch two-sample t-tests (note that for GSG1L all RI values were 0). (d) Difference plots showing the shift in rectification index (Δ RI+60/−60) in the presence of NASPM compared with spermine. Symbols show mean unpaired differences, and the error bars indicate the bootstrapped 95% confidence intervals. 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 Download elife-66765-fig4-data1-v2.zip 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 Download elife-66765-fig4-data2-v2.zip We next compared the effects of spermine and NASPM on I-V relationships for GluA1 expressed alone or with different auxiliary subunits representing a broad cross-section of those known to differentially modulate the function of native AMPARs (Figure 4b–d). Thus, we expressed GuA1 with TARPs (γ2, γ7 or γ8), cornichons (CNIH2 or CNIH3), GSG1L, or CKAMP59. We also examined I-V relationships in the presence of γ8 together with CNIH2 or CKAMP44. With GSG1L, full rectification was seen with both 100 μM spermine and 100 μM NASPM (RI+60/−60=0). For all other combinations, in the presence of spermine RI+60/−60 varied (from 0.037 to 0.29) depending on the auxiliary subunit present, but with NASPM rectification was essentially complete in all cases (RI+60/−60 varied from 0 to 0.022) (Figure 4c). Thus, unlike 100 μM spermine, 100 μM NASPM produces a near-total block of outward CP-AMPAR-mediated currents that appears to be independent of the type of auxiliary subunit. The effects of intracellular NASPM on native AMPARs To determine whether the difference between spermine and NASPM seen with recombinant receptors was preserved in different native receptor populations, we next compared the actions of the polyamines on extrasynaptic and synaptic receptors in neurons from wild-type and GluA2 KO (Gria2tm1Rod/J) mice (Jia et al., 1996). First, we compared the rectification conferred by 100 μM spermine or NASPM in patches pulled from visually identified dentate gyrus granule cells (DGGC) in acute hippocampal slices (Figure 5a and b). These cells strongly express auxiliary subunits CKAMP44 and γ8 (Fukaya et al., 2006; Khodosevich et al., 2014; Tomita et al., 2003) and AMPAR-mediated currents from excised patches have previously been shown to display partial rectification, potentially indicative of a mixed complement of CP- and CI-AMPARs (Khodosevich et al., 2014). In wild-type patches, our data were consistent with this. With spermine, the RI+60/−60 was 0.62 ± 0.04 (n=6) (range 0.50–0.72) and with NASPM it was 0.58 ± 0.05 (n=8) (range 0.45–0.95). The unpaired mean difference was −0.037 [−0.14, 0.10] (p=0.59, two-sided Welch two-sample t-test) (Figure 5c). For GluA2 KO DGGC, although both polyamines conferred enhanced rectification, there was a clear difference in its extent. In the presence of spermine, outward currents were present at +60 mV and the RI+60/−60 was 0.26 ± 0.08 (n=5) (range 0.15–0.56). By contrast, outward currents were negligible in the presence of NASPM and the RI+60/−60 was 0.02 ± 0.01 (n=6) (range 0.003–0.05). The unpaired mean difference was −0.24 [−0.47,–0.15] (p=0.036, two-sided Welch two-sample t-test). These data from DGGC patches are consistent with our recombinant data and demonstrate that on native CP-AMPARs NASPM also produces more pronounced rectification than spermine. Figure 5 Download asset Open asset Intracellular 1-naphthylacetyl spermine (NASPM) (100 μM) produces full r