Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) play essential roles in synaptic transmission and plasticity in the CNS. Type 1 transmembrane AMPAR regulatory proteins (TARPs) are CP-AMPAR auxiliary subunits that slow gating, increase single-channel conductance and relieve channel block by intracellular polyamines (Soto et al., 2007). Conversely, the atypical auxiliary subunit GSG1L (germ cell-specific gene 1-like protein) (Shanks et al., 2012; Schwenk., et al 2012) decreases single-channel conductance and increases polyamine block, suppressing current flow and excitatory synaptic transmission (McGee et al., 2015; Gu et al., 2016). The aim of this study was to investigate the role of polyamines in the opposing effects of these different auxiliary subunits by examining the effect of spermine on CP-AMPARs expressed with and without GSG1L and the type 1 TARP γ2.  AMPARs were transiently transfected into Human Embryonic Kidney (HEK 293) cells and currents were recorded from outside-out patches in response to rapid application of glutamate (10 mM) achieved through piezoelectric translation of a theta-barrel application tool. Macroscopic current-voltage relationships were examined from −120 to +100 mV to characterise spermine-dependent rectification.  To estimate the weighted mean single-channel conductance we used nonstationary fluctuation analysis (NSFA). We also analysed directly resolved channel openings in the tail of macroscopic currents at −120 mV. In the absence of auxiliary subunits, elevating intracellular spermine from 100 µM to 1 mM unexpectedly, decreased single-channel conductance of GluA2(Q), as measured with NSFA, by ~50% (from 20.6 ± 1.6 to 9.1 ± 2.2 pS; p < 0.0001; Welch t test; n = 12 and 5, respectively). As TARP γ2 greatly reduces the block of CP-AMPARs by intracellular spermine, we predicted that in its presence the polyamine-dependent reduction of GluA2(Q) conductance would also be reduced. This was indeed the case. With co-expression of γ2 the effect spermine was eliminated (30.9 ± 2.3 and 27.4 ± 3.7 pS with 100 µM and 1 mM spermine, respectively; p = 0.45; unpaired Welch two-sample t test; n = 10 and 4).  In contrast, GSG1L increased the effect of spermine on channel conductance.  In the presence of just 100 µM intracellular spermine, the single-channel conductance was 12.7 ± 0.6 pS (n =18; p = 0.00031 compared to GluA2(Q) alone and p = 0.011 compared to the spermine-free condition; n = 14). This reduction in NSFA-estimated single-channel conductance of GluA2(Q)/GSG1L by 100 µM spermine was mirrored by its effect on the mean amplitude of directly resolved single-channel events (reduced from 21.3 ± 1.4 to 15.7 ± 0.3 pS; p = 0.016; unpaired Welch two-sample t test; n = 5 and 5).  Finally, we observed that GSG1L and spermine reduced channel conductance only when aspartate was present at the AMPARs Q/R +4 site in the channel's ion selectivity filter.  Together, our results demonstrate that polyamines and GSG1L cooperate to attenuate CP-AMPAR conductance.  Crucially, this unexpected property of intracellular polyamines is apparent at physiologically relevant negative membrane potentials.