Advertisement

Polyamine-mediated channel block of ionotropic glutamate receptors and its regulation by auxiliary proteins

  • Derek Bowie
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, Bellini Bldg., Rm. 164, McGill University, 3649 Promenade Sir William Osler, Montreal, Québec H3G 0B1, Canada. Tel.:514-398-1581; Fax:514- 398-6690
    Affiliations
    Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 0B1, Canada
    Search for articles by this author
  • Author Footnotes
    2 The abbreviations used are: SpmspermineSpdspermidineiGluRionotropic glutamate receptorAMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidAMPARα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptorNMDARN-methyl-d-aspartic acid receptorTARPtransmembrane AMPA receptor regulatory proteinNETOneuropilin and tolloid-like proteinPutputrescineKARkainate; receptorNASPM1-naphthyl acetyl spermineCNIHcornichonCNScentral nervous systempSpicosiemenCTZcyclothiazide.
Open AccessPublished:October 17, 2018DOI:https://doi.org/10.1074/jbc.TM118.003794
      Most excitatory neurotransmission in the mammalian brain is mediated by a family of plasma membrane–bound signaling proteins called ionotropic glutamate receptors (iGluRs). iGluRs assemble at central synapses as tetramers, forming a central ion-channel pore whose primary function is to rapidly transport Na+ and Ca2+ in response to binding the neurotransmitter l-glutamic acid. The pore of iGluRs is also accessible to bulkier cytoplasmic cations, such as the polyamines spermine, spermidine, and putrescine, which are drawn into the permeation pathway, but get stuck and block the movement of other ions. The degree of this polyamine-mediated channel block is highly regulated by processes that control the free cytoplasmic polyamine concentration, the membrane potential, or the iGluR subunit composition. Recently, an additional regulation by auxiliary proteins, most notably transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor regulatory proteins (TARPs), cornichons, and neuropilin and tolloid-like proteins (NETOs), has been identified. Here, I review what we have learned of polyamine block of iGluRs and its regulation by auxiliary subunits. TARPs, cornichons, and NETOs attenuate the channel block by enabling polyamines to exit the pore. As a result, polyamine permeation occurs at more negative and physiologically relevant membrane potentials. The structural basis for enhanced polyamine transport remains unresolved, although alterations in both channel architecture and charge-screening mechanisms have been proposed. That auxiliary subunits can attenuate the polyamine block reveals an unappreciated impact of polyamine permeation in shaping the signaling properties of neuronal AMPA- and kainate-type iGluRs. Moreover, enhanced polyamine transport through iGluRs may have a role in regulating cellular polyamine levels.

      Introduction

      The first documented evidence linking polyamines to an experimental observation can probably be attributed to the work of pioneering Dutch microscopist, Antonie Van Leeuwenhoek (
      • Anderson D.
      Still going strong: Leeuwenhoek at eighty.
      ), who in 1678, while observing spermatozoa, inadvertently noted crystal formation of polyamines in a specimen of drying semen (
      • Lewenhoeck A.
      Observationes D. Anthonii Lewenhoeck, de natis è semine genitali animalculis.
      ). The three naturally occurring polyamines, namely spermine (Spm)
      The abbreviations used are: Spm
      spermine
      Spd
      spermidine
      iGluR
      ionotropic glutamate receptor
      AMPA
      α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
      AMPAR
      α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
      NMDAR
      N-methyl-d-aspartic acid receptor
      TARP
      transmembrane AMPA receptor regulatory protein
      NETO
      neuropilin and tolloid-like protein
      Put
      putrescine
      KAR
      kainate; receptor
      NASPM
      1-naphthyl acetyl spermine
      CNIH
      cornichon
      CNS
      central nervous system
      pS
      picosiemen
      CTZ
      cyclothiazide.
      (
      • Ladenburg A.
      • Abel J.
      Ueber das aethylenimin (Spermin?).
      ,
      • Dudley H.W.
      • Rosenheim M.C.
      • Rosenheim O.
      The chemical constitution of spermine. I. The isolation of spermine from animal tissues, and the preparation of its salts.
      ), spermidine (Spd) (
      • Dudley H.W.
      • Rosenheim O.
      • Starling W.W.
      The constitution and synthesis of spermidine, a newly discovered base isolated from animal tissues.
      ), and putrescine (Put), would not be isolated nor formally identified until several centuries later with much of the early seminal contributions, especially in the area of the microbial biosynthetic pathways, made by Dr. Herbert (or Herb) Tabor and his wife, Dr. Celia Tabor (
      • Kresge N.S.
      • Simoni R.D.
      • Hill R.L.
      The biosynthesis of polyamines: the work of Herbert Tabor and Celia White Tabor.
      • Tabor H.
      • Tabor C.W.
      Spermidine, spermine, and related amines. Pharmacol.
      ,
      • Tabor C.W.
      • Tabor H.
      1,4-Diaminobutane (putrescine), spermidine, and spermine.
      ,
      • Tabor C.W.
      • Tabor H.
      Polyamines.
      ,
      • Tabor C.W.
      • Tabor H.
      Polyamines in microorganisms. Microbiol.
      • Tabor C.W.
      • Tabor H.
      It all started on a streetcar in Boston.
      ). Dr. Herb Tabor, who will celebrate his 100th birthday in November, 2018, could not have envisaged the many areas of research impacted by polyamines, although he and his wife appreciated early on, while working with Dr. Sanford Rosenthal at the National Institutes of Health, the ubiquitous cytoplasmic expression of polyamines and their potential role in disease (
      • Rosenthal S.M.
      • Tabor C.W.
      The pharmacology of spermine and spermidine; distribution and excretion.
      ,
      • Tabor C.W.
      • Rosenthal S.M.
      Pharmacology of spermine and spermidine; some effects on animals and bacteria.
      • Tabor H.
      • Rosenthal S.M.
      • Tabor C.W.
      The biosynthesis of spermidine and spermine from putrescine and methionine.
      ). Dr. Herb Tabor also contributed significantly to the growth of the Journal of Biological Chemistry over many decades (
      • Kresge N.S.
      • Simoni R.D.
      • Hill R.L.
      The biosynthesis of polyamines: the work of Herbert Tabor and Celia White Tabor.
      ,
      • Tabor C.W.
      • Tabor H.
      It all started on a streetcar in Boston.
      ); therefore, it is fitting that the Journal has commissioned a series of minireviews on the topic of polyamines to celebrate and reflect upon his life and career.
      Our understanding of the impact of polyamines, particularly on membrane excitability, has advanced significantly in the last 2 decades beginning with a series of observations showing that cytoplasmic polyamines block the pore regions of several voltage- (
      • Fu L.Y.
      • Cummins T.R.
      • Moczydlowski E.G.
      Sensitivity of cloned muscle, heart and neuronal voltage-gated sodium channels to block by polyamines: a possible basis for modulation of excitability in vivo.
      • Gomez M.
      • Hellstrand P.
      Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle.
      ,
      • Kerschbaum H.H.
      • Kozak J.A.
      • Cahalan M.D.
      Polyvalent cations as permeant probes of MIC and TRPM7 pores.
      • Lopatin A.N.
      • Makhina E.N.
      • Nichols C.G.
      Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification.
      ) and ligand-gated (
      • Kerschbaum H.H.
      • Kozak J.A.
      • Cahalan M.D.
      Polyvalent cations as permeant probes of MIC and TRPM7 pores.
      ,
      • Bowie D.
      • Mayer M.L.
      Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block.
      ,
      • Haghighi A.P.
      • Cooper E.
      Neuronal nicotinic acetylcholine receptors are blocked by intracellular spermine in a voltage-dependent manner. J.
      • Lu Z.
      • Ding L.
      Blockade of a retinal cGMP-gated channel by polyamines. J.
      ) ion channel families that are all cation-selective. Because Spm, Spd, and Put are positively charged at physiological pH, they are preferentially attracted into the water-filled pore regions of cation-selective channels where, due to their larger cross-sectional diameter (cf. Refs.
      • Bähring R.
      • Bowie D.
      • Benveniste M.
      • Mayer M.L.
      Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines.
      ,
      • Guo D.
      • Lu Z.
      Mechanism of cGMP-gated channel block by intracellular polyamines.
      ), they obstruct the transport of smaller permeating ions, such as Na+, K+ and/or Ca2+ (
      • Nichols C.G.
      • Lopatin A.N.
      Inward rectifier potassium channels.
      ,
      • Bowie D.
      • Bahring R.
      • Mayer M.L.
      ). The degree of channel block is voltage-dependent; and as a result, the action of cytoplasmic polyamines can finely tune cellular excitability within the range of the resting membrane potential by regulating the number of channels available for activation. This property of cytoplasmic polyamines is exemplified by their modulation of G-protein–gated inward rectifier K+ (GIRK) channels, which slows the cardiac action potential (
      • Sakmann B.
      • Noma A.
      • Trautwein W.
      Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of the mammalian heart.
      ,
      • Hibino H.
      • Inanobe A.
      • Furutani K.
      • Murakami S.
      • Findlay I.
      • Kurachi Y.
      Inwardly rectifying potassium channels: their structure, function, and physiological roles.
      ). Additional studies have revealed other dynamic aspects of polyamine channel block that include the following: (i) the regulated synthesis of cellular polyamine levels through the activity of ornithine decarboxylase (
      • Aizenman C.D.
      • Muñoz-Elias G.
      • Cline H.T.
      Visually driven modulation of glutamatergic synaptic transmission is mediated by the regulation of intracellular polyamines.
      ); (ii) the relief of channel block by activity-driven unblocking of polyamines (
      • Rozov A.
      • Zilberter Y.
      • Wollmuth L.P.
      • Burnashev N.
      Facilitation of currents through rat Ca2+-permeable AMPA receptor channels by activity-dependent relief from polyamine block.
      ,
      • Rozov A.
      • Burnashev N.
      Polyamine-dependent facilitation of postsynaptic AMPA receptors counteracts paired-pulse depression.
      • Bowie D.
      • Lange G.D.
      • Mayer M.L.
      Activity-dependent modulation of glutamate receptors by polyamines.
      ); and (iii) by trafficking polyamine-sensitive and -insensitive channels into and out of the plasma membrane (
      • Liu S.J.
      • Cull-Candy S.G.
      Activity-dependent change in AMPA receptor properties in cerebellar stellate cells.
      ,
      • Liu S.Q.
      • Cull-Candy S.G.
      Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype.
      • Plant K.
      • Pelkey K.A.
      • Bortolotto Z.A.
      • Morita D.
      • Terashima A.
      • McBain C.J.
      • Collingridge G.L.
      • Isaac J.T.
      Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation.
      ). More recent work has identified an additional level of regulation of polyamine block of ionotropic glutamate receptors (iGluRs) through their association with several families of auxiliary proteins called transmembrane AMPA receptor regulatory proteins (or TARPs), cornichons (CNIHs), and neuropilin and tolloid-like proteins (NETOs) (
      • Coombs I.D.
      • Soto D.
      • Zonouzi M.
      • Renzi M.
      • Shelley C.
      • Farrant M.
      • Cull-Candy S.G.
      Cornichons modify channel properties of recombinant and glial AMPA receptors.
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ,
      • Soto D.
      • Coombs I.D.
      • Kelly L.
      • Farrant M.
      • Cull-Candy S.G.
      Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors.
      ,
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ).
      In this Minireview, I will examine what we have learned about the mechanism of polyamine block of iGluRs in the last 2 decades. A detailed treatise on the biophysical mechanism(s) of channel block has been reviewed elsewhere (
      • Bowie D.
      • Bahring R.
      • Mayer M.L.
      ,
      • Bowie D.
      Redefining the classification of AMPA-selective ionotropic glutamate receptors.
      ,
      • Cull-Candy S.
      • Kelly L.
      • Farrant M.
      Regulation of Ca2+-permeable AMPA receptors: synaptic plasticity and beyond.
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ). Consequently, more emphasis will be placed on recent work examining the structural make-up of the AMPAR and KAR ion channel pore and how different mechanisms, particularly via auxiliary proteins, modify their architecture to shape ion permeation and polyamine block of native channels.
      What has emerged from this work is that most native AMPARs and KARs have evolved distinct mechanisms to circumvent polyamine channel block. The two distinct mechanisms that prevail in AMPARs include (i) the formation of an electrostatic repulsion site at the apex of the pore, called the Q/R site, and (ii) the co-assembly of AMPAR subunits with auxiliary proteins, such as TARPs and CNIHs. Kainate receptors have evolved three distinct mechanisms to attenuate polyamine block that include (i) electrostatic repulsion at the Q/R site, (ii) structural instability of pore helices via proline residues, and (iii) the modulatory effect of NETO proteins. Given the persistent presence of polyamines in the cytoplasm of almost all cells, these mechanisms ensure unfettered signaling by AMPARs and KARs in the mammalian CNS.

      Ionotropic glutamate receptors are differentially sensitive to polyamine channel block

      Ionotropic glutamate receptors mediate the vast majority of fast excitatory signaling in the mammalian brain through the activity of three major ion channel families that include AMPAR receptors (AMPARs), N-methyl-d-aspartic acid receptors (NMDARs), and kainate receptors (KARs) (Fig. 1) (
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ,
      • Traynelis S.F.
      • Wollmuth L.P.
      • McBain C.J.
      • Menniti F.S.
      • Vance K.M.
      • Ogden K.K.
      • Hansen K.B.
      • Yuan H.
      • Myers S.J.
      • Dingledine R.
      Glutamate receptor ion channels: structure, regulation, and function.
      ). The iGluR family also includes an additional orphan-class family composed of δ1 and δ2 subunits (Fig. 1) that signal via a metabotropic pathway, rather than an ion channel pore (
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ), through the formation of a trans-synaptic scaffolding complex (
      • Elegheert J.
      • Kakegawa W.
      • Clay J.E.
      • Shanks N.F.
      • Behiels E.
      • Matsuda K.
      • Kohda K.
      • Miura E.
      • Rossmann M.
      • Mitakidis N.
      • Motohashi J.
      • Chang V.T.
      • Siebold C.
      • Greger I.H.
      • Nakagawa T.
      • Yuzaki M.
      • Aricescu A.R.
      Structural basis for integration of GluD receptors within synaptic organizer complexes.
      ). Whereas AMPARs and KARs can be strongly regulated by cytoplasmic polyamines, NMDARs are relatively insensitive to intracellular channel block (
      • Bowie D.
      • Bahring R.
      • Mayer M.L.
      ). Curiously, however, NMDARs are antagonized in a voltage-dependent manner by external Spm and polyamine spider toxins (
      • Priestley T.
      • Woodruff G.N.
      • Kemp J.A.
      Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin636.
      • Reynolds I.J.
      The spider toxin, argiotoxin636, binds to a Mg2+ site on the N-methyl-d-aspartate receptor complex.
      ,
      • Raditsch M.
      • Ruppersberg J.P.
      • Kuner T.
      • Günther W.
      • Schoepfer R.
      • Seeburg P.H.
      • Jahn W.
      • Witzemann V.
      Subunit-specific block of cloned NMDA receptors by argiotoxin636.
      ,
      • Benveniste M.
      • Mayer M.L.
      Multiple effects of spermine on N-methyl-d-aspartic acid receptor responses of rat cultured hippocampal neurones.
      ,
      • Williams K.
      Interactions of polyamines with ion channels.
      • Huettner J.E.
      Glutamate receptor pores.
      ). Why NMDARs are insensitive to cytoplasmic polyamines is unclear, although it may relate to the narrower diameter (5.5 Å) and the multi-ion occupancy of the NMDAR pore (
      • Villarroel A.
      • Burnashev N.
      • Sakmann B.
      Dimensions of the narrow portion of a recombinant NMDA receptor channel.
      ) compared with the larger diameter (7–7.8 Å) (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ) and the single ion-pore of AMPARs and KARs (
      • Huettner J.E.
      Glutamate receptor pores.
      ,
      • Wollmuth L.P.
      • Sakmann B.
      Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels.
      ) that may be more suitable for the blocker. Given the estimated radii of a sodium ion and a water molecule to be 1.02 and 2.75 Å, respectively (
      • Briand C.L.
      • Burton J.J.
      Molecular dynamics study of the effects of ions on water microclusters.
      ), and the internuclear distance between sodium and oxygen to be 2.35 Å (
      • Marcus Y.
      Ionic radii in aqueous solutions.
      ), these dimensions suggest that the 7–7.8 Å pore of AMPARs and KARs is capable of transporting hydrated alkali metal ions, such as Na+. As discussed later, subtle changes in the geometry of the pore may be a critical factor in governing the degree of block of iGluRs by polyamines especially as it relates to the regulatory effect of auxiliary subunits on AMPARs and KARs.
      Figure thumbnail gr1
      Figure 1Ionotropic glutamate receptor families share a common tetrameric architecture. Left, X-ray crystal structure showing the tetrameric subunit arrangement of the homomeric GluA2 AMPAR, which consists of four distinct domains that include (i) an N-terminal domain (ATD), which directs subunit assembly and receptor clustering at synapses (
      • García-Nafría J.
      • Herguedas B.
      • Watson J.F.
      • Greger I.H.
      The dynamic AMPA receptor extracellular region: a platform for synaptic protein interactions.
      ); (ii) four clamshell-like ligand-binding pockets (LBD) (
      • Mayer M.L.
      • Armstrong N.
      Structure and function of glutamate receptor ion channels.
      ); (iii) a TMD that forms a central ion channel pore that transports Na+ and Ca2+ ions in response to neurotransmitter binding (
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ,
      • Traynelis S.F.
      • Wollmuth L.P.
      • McBain C.J.
      • Menniti F.S.
      • Vance K.M.
      • Ogden K.K.
      • Hansen K.B.
      • Yuan H.
      • Myers S.J.
      • Dingledine R.
      Glutamate receptor ion channels: structure, regulation, and function.
      ); and (iv) a cytoplasmic C-terminal domain (not shown) that directs receptor trafficking and synaptic anchoring (
      • Shepherd J.D.
      • Huganir R.L.
      The cell biology of synaptic plasticity: AMPA receptor trafficking.
      ). Image of structure was adapted from Ref.
      • Sobolevsky A.I.
      • Rosconi M.P.
      • Gouaux E.
      X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor.
      . Right, table of the four classes of iGluR arranged with each of their respective subunits.

      Native AMPA receptor heteromers either possess or lack the GluA2 subunit

      AMPARs are found at almost all glutamatergic synapses and in all brain regions (
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ,
      • Traynelis S.F.
      • Wollmuth L.P.
      • McBain C.J.
      • Menniti F.S.
      • Vance K.M.
      • Ogden K.K.
      • Hansen K.B.
      • Yuan H.
      • Myers S.J.
      • Dingledine R.
      Glutamate receptor ion channels: structure, regulation, and function.
      ). Consequently, understanding how they are regulated, including block by cytoplasmic polyamines, has been an area of active investigation.
      AMPARs are primarily responsible for the rapid millisecond response rise time to the neurotransmitter, l-glutamate (l-Glu) (
      • Bowie D.
      Redefining the classification of AMPA-selective ionotropic glutamate receptors.
      ,
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ). However, they also strengthen or weaken glutamatergic transmission by cycling into and out of synapses during periods of sustained patterned activity or altered homeostasis (
      • Lisman J.
      Glutamatergic synapses are structurally and biochemically complex because of multiple plasticity processes: long-term potentiation, long-term depression, short-term potentiation and scaling.
      • Henley J.M.
      • Wilkinson K.A.
      Synaptic AMPA receptor composition in development, plasticity and disease.
      ,
      • Turrigiano G.G.
      The dialectic of Hebb and homeostasis.
      • Herring B.E.
      • Nicoll R.A.
      Long-term potentiation: from CaMKII to AMPA receptor trafficking.
      ). AMPARs can, in principle, assemble as homomeric and/or heteromeric tetramers from any one of four possible receptor subunits, namely GluA1 to GluA4, that were cloned in the 1990s (
      • Seeburg P.H.
      The TiPS/TINS lecture: the molecular biology of mammalian glutamate receptor channels.
      ,
      • Hollmann M.
      • Heinemann S.
      Cloned glutamate receptors.
      ). However, in situ hybridization (
      • Keinänen K.
      • Wisden W.
      • Sommer B.
      • Werner P.
      • Herb A.
      • Verdoorn T.A.
      • Sakmann B.
      • Seeburg P.H.
      A family of AMPA-selective glutamate receptors.
      ) and single-cell PCR studies (
      • Geiger J.R.
      • Melcher T.
      • Koh D.S.
      • Sakmann B.
      • Seeburg P.H.
      • Jonas P.
      • Monyer H.
      Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.
      ,
      • Angulo M.C.
      • Lambolez B.
      • Audinat E.
      • Hestrin S.
      • Rossier J.
      Subunit composition, kinetic, and permeation properties of AMPA receptors in single neocortical nonpyramidal cells.
      • Bochet P.
      • Audinat E.
      • Lambolez B.
      • Crépel F.
      • Rossier J.
      • Iino M.
      • Tsuzuki K.
      • Ozawa S.
      Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel.
      ) established during that time that most brain regions and individual neurons express more than one AMPAR subunit that usually includes the GluA2 subunit. The current consensus is that neurons express a preferred family of AMPARs (
      • Bowie D.
      Redefining the classification of AMPA-selective ionotropic glutamate receptors.
      ,
      • Henley J.M.
      • Wilkinson K.A.
      Synaptic AMPA receptor composition in development, plasticity and disease.
      ,
      • Jacobi E.
      • von Engelhardt J.
      Diversity in AMPA receptor complexes in the brain.
      ). For example, studies in the hippocampal and cortical brain regions suggest that most native AMPARs assemble as GluA1/GluA2 heteromers or GluA2/GluA3 heteromers (
      • Lu W.
      • Shi Y.
      • Jackson A.C.
      • Bjorgan K.
      • During M.J.
      • Sprengel R.
      • Seeburg P.H.
      • Nicoll R.A.
      Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach.
      ,
      • Wenthold R.J.
      • Petralia R.S.
      • Blahos II, J.
      • Niedzielski A.S.
      Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons.
      • Shi S.
      • Hayashi Y.
      • Esteban J.A.
      • Malinow R.
      Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.
      ). GluA1/A2 heteromers are proposed to traffic into central synapses during periods of patterned activity triggered by the activation of Ca2+/calmodulin-dependent protein kinase II, whereas GluA2/A3 receptors are thought to constitutively cycle into and out of synapses (
      • Shi S.
      • Hayashi Y.
      • Esteban J.A.
      • Malinow R.
      Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.
      ). Because EM studies estimate that the GluA3 subunit is expressed at about a tenth of the level of GluA1 and GluA2 (
      • Sans N.
      • Vissel B.
      • Petralia R.S.
      • Wang Y.X.
      • Chang K.
      • Royle G.A.
      • Wang C.Y.
      • O'Gorman S.
      • Heinemann S.F.
      • Wenthold R.J.
      Aberrant formation of glutamate receptor complexes in hippocampal neurons of mice lacking the GluR2 AMPA receptor subunit.
      ), it suggests that a significant proportion of all AMPARs are earmarked for synaptic plasticity mechanisms (
      • Kessels H.W.
      • Kopec C.D.
      • Klein M.E.
      • Malinow R.
      Roles of stargazin and phosphorylation in the control of AMPA receptor subcellular distribution.
      ). Other combinations are also possible, for example, migrating and mature cerebellar granule cells are thought to express GluA2/A4 heteromeric receptors (
      • Smith T.C.
      • Howe J.R.
      Concentration-dependent substate behavior of native AMPA receptors.
      ,
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ) as well as femtosiemens conductance channels that are consistent with GluA2 homomers (
      • Smith T.C.
      • Howe J.R.
      Concentration-dependent substate behavior of native AMPA receptors.
      ).
      There are, of course, exceptions to this general rule of the inclusion of GluA2. For example, several studies using single-cell PCR and/or antibody staining to document the expression level of different AMPAR subunits have identified some cells that either lack the GluA2 subunit entirely or express it at low levels. Cerebellar Bergmann glia cells (
      • Geiger J.R.
      • Melcher T.
      • Koh D.S.
      • Sakmann B.
      • Seeburg P.H.
      • Jonas P.
      • Monyer H.
      Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.
      ,
      • Burnashev N.
      • Khodorova A.
      • Jonas P.
      • Helm P.J.
      • Wisden W.
      • Monyer H.
      • Seeburg P.H.
      • Sakmann B.
      Calcium-permeable AMPA-kainate receptors in fusiform cerebellar glial cells.
      ) and inhibitory interneurons of the basolateral amygdala (
      • Mahanty N.K.
      • Sah P.
      Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala.
      ,
      • McDonald A.J.
      Localization of AMPA glutamate receptor subunits in subpopulations of non-pyramidal neurons in the rat basolateral amygdala.
      ) are apparently devoid of GluA2, whereas dentate gyrus basket cells and some interneurons of the hippocampus, such as parvalbumin-positive (PV+) basket cells (
      • Geiger J.R.
      • Melcher T.
      • Koh D.S.
      • Sakmann B.
      • Seeburg P.H.
      • Jonas P.
      • Monyer H.
      Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.
      ,
      • Bochet P.
      • Audinat E.
      • Lambolez B.
      • Crépel F.
      • Rossier J.
      • Iino M.
      • Tsuzuki K.
      • Ozawa S.
      Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel.
      ), and a fraction of neocortical fast-spiking nonpyramidal (
      • Angulo M.C.
      • Lambolez B.
      • Audinat E.
      • Hestrin S.
      • Rossier J.
      Subunit composition, kinetic, and permeation properties of AMPA receptors in single neocortical nonpyramidal cells.
      ) and pyramidal layer V cells (
      • Kumar S.S.
      • Bacci A.
      • Kharazia V.
      • Huguenard J.R.
      A developmental switch of AMPA receptor subunits in neocortical pyramidal neurons.
      ) all express low levels of GluA2. It is possible that other neurons and/or glial cells express GluA2 at diminished levels; however, their distribution has yet to be formally investigated. To complicate matters further, individual neurons may express both GluA2-lacking and GluA2-containing receptors which, in some cases, have been shown to be segregated to different synapses of the same neuron. For example, synapses of individual stratum lucidum hippocampal interneurons express GluA2-containing AMPARs when innervated by axon collaterals from CA3 pyramidal neurons and GluA2-lacking AMPARs when innervated by mossy-fiber axons of dentate gyrus granule cells (
      • Tóth K.
      • McBain C.J.
      Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurons.
      ). Interestingly, cells with reduced levels of GluA2 often express GluA1 and GluA4 receptor subunits. Whether GluA1 and GluA4 assemble as distinct populations of homomeric channels or heteromers in these cells is still not clear; although gene knockout studies reveal that both subunits contribute similarly to the ensemble AMPAR response of hippocampal PV+ basket cells (
      • Fuchs E.C.
      • Zivkovic A.R.
      • Cunningham M.O.
      • Middleton S.
      • Lebeau F.E.
      • Bannerman D.M.
      • Rozov A.
      • Whittington M.A.
      • Traub R.D.
      • Rawlins J.N.
      • Monyer H.
      Recruitment of parvalbumin-positive interneurons determines hippocampal function and associated behavior.
      ). GluA4-containing AMPARs are also one of the most prominent receptors expressed in the developing brain especially in inhibitory interneurons (
      • Bernard V.
      • Somogyi P.
      • Bolam J.P.
      Cellular, subcellular, and subsynaptic distribution of AMPA-type glutamate receptor subunits in the neostriatum of the rat.
      ,
      • Pelkey K.A.
      • Barksdale E.
      • Craig M.T.
      • Yuan X.
      • Sukumaran M.
      • Vargish G.A.
      • Mitchell R.M.
      • Wyeth M.S.
      • Petralia R.S.
      • Chittajallu R.
      • Karlsson R.M.
      • Cameron H.A.
      • Murata Y.
      • Colonnese M.T.
      • Worley P.F.
      • McBain C.J.
      Pentraxins coordinate excitatory synapse maturation and circuit integration of parvalbumin interneurons.
      • Akgül G.
      • McBain C.J.
      Diverse roles for ionotropic glutamate receptors on inhibitory interneurons in developing and adult brain.
      ). Compared with the delivery mechanisms described for GluA1/A2 heteromers, GluA4-containing AMPARs appear to follow a different set of trafficking rules that is independent of Ca2+/calmodulin-dependent protein kinase II and relies on spontaneous synaptic transmission rather than patterned neuronal activity (
      • Zhu J.J.
      • Esteban J.A.
      • Hayashi Y.
      • Malinow R.
      Postnatal synaptic potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity.
      ). Consequently, receptor trafficking into and out of individual synapses is governed by the repertoire of AMPAR subunits that make up individual AMPAR heteromers. As explained below, the separation of native AMPAR populations into GluA2-containing or -lacking receptors not only affects receptor trafficking but also impacts the channel’s functional behavior, including block by cytoplasmic polyamines.

      GluA2 subunit determines ion-flow through the AMPA receptor pore

      AMPARs have been grouped into two functionally distinct families based on the presence or absence of the GluA2 receptor subunit (
      • Bowie D.
      • Bahring R.
      • Mayer M.L.
      ,
      • Bowie D.
      Redefining the classification of AMPA-selective ionotropic glutamate receptors.
      ,
      • Cull-Candy S.
      • Kelly L.
      • Farrant M.
      Regulation of Ca2+-permeable AMPA receptors: synaptic plasticity and beyond.
      ,
      • Isaac J.T.
      • Ashby M.C.
      • McBain C.J.
      The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity.
      ). GluA2-lacking AMPARs possess ion channels with a large single-channel conductance (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ), appreciable Ca2+ permeability (
      • Burnashev N.
      • Monyer H.
      • Seeburg P.H.
      • Sakmann B.
      Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.
      ,
      • Hollmann M.
      • Hartley M.
      • Heinemann S.
      Ca2+ permeability of KA-AMPA–gated glutamate receptor channels depends on subunit composition.
      • Hume R.I.
      • Dingledine R.
      • Heinemann S.F.
      Identification of a site in glutamate receptor subunits that controls calcium permeability.
      ), and high affinity to polyamine block (
      • Bowie D.
      • Mayer M.L.
      Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block.
      ,
      • Donevan S.D.
      • Rogawski M.A.
      Intracellular polyamines mediate inward rectification of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.
      ,
      • Kamboj S.K.
      • Swanson G.T.
      • Cull-Candy S.G.
      Intracellular spermine confers rectification on rat calcium-permeable AMPA and kainate receptors.
      • Koh D.S.
      • Burnashev N.
      • Jonas P.
      Block of native Ca2+-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification.
      ). In contrast, GluA2-containing receptors typically have much smaller unitary events (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ), divalent impermeability (
      • Burnashev N.
      • Monyer H.
      • Seeburg P.H.
      • Sakmann B.
      Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.
      ,
      • Hollmann M.
      • Hartley M.
      • Heinemann S.
      Ca2+ permeability of KA-AMPA–gated glutamate receptor channels depends on subunit composition.
      • Hume R.I.
      • Dingledine R.
      • Heinemann S.F.
      Identification of a site in glutamate receptor subunits that controls calcium permeability.
      ), and insensitivity to polyamine block (
      • Bowie D.
      • Mayer M.L.
      Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block.
      ,
      • Verdoorn T.A.
      • Burnashev N.
      • Monyer H.
      • Seeburg P.H.
      • Sakmann B.
      Structural determinants of ion flow through recombinant glutamate receptor channels.
      ). The exact copy number of GluA2 per AMPAR tetramer has been a matter of debate with some suggesting that the GluA2 content is variable (
      • Washburn M.S.
      • Numberger M.
      • Zhang S.
      • Dingledine R.
      Differential dependence on GluR2 expression of three characteristic features of AMPA receptors.
      ) or fixed (
      • Mansour M.
      • Nagarajan N.
      • Nehring R.B.
      • Clements J.D.
      • Rosenmund C.
      Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement.
      ). However, a recent cryo-EM structure of the intact GluA2/A3 AMPAR suggests that they can assemble in a 2:2 fixed format with the GluA2 subunits occupying positions proximal to the pore (
      • Herguedas B.
      • García-Nafría J.
      • Cais O.
      • Fernández-Leiro R.
      • Krieger J.
      • Ho H.
      • Greger I.H.
      Structure and organization of heteromeric AMPA-type glutamate receptors.
      ). There is also evidence for the existence of a third class of AMPAR which, although similarly Ca2+-permeable, is characterized by its near-insensitivity to internal and external channel block by polyamines (
      • Bowie D.
      Redefining the classification of AMPA-selective ionotropic glutamate receptors.
      ). Although its exact molecular composition still remains unknown, AMPARs with these characteristics have been observed in the retina (
      • Osswald I.K.
      • Galan A.
      • Bowie D.
      Light triggers expression of philanthotoxin-insensitive Ca2+-permeable AMPA receptors in the developing rat retina.
      ,
      • Mørkve S.H.
      • Veruki M.L.
      • Hartveit E.
      Functional characteristics of non-NMDA-type ionotropic glutamate receptor channels in AII amacrine cells in rat retina.
      ), brain stem (
      • Otis T.S.
      • Raman I.M.
      • Trussell L.O.
      AMPA receptors with high Ca2+ permeability mediate synaptic transmission in the avian auditory pathway.
      ), cerebellum (
      • Liu S.Q.
      • Cull-Candy S.G.
      Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype.
      ,
      • Soler-Llavina G.J.
      • Sabatini B.L.
      Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells.
      ), and glia progenitor cells (
      • Bowie D.
      Redefining the classification of AMPA-selective ionotropic glutamate receptors.
      ,
      • Meucci O.
      • Fatatis A.
      • Holzwarth J.A.
      • Miller R.J.
      Developmental regulation of the toxin sensitivity of Ca2+-permeable AMPA receptors in cortical glia.
      ).
      The critical role of the GluA2 subunit in determining sensitivity to polyamine block is due to RNA editing of an exon that encodes a critical residue in the GluA2 AMPAR pore region, called the Q/R site (Fig. 2) (
      • Higuchi M.
      • Single F.N.
      • Köhler M.
      • Sommer B.
      • Sprengel R.
      • Seeburg P.H.
      RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency.
      ,
      • Sommer B.
      • Köhler M.
      • Sprengel R.
      • Seeburg P.H.
      RNA editing in brain controls a determinant of ion flow in glutamate-gated channels.
      • Melcher T.
      • Maas S.
      • Higuchi M.
      • Keller W.
      • Seeburg P.H.
      Editing of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA in vitro reveals site-selective adenosine to inosine conversion.
      ). The resulting codon change in GluA2 RNA transcripts, from an adenosine to inosine due to adenosine deaminase acting on RNA type 2 (ADAR2) activity (
      • Rueter S.M.
      • Burns C.M.
      • Coode S.A.
      • Mookherjee P.
      • Emeson R.B.
      Glutamate receptor RNA editing in vitro by enzymatic conversion of adenosine to inosine.
      ,
      • Yang J.H.
      • Sklar P.
      • Axel R.
      • Maniatis T.
      Editing of glutamate receptor subunit B pre-mRNA in vitro by site-specific deamination of adenosine.
      • Higuchi M.
      • Maas S.
      • Single F.N.
      • Hartner J.
      • Rozov A.
      • Burnashev N.
      • Feldmeyer D.
      • Sprengel R.
      • Seeburg P.H.
      Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2.
      ), results in a switch in the amino acid sequence from a glutamine (Gln) to an arginine (Arg) (
      • Rosenthal J.J.
      • Seeburg P.H.
      A-to-I RNA editing: effects on proteins key to neural excitability.
      ,
      • Seeburg P.H.
      A-to-I editing: new and old sites, functions and speculations.
      ). Almost all transcripts of GluA2 are fully edited (>99% efficiency), which means that the Q/R site of all GluA2 receptor subunits contains a positively charged Arg (
      • Burnashev N.
      • Monyer H.
      • Seeburg P.H.
      • Sakmann B.
      Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.
      ,
      • Sommer B.
      • Köhler M.
      • Sprengel R.
      • Seeburg P.H.
      RNA editing in brain controls a determinant of ion flow in glutamate-gated channels.
      ). The activity of ADAR2 is particularly remarkable in this regard because RNA editing of most other signaling proteins, such as potassium channels and sodium pumps, is much less efficient (
      • Rosenthal J.J.
      • Seeburg P.H.
      A-to-I RNA editing: effects on proteins key to neural excitability.
      ). In contrast, transcripts of GluA1, -A3, and -A4 are not subject to editing at this site; consequently, these subunits contain a Gln at the Q/R site instead. The specificity of ADAR2’s actions on GluA2 is due to the cis-acting intron downstream of the unedited exonic site that is absent from GluA1, -A3, and -A4 and forms a dsRNA structure needed for editing (
      • Higuchi M.
      • Single F.N.
      • Köhler M.
      • Sommer B.
      • Sprengel R.
      • Seeburg P.H.
      RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency.
      ,
      • Yang J.H.
      • Sklar P.
      • Axel R.
      • Maniatis T.
      Editing of glutamate receptor subunit B pre-mRNA in vitro by site-specific deamination of adenosine.
      ,
      • Higuchi M.
      • Maas S.
      • Single F.N.
      • Hartner J.
      • Rozov A.
      • Burnashev N.
      • Feldmeyer D.
      • Sprengel R.
      • Seeburg P.H.
      Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2.
      ,
      • Seeburg P.H.
      A-to-I editing: new and old sites, functions and speculations.
      ).
      Figure thumbnail gr2
      Figure 2Sequence alignment of M2 re-entrant loop and M3 helix of different iGluR families. A, sequence comparison of the GluN1 and GluN2A NMDAR subunits with GluA2(R) AMPAR and the GluK2(Q) KAR subunits. The apex of the M2 pore loop of all iGluR families is capped by the Q/R/N site. In AMPARs and KARs, the Q/R site together with the +4 site (Asp in AMPARs and Glu in KARs) govern the degree of polyamine block. All iGluR families possess a conserved Asn residue in the M3 helix that contributes to the high-divalent permeability of the pore. B, cryo-EM structure of the GluA2(Q) AMPAR pore adapted from Protein Data Bank code 5WEO.
      To complicate matters, unedited AMPARs possess at least 3–4 subconductance levels that range in amplitude from 5 to 7, 10 to 15, 20 to 25, and 30 to 40 pS (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ,
      • Rosenmund C.
      • Stern-Bach Y.
      • Stevens C.F.
      The tetrameric structure of a glutamate receptor channel.
      • Banke T.G.
      • Bowie D.
      • Lee H.
      • Huganir R.L.
      • Schousboe A.
      • Traynelis S.F.
      Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase.
      ,
      • Dawe G.B.
      • Musgaard M.
      • Aurousseau M.R.P.
      • Nayeem N.
      • Green T.
      • Biggin P.C.
      • Bowie D.
      Distinct structural pathways coordinate the activation of AMPA receptor-auxiliary subunit complexes.
      • Zhang W.
      • Cho Y.
      • Lolis E.
      • Howe J.R.
      Structural and single-channel results indicate that the rates of ligand-binding domain closing and opening directly impact AMPA receptor gating.
      ). In conditions of reduced AMPAR desensitization, the distinct sublevels correspond to the fractional occupancy of each of the four ligand-binding sites with agonist molecule (
      • Smith T.C.
      • Howe J.R.
      Concentration-dependent substate behavior of native AMPA receptors.
      ,
      • Rosenmund C.
      • Stern-Bach Y.
      • Stevens C.F.
      The tetrameric structure of a glutamate receptor channel.
      ,
      • Zhang W.
      • Eibl C.
      • Weeks A.M.
      • Riva I.
      • Li Y.J.
      • Plested A.J.R.
      • Howe J.R.
      Unitary properties of AMPA receptors with reduced desensitization.
      ). However, when desensitization is intact, the subconductance levels have been proposed to correspond to the number of desensitized subunits per tetramer (
      • Bowie D.
      • Lange G.D.
      Functional stoichiometry of glutamate receptor desensitization.
      ). Under these conditions, the sublevels have been linked to conformations occurring within the ligand-binding domain dimer interface (
      • Daniels B.A.
      • Andrews E.D.
      • Aurousseau M.R.
      • Accardi M.V.
      • Bowie D.
      Crosslinking the ligand-binding domain dimer interface locks kainate receptors out of the main open state.
      ,
      • Musgaard M.
      • Biggin P.C.
      Steered molecular dynamics simulations predict conformational stability of glutamate receptors.
      ) and/or the extracellular portion of the α-helix of transmembrane domain 3 (M3) (
      • Zhang W.
      • Eibl C.
      • Weeks A.M.
      • Riva I.
      • Li Y.J.
      • Plested A.J.R.
      • Howe J.R.
      Unitary properties of AMPA receptors with reduced desensitization.
      ). Based on what we know about other tetrameric ion channels (
      • Root M.J.
      • MacKinnon R.
      Two identical noninteracting sites in an ion channel revealed by proton transfer.
      ), it is unlikely that the distinct AMPAR open states of the channel correspond to significant structural re-arrangements of the pore region. It is possible, however, that each subconductance state may be distinguishable by their relative permeability to different cations. For example, in Shaker K+ channels where inactivation has been slowed, the different sublevels observed during sequential channel activation have different selectively for alkali metal ions (
      • Zheng J.
      • Sigworth F.J.
      Selectivity changes during activation of mutant Shaker potassium channels.
      ,
      • Zheng J.
      • Sigworth F.J.
      Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels.
      ). The smaller and intermediate open states are more permeable to Rb+ ions, whereas the main open state is more permeable to K+ (
      • Zheng J.
      • Sigworth F.J.
      Selectivity changes during activation of mutant Shaker potassium channels.
      ,
      • Zheng J.
      • Sigworth F.J.
      Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels.
      ). Whether the different AMPAR subconductance levels are similarly distinguishable in their ability to transport different cations remains to be investigated.
      In contrast, AMPARs containing four positively-charged Arg residues at the Q/R site, as would occur with homomeric GluA2(R) homomers, exhibit a dramatically reduced unitary conductance (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ) and lose their selectivity for cations (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ). Unitary events mediated by GluA2(R) AMPARs are too small to be resolved by conventional single-channel recordings and thus have been estimated to be 300 femtosiemens using stationary noise analysis (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ). Experiments on homomeric GluA2(R) channels suggest that the channel is not exclusively cation-selective but also exhibits anion permeability with the relative permeability of Cl to Cs+ estimated to be 0.17 (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ). The impact of the inclusion of an Arg at the Q/R site on unitary conductance and/or cation versus anion selectivity cannot be explained by differences in the cross-sectional diameter of the pore that are similar for both Q- and R-forms (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ). Instead, the positively-charged Arg ring structure at the selectivity filter is proposed to attract anion binding while repelling divalent ions (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ). The electropositive nature of the pore would also disfavor monovalent cation transport accounting for the much-reduced unitary channel conductance (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ).
      Less is known about the impact of the Q/R site on recombinant heteromeric AMPARs, such as GluA1/A2 or GluA2/A3 receptors, due to the difficulty of obtaining single-channel recordings that are not contaminated by the expression of homomeric channels. The recent GluA2/A3 AMPAR structure suggests that the pore region of heteromers has a 2Q:2R arrangement (
      • Herguedas B.
      • García-Nafría J.
      • Cais O.
      • Fernández-Leiro R.
      • Krieger J.
      • Ho H.
      • Greger I.H.
      Structure and organization of heteromeric AMPA-type glutamate receptors.
      ), which would be expected to impact cation transport. In keeping with this, earlier work by Swanson et al. (
      • Swanson G.T.
      • Kamboj S.K.
      • Cull-Candy S.G.
      Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.
      ) had shown that the single-channel events of GluA2(R)/A4(Q) heteromers had a main conductance of 4 pS when compared with the 8-pS main open state of GluA2(Q)/A4(Q) heteromers, which is similar to observations reported for GluA1(Q)/A2(R) heteromeric channels (
      • Mansour M.
      • Nagarajan N.
      • Nehring R.B.
      • Clements J.D.
      • Rosenmund C.
      Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement.
      ). Heteromeric channels are strictly cation-selective (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ) suggesting that, unlike homomeric GluA2(R) channels, the presence of only two Arg residues at the Q/R site is insufficient to attract anions into the pore.

      Molecular architecture and block of the AMPA receptor pore

      Prior to any direct structural insight, the Q/R site was already recognized as being at the apex of a re-entrant loop facing the extracellular vestibule of the channel (
      • Kuner T.
      • Beck C.
      • Sakmann B.
      • Seeburg P.H.
      Channel-lining residues of the AMPA receptor M2 segment: structural environment of the Q/R site and identification of the selectivity filter.
      ,
      • Bennett J.A.
      • Dingledine R.
      Topology profile for a glutamate receptor: three transmembrane domains and a channel-lining reentrant membrane loop.
      • Panchenko V.A.
      • Glasser C.R.
      • Partin K.M.
      • Mayer M.L.
      Amino acid substitutions in the pore of rat glutamate receptors at sites influencing block by polyamines.
      ). This structural arrangement is comparable with the pore architecture of other ion channels, such as K+ channels, Na+ channels, Ca2+ channels, and cyclic nucleotide-gated ion channels, which are now collectively known as pore–loop channels (
      • MacKinnon R.
      Pore loops: an emerging theme in ion channel structure.
      • Panchenko V.A.
      • Glasser C.R.
      • Mayer M.L.
      Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis.
      ,
      • Chen G.Q.
      • Cui C.
      • Mayer M.L.
      • Gouaux E.
      Functional characterization of a potassium-selective prokaryotic glutamate receptor.
      • Kuner T.
      • Seeburg P.H.
      • Guy H.R.
      A common architecture for K+ channels and ionotropic glutamate receptors?.
      ). The commanding position of the Q/R site makes it suitable to act as a selectivity filter of cations entering the extracellular or intracellular vestibules of the pore. When the pore region of AMPARs contains four neutrally-charged glutamine (Gln) residues at the Q/R site, such as GluA1/A4 heteromers, they are thought to form a single ion–binding site at the narrowest region of the pore that controls the transport of both monovalent and divalent cations (
      • Wollmuth L.P.
      • Sakmann B.
      Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels.
      ). Because the permeability of all alkali metal ions, such as Na+ and K+, is similar for unedited Q-form AMPARs (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ), cations are thought to be transported without losing their inner water shells, which is probably not the case for the NMDAR pore that possesses multiple ion–binding sites (
      • Wollmuth L.P.
      • Sakmann B.
      Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels.
      ,
      • Schneggenburger R.
      • Ascher P.
      Coupling of permeation and gating in an NMDA-channel pore mutant.
      ). As mentioned above, this property is in keeping with the cross-sectional diameter of the AMPAR and KAR pores that have been estimated to be 7–7.8 Å (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ). Exactly how the electrostatic environment of the AMPAR pore is altered when the Q/R residues contain both Gln and Arg residues, as would occur with GluA1/A2 or GluA2/A3, is not known.
      The Q/R site is not the only residue that affects divalent permeability and polyamine block of AMPARs. Jatzke et al. (
      • Jatzke C.
      • Hernandez M.
      • Wollmuth L.P.
      Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.
      ) examined the role of several residues in the third transmembrane domain (M3) of AMPARs, including the conserved asparagine residue (N or Asn) of the SYTANLAAF motif and the highly-charged, extracellular vestibule formed by the DRPEER motif of the NR1 subunit, each of which have been linked to high Ca2+ permeability of NMDARs (Fig. 2) (
      • Watanabe J.
      • Beck C.
      • Kuner T.
      • Premkumar L.S.
      • Wollmuth L.P.
      DRPEER: a motif in the extracellular vestibule conferring high Ca2+ flux rates in NMDA receptor channels.
      ). As anticipated, mutation (Asn to Lys or Cys) of the conserved Asn in M3 of GluA2 AMPAR channels strongly attenuated divalent permeability establishing that this residue makes a significant contribution to Ca2+ influx in both AMPARs and NMDARs (
      • Jatzke C.
      • Hernandez M.
      • Wollmuth L.P.
      Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.
      ). Importantly, mutation of the Asn did not affect polyamine block (
      • Jatzke C.
      • Hernandez M.
      • Wollmuth L.P.
      Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.
      ) demonstrating that the ability of AMPARs to transport divalent ions can be uncoupled from their susceptibility to channel block. In contrast to NMDARs, the Glu and Arg residues C-terminal to M3 had only a minor effect on the divalent permeability of AMPARs and had no effect on KARs (
      • Jatzke C.
      • Hernandez M.
      • Wollmuth L.P.
      Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.
      ), although this region has been linked to regulation of KARs by auxiliary NETO proteins (
      • Griffith T.N.
      • Swanson G.T.
      Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2.
      ). Interestingly, another study also noted the uncoupling of Ca2+ permeability and polyamine block by mutating the negatively-charged Asp (to Asn) or Glu residue of AMPARs or KARs, respectively, which is four residues downstream of the Q/R site in the M2 (Fig. 2) (
      • Dingledine R.
      • Hume R.I.
      • Heinemann S.F.
      Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels.
      ). In this case, however, the +4 site attenuated polyamine channel block while apparently retaining high-divalent permeability (
      • Dingledine R.
      • Hume R.I.
      • Heinemann S.F.
      Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels.
      ). Panchenko et al. (
      • Panchenko V.A.
      • Glasser C.R.
      • Partin K.M.
      • Mayer M.L.
      Amino acid substitutions in the pore of rat glutamate receptors at sites influencing block by polyamines.
      ) explained this finding by proposing that there are multiple binding sites for the distributed charged amine groups in each polyamine molecule with the +4 site being located close to the cytoplasmic entrance of the channel pore. In keeping with this, mutation of the Q/R site (e.g. Gln to Asn (
      • Dingledine R.
      • Hume R.I.
      • Heinemann S.F.
      Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels.
      )) was also shown to eliminate polyamine block while retaining divalent permeability (
      • Burnashev N.
      • Monyer H.
      • Seeburg P.H.
      • Sakmann B.
      Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.
      ,
      • Dingledine R.
      • Hume R.I.
      • Heinemann S.F.
      Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels.
      ,
      • Curutchet P.
      • Bochet P.
      • Prado de Carvalho L.
      • Lambolez B.
      • Stinnakre J.
      • Rossier J.
      In the GluR1 glutamate receptor subunit a glutamine to histidine point mutation suppresses inward rectification but not calcium permeability.
      ) suggesting that polyamine molecules traverse multiple regulatory contact points in the pore.
      To examine the structure of the ion-permeation pathway, Twomey et al. (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ) compared the GluA2 AMPAR in conditions that promote the open or closed state of the channel. The closed conformation was favored by purifying the ZK200775 antagonist-bound GluA2 AMPAR in a covalent fusion construct with germline-specific gene 1-like (or GSG1L). GSG1L is an AMPAR auxiliary subunit that slows recovery from desensitization (
      • Shanks N.F.
      • Savas J.N.
      • Maruo T.
      • Cais O.
      • Hirao A.
      • Oe S.
      • Ghosh A.
      • Noda Y.
      • Greger I.H.
      • Yates 3rd., J.R.
      • Nakagawa T.
      Differences in AMPA and kainate receptor interactomes facilitate identification of AMPA receptor auxiliary subunit GSG1L.
      ), and thus, the authors reasoned that it would promote the closed state of the channel (either resting and/or desensitized) (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ,
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Structural bases of desensitization in AMPA receptor-auxiliary subunit complexes.
      ). The open conformation was achieved by purifying the agonist-bound GluA2 AMPAR in complex with the transmembrane AMPA receptor auxiliary protein (or TARP), stargazin, or γ2 (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ), which, unlike GSG1l, promotes the channel open state (
      • Tomita S.
      • Adesnik H.
      • Sekiguchi M.
      • Zhang W.
      • Wada K.
      • Howe J.R.
      • Nicoll R.A.
      • Bredt D.S.
      Stargazin modulates AMPA receptor gating and trafficking by distinct domains.
      ,
      • Priel A.
      • Kolleker A.
      • Ayalon G.
      • Gillor M.
      • Osten P.
      • Stern-Bach Y.
      Stargazin reduces desensitization and slows deactivation of the AMPA-type glutamate receptors.
      ). The stability of the open state was further promoted by obtaining cryo-EM structures in the presence of the positive allosteric modulator, cyclothiazide (CTZ), which attenuates AMPAR desensitization (
      • Patneau D.K.
      • Vyklicky Jr., L.
      • Mayer M.L.
      Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate.
      ). As elaborated below, these structural studies support the idea that the Q/R site, the +4 site, and conserved Asn of M3 are all ideally positioned to influence ion flow through the pore region of AMPARs (Figure 3, Figure 4).
      Figure thumbnail gr3
      Figure 3Closed conformation of the GluA2 AMPAR. A, cryo-EM structure of the GluA2 AMPAR in complex with the auxiliary protein, GSG1L, and the antagonist, ZK200775, which promote channel closure. B, closer view of the ion-conduction pathway (violet) with pore-lining residues in the M2 and M3 segments of subunits A and C shown in yellow highlighting the Q/R site (Gln-586) and +4 site (Asp-590). Adapted from Ref.
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      with permission.
      Figure thumbnail gr4
      Figure 4Open conformation of the GluA2 AMPAR. A, cryo-EM structure of the GluA2 AMPAR in complex with the auxiliary protein, γ2, the agonist, l-Glu, and the positive allosteric modulator, CTZ, which promote channel opening. B, closer view of the ion-conduction pathway (violet) with pore-lining residues in the M2 and M3 segments of subunits A and C shown in yellow highlighting the Q/R site (Gln-586) and +4 site (Asp-590). C, pore radius calculated using HOLE highlighting differences between the open state (pink), closed state (blue), and desensitized state (orange). Adapted from Ref.
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      with permission.
      As can be readily appreciated from these structures, access of ions to the Q/R site is regulated by movement of the M3 bundle crossing composed of the Thr-617, Ala-621, Thr-625, and Met-629 residues (Figure 3, Figure 4). In the closed position, the bundle crossing occludes access to the Q/R site (i.e. Gln-586, Fig. 3) whereas in the open conformation, it kinks out into a hydrophobic cavity in the middle of the channel pore (Fig. 4). A second constriction is formed below this cavity by the re-entrant loop of M2, which may operate as a lower gate much like other tetrameric ion channels, such as K+ (
      • Doyle D.A.
      • Morais Cabral J.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • MacKinnon R.
      The structure of the potassium channel: molecular basis of K+ conduction and selectivity.
      ) and TRPV1 channels (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ). In the closed conformation, the M2 helix below the Q/R site has an extensive network of hydrophobic interactions that are formed between M1 and M3 regions of the same subunit as well as M3 of the adjacent subunit (Figure 3, Figure 4) (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ). The open-pore dimensions shown in Fig. 4 fall short of the 7–7.8 Å measurement estimated for AMPARs and KARs (
      • Burnashev N.
      • Villarroel A.
      • Sakmann B.
      Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
      ), and therefore, our current model would not be expected to transport hydrated monovalent cations, such as Na+ (see text above). Although molecular dynamics simulations of the open channel structure (
      • Yelshanskaya M.V.
      • Mesbahi-Vasey S.
      • Kurnikova M.G.
      • Sobolevsky A.I.
      Role of the ion channel extracellular collar in AMPA receptor gating.
      ) and electron density measurements of the selectivity filter (see extended data, Fig. 5, B and C, of Ref.
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ) suggest that the pore is in an open configuration, it is likely that more information is still needed to understand the molecular architecture and dimensions of the fully open AMPAR pore. Twomey et al. (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ) also note that the pore loop is apparently more flexible in closed-state structures but more ordered in the open state to facilitate ion transport through the pore. As discussed below, flexibility of the pore loop is a recurring theme in iGluRs. The loss of polyamine block in GluK2/K5 KAR heteromers has also been proposed to be due to the destabilizing effect of proline residues on the M2 helix of GluK5 subunits (
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ).
      Figure thumbnail gr5
      Figure 5AMPAR pore occupied by polyamine channel blocker. A, extended structures of the endogenous polyamines, spermine and spermidine. B, cross-section of the GluA2(Q) AMPAR pore highlighting the electroneutral cavity (white) above the Q/R site and electronegative cavity (red) of the inner pore where endogenous polyamines, Spm and Spd (yellow for carbon and blue for nitrogen), are proposed to bind. C, more detailed structure of the pore highlighting the residues in M2 that contribute to polyamine binding and the position of the M3 helices in the open state. Adapted from Ref.
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Vassilevski A.A.
      • Sobolevsky A.I.
      Mechanisms of channel block in calcium-permeable AMPA receptors.
      with permission.
      The position of the polyamine blocker in the ion-permeation pathway was recently resolved by imaging cryo-EM structures of the GluA2(Q) AMPAR pore occupied by several known channel blockers. Specifically, the authors used the orb weaver spider toxin, argiotoxin 636 (AgTx-636) (
      • Herlitze S.
      • Raditsch M.
      • Ruppersberg J.P.
      • Jahn W.
      • Monyer H.
      • Schoepfer R.
      • Witzemann V.
      Argiotoxin detects molecular differences in AMPA receptor channels.
      ), the spider toxin analog, 1-naphthyl acetyl spermine (NASPM) (
      • Koike M.
      • Iino M.
      • Ozawa S.
      Blocking effect of 1-naphthyl acetyl spermine on Ca2+-permeable AMPA receptors in cultured rat hippocampal neurons.
      ), and the adamantane derivative, 1-trimethylammonio-5-(1-adamantane-methyl-ammoniopentane dibromide) (IEM-1460) (
      • Magazanik L.G.
      • Buldakova S.L.
      • Samoilova M.V.
      • Gmiro V.E.
      • Mellor I.R.
      • Usherwood P.N.
      Block of open channels of recombinant AMPA receptors and native AMPA/kainate receptors by adamantane derivatives.
      ), each of which block Ca2+-permeable, GluA2(R)-lacking AMPARs in a use-dependent manner. To promote channel opening, Twomey et al. (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ) tethered the AMPAR to the TARP, γ2, and bathed the samples in the agonist, l-Glu, and the positive allosteric modulator, CTZ (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Vassilevski A.A.
      • Sobolevsky A.I.
      Mechanisms of channel block in calcium-permeable AMPA receptors.
      ). AgTX-636, NASPM, and IEM-1460 all contain extended polyamine tails and a bulky hydrophobic headgroup that gets trapped in the narrow constriction of the permeation pathway allowing the authors to identify which pore residues interact with the polyamine tail.
      Using this approach, the overall GluA2 AMPAR structure with the pore blocker in place was revealed to be comparable with previous structures of the open-channel tetramer (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ). The upper portion of the pore was characterized by an electroneutral surface and central cavity that sits atop a narrowing caused by the glutamines of the Q/R site, which then opens into the lower electronegative portion of the channel (Fig. 5). The electronegativity of this area most likely contributes to the cation selectivity of AMPARs, which occurs primarily through the backbone carbonyl oxygens of Gln-586, Gly-588, Cys-589, and Asp-590 (Fig. 5). These backbone residues presumably contribute to the membrane electric field that gives rise to the steep voltage dependence of polyamine block through the exit rates (unblock and permeation) of polyamine blocker from the pore (
      • Bowie D.
      • Lange G.D.
      • Mayer M.L.
      Activity-dependent modulation of glutamate receptors by polyamines.
      ,
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). In keeping with this, Twomey et al. (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Grassucci R.A.
      • Frank J.
      • Sobolevsky A.I.
      Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
      ) suggest that the endogenous polyamines, Spm and Spd, block the pore at this point in the selectivity filter between residues Gln-586 and Asp-590. Because polyamines acting on AMPAR–TARP complexes induce both channel block and appreciable blocker permeation, it has been speculated that the polyamine molecule may adopt two conformations in the pore, a linear structure, as noted here, that would favor permeation and a kinked structure that would lead to channel block (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ). Whether polyamines entering the pore adopt two distinct structures, as also proposed for channel block of cyclic nucleotide-gated channels (
      • Lu Z.
      • Ding L.
      Blockade of a retinal cGMP-gated channel by polyamines. J.
      ) or K+ channels (
      • Miller C.
      Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K+ channel.
      ), awaits future investigation.

      AMPA and kainate receptor auxiliary proteins attenuate polyamine channel block

      An important development in the iGluR field has been the identification of a diverse family of auxiliary proteins that bind and modulate both AMPARs and KARs (
      • Jackson A.C.
      • Nicoll R.A.
      The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits.
      • Greger I.H.
      • Watson J.F.
      • Cull-Candy S.G.
      Structural and functional architecture of AMPA-type glutamate receptors and their auxiliary proteins.
      ,
      • Copits B.A.
      • Swanson G.T.
      Dancing partners at the synapse: auxiliary subunits that shape kainate receptor function.
      ,
      • Howe J.R.
      Modulation of non-NMDA receptor gating by auxiliary subunits.
      ,
      • Tomita S.
      • Castillo P.E.
      Neto1 and Neto2: auxiliary subunits that determine key properties of native kainate receptors.
      • Haering S.C.
      • Tapken D.
      • Pahl S.
      • Hollmann M.
      Auxiliary subunits: shepherding AMPA receptors to the plasma membrane.
      ). TARP, stargazin (γ2), was the first auxiliary protein to be identified that was shown to be critical for the transport and synaptic targeting of native AMPARs (
      • Schnell E.
      • Sizemore M.
      • Karimzadegan S.
      • Chen L.
      • Bredt D.S.
      • Nicoll R.A.
      Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number.
      ). Additional studies uncovered that stargazin and other TARP isoforms modulate the gating properties of AMPARs (
      • Kato A.S.
      • Siuda E.R.
      • Nisenbaum E.S.
      • Bredt D.S.
      AMPA receptor subunit-specific regulation by a distinct family of type II TARPs.
      ) as well as their responsiveness to allosteric modulators (
      • Tomita S.
      • Sekiguchi M.
      • Wada K.
      • Nicoll R.A.
      • Bredt D.S.
      Stargazin controls the pharmacology of AMPA receptor potentiators.
      ) and antagonists (
      • Menuz K.
      • Stroud R.M.
      • Nicoll R.A.
      • Hays F.A.
      TARP auxiliary subunits switch AMPA receptor antagonists into partial agonists.
      ,
      • Maclean D.M.
      • Bowie D.
      Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation.
      ). Subsequent proteomic analyses then identified the cornichon family (
      • Schwenk J.
      • Harmel N.
      • Zolles G.
      • Bildl W.
      • Kulik A.
      • Heimrich B.
      • Chisaka O.
      • Jonas P.
      • Schulte U.
      • Fakler B.
      • Klöcker N.
      Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors.
      ) as well as other auxiliary proteins such as GSG1L and CKAMP44 (
      • Schwenk J.
      • Harmel N.
      • Brechet A.
      • Zolles G.
      • Berkefeld H.
      • Müller C.S.
      • Bildl W.
      • Baehrens D.
      • Hüber B.
      • Kulik A.
      • Klöcker N.
      • Schulte U.
      • Fakler B.
      High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes.
      ) that differentially associate with AMPARs in a developmental- and regional-specific manner (
      • Collins M.O.
      AMPA receptor complex dynamics in time and space.
      ). At about the same time, the neuropilin and tolloid-like proteins, NETO1 and NETO2, were also identified and revealed to bind to native KARs and to modulate their trafficking and gating behavior (
      • Howe J.R.
      Modulation of non-NMDA receptor gating by auxiliary subunits.
      ,
      • Zhang W.
      • St-Gelais F.
      • Grabner C.P.
      • Trinidad J.C.
      • Sumioka A.
      • Morimoto-Tomita M.
      • Kim K.S.
      • Straub C.
      • Burlingame A.L.
      • Howe J.R.
      • Tomita S.
      A transmembrane accessory subunit that modulates kainate-type glutamate receptors.
      • Ng D.
      • Pitcher G.M.
      • Szilard R.K.
      • Sertié A.
      • Kanisek M.
      • Clapcote S.J.
      • Lipina T.
      • Kalia L.V.
      • Joo D.
      • McKerlie C.
      • Cortez M.
      • Roder J.C.
      • Salter M.W.
      • McInnes R.R.
      Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.
      ,
      • Copits B.A.
      • Robbins J.S.
      • Frausto S.
      • Swanson G.T.
      Synaptic targeting and functional modulation of GluK1 kainate receptors by the auxiliary neuropilin and tolloid-like (NETO) proteins.
      ,
      • Straub C.
      • Hunt D.L.
      • Yamasaki M.
      • Kim K.S.
      • Watanabe M.
      • Castillo P.E.
      • Tomita S.
      Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1.
      • Tang M.
      • Pelkey K.A.
      • Ng D.
      • Ivakine E.
      • McBain C.J.
      • Salter M.W.
      • McInnes R.R.
      Neto1 is an auxiliary subunit of native synaptic kainate receptors.
      ). A feature common to both TARP, CNIH, and NETO auxiliary protein families has been their ability to attenuate the degree of polyamine block of AMPARs and KARs.
      Both TARPs and CNIHs attenuate the polyamine block of AMPARs by causing a reduction in the onset of block and a greater relief of block observed at both negative and positive membrane potentials, respectively (
      • Coombs I.D.
      • Soto D.
      • Zonouzi M.
      • Renzi M.
      • Shelley C.
      • Farrant M.
      • Cull-Candy S.G.
      Cornichons modify channel properties of recombinant and glial AMPA receptors.
      ,
      • Soto D.
      • Coombs I.D.
      • Kelly L.
      • Farrant M.
      • Cull-Candy S.G.
      Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors.
      ,
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ). NETOs attenuate the polyamine block of KARs in a comparable manner (
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ) suggesting that the underlying mechanism may be similar (Fig. 6). Unlike block of the multi-ion pore of K+ channels (
      • Nichols C.G.
      • Lopatin A.N.
      Inward rectifier potassium channels.
      ,
      • Hibino H.
      • Inanobe A.
      • Furutani K.
      • Murakami S.
      • Findlay I.
      • Kurachi Y.
      Inwardly rectifying potassium channels: their structure, function, and physiological roles.
      ,
      • Kubo Y.
      Two aspects of the inward rectification mechanism. Effects of cytoplasmic blockers and extracellular K+ on the inward rectifier K+ channel.
      ,
      • Baronas V.A.
      • Kurata H.T.
      Inward rectifiers and their regulation by endogenous polyamines.
      ), the onset of block of AMPARs and KARs is caused by a single polyamine molecule entering and occluding the pore from the cytoplasm (
      • Bowie D.
      • Lange G.D.
      • Mayer M.L.
      Activity-dependent modulation of glutamate receptors by polyamines.
      ), whereas relief of block is due to polyamine permeation to the extracellular side of the pore (Fig. 6) (
      • Bähring R.
      • Bowie D.
      • Benveniste M.
      • Mayer M.L.
      Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines.
      ,
      • Bowie D.
      • Bahring R.
      • Mayer M.L.
      ). At 0 mV, the apparent Spm affinity for unedited GluA2(Q) AMPARs is reduced by about 3- or 10-fold by association with CNIH3 or stargazin (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ), respectively, whereas block of GluK2 KARs is attenuated by 8–20-fold through NETO1 and NETO2 association (
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). Because the rate of polyamine binding to the open AMPAR or KAR pore is unaffected by auxiliary proteins, the attenuation of block is caused almost entirely by faster exit rates of the blocker from the pore (Fig. 6) (
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ,
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). Spm resides less time at its block site, and consequently, the blocker returns to the cytoplasm or permeates all the way through the pore more readily. In keeping with this, Brown et al. (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ) demonstrated that Spm permeates the AMPAR and KAR pores to a much greater extent when bound to auxiliary proteins. For example, the permeability of Spm relative to Na+ was estimated to be 4–16-fold more permeable in GluA2 AMPARs associated with TARPs or CNIHs than GluA2 AMPAR alone (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ). A similar observation was also noted when comparing Spm permeability of GluK2 KARs in the presence and absence of NETOs (
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ).
      Figure thumbnail gr6
      Figure 6Auxiliary proteins speed up polyamine exit rates from the pore. A, conductance voltage plot of the voltage-dependent Spm block of GluK2 KARs. Note that the onset of block occurs at negative membrane potentials, whereas relief of block occurs at positive membrane potentials. B, plot showing how the rate of Spm binding (kon), unbinding (koff), and permeation (kperm) to the GluK2 KAR pore changes at different membrane potentials. Note that although Spm-binding rate is fairly voltage-insensitive, exit rates from the pore (koff and kperm) are steeply voltage-dependent. The solid black lines correspond to the sum of all block rates at different membrane potentials. C and D, plots showing how the rate of Spm unbinding (C) and permeation (D) is shifted by NETO1 and NET2 auxiliary proteins as well as by heteromerization. Adapted from Ref.
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      .
      The structural mechanism by which AMPAR and KAR auxiliary proteins attenuate polyamine block is still unresolved. However, because their primary effect is to curtail the time the blocking molecule resides at its binding site (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ), it is likely that TARPs, CNIHs, and NETOs alter the pore architecture. An attractive possibility is that auxiliary proteins re-shape the electronegative cavity between the Q/R and +4 sites where Spm and other endogenous polyamines are proposed to reside (
      • Twomey E.C.
      • Yelshanskaya M.V.
      • Vassilevski A.A.
      • Sobolevsky A.I.
      Mechanisms of channel block in calcium-permeable AMPA receptors.
      ). In keeping with this, Perrais et al. (
      • Perrais D.
      • Coussen F.
      • Mulle C.
      Atypical functional properties of GluK3-containing kainate receptors.
      ) have shown that the high nanomolar block affinity of GluK3 KARs is due to methionine (Met) and serine (Ser) residues in the M2 helix that are absent from GluK1 and GluK2 KARs, which contain Val and Ala residues instead (Fig. 7). The loss of nanomolar Spm affinity by replacement of the Met and Ser residues of GluK3 with Val and Ala, respectively, of GluK1 and GluK2 would not be expected to change the electrostatic environment of the pore; however, it may alter the structural flexibility of the M2 helix. In support of this, attenuation of polyamine block in GluK2/K5 KAR heteromers has been attributed to a conserved proline residue also found in the M2 helix, in this case of the GluK5 (and GluK4) subunit (Fig. 7) (
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). Molecular dynamic simulations suggest that the two proline residues, which are arranged on opposing subunits of the GluK2/K5 tetramer (
      • Reiner A.
      • Arant R.J.
      • Isacoff E.Y.
      Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex.
      ), increase structural flexibility in the pore making it less favorable for polyamine block and more favorable for permeation (Fig. 7) (
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). This is an important point because it reveals that GluK4- and/or GluK5-containing KAR heteromers attenuate polyamine block, not by an electrostatic repulsion mechanism, as is proposed for GluA2-containing AMPAR heteromers, but rather through a different mechanism that introduces structural flexibility in the pore helices. The fact that the near loss of polyamine block in GluK2/K5 heteromers is not accompanied by changes in divalent permeability (
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ) again suggests that the primary disruption in the ion-permeation pathway is at the level of the Q/R site and M2 helix rather than in the extended M3 helix where the conserved Asn residue contributes to Ca2+ permeability (
      • Jatzke C.
      • Hernandez M.
      • Wollmuth L.P.
      Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.
      ). In keeping with this, TARPs acting on GluA4 AMPARs (
      • Soto D.
      • Coombs I.D.
      • Kelly L.
      • Farrant M.
      • Cull-Candy S.G.
      Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors.
      ) and NETOs acting on GluK2 KARs (
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ) attenuate polyamine block without having any effect on divalent permeability.
      Figure thumbnail gr7
      Figure 7Asymmetric pore of heteromeric KAR channels. A, sequence alignment of the five KAR subunits highlighting the Gly residue of GluK1–3 subunits and the Pro of GluK4 and GluK5 found in the M2 helix. High nanomolar affinity block of GluK3 is conferred by the Met and Ser residues that are Val or Ala/Gly residues, respectively, in the other KAR subunits. B, left, NaK open channel structure (
      • Alam A.
      • Jiang Y.
      High-resolution structure of the open NaK channel.
      ) was used as a template for GluK2/K5 heteromers introducing the Pro residues on subunits A and C. B, right, side view of the A/C (cyan) and B/D (orange) subunits of the inverted NaK pore before (gray) and after (colored) 257-ns simulations reveal that the Pro residues introduce asymmetry into the pore. Adapted from Ref.
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      .
      The situation, however, is likely to be more complicated for several reasons. First, an Arg scan of the M3 helix atop of the Q/R site in GluK2 KARs identified residues that attenuate polyamine block (
      • Wilding T.J.
      • Chen K.
      • Huettner J.E.
      Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.
      ) suggesting that alterations in this electroneutral cavity by auxiliary proteins may also affect the degree of channel block. A potential caveat, however, is that Arg replacement in the extracellular vestibule may create a local electrostatic environment that alters permeant ion flux in the pore, which will indirectly attenuate polyamine block (see Fig. 4D of Ref.
      • Bowie D.
      • Lange G.D.
      • Mayer M.L.
      Activity-dependent modulation of glutamate receptors by polyamines.
      ), a possibility that may be in keeping with the enhanced anion permeability reported for the Arg mutants (
      • Wilding T.J.
      • Chen K.
      • Huettner J.E.
      Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.
      ). Second, both CNIHs and TARPs attenuate polyamine block of GluA1 AMPARs while increasing divalent permeability (
      • Coombs I.D.
      • Soto D.
      • Zonouzi M.
      • Renzi M.
      • Shelley C.
      • Farrant M.
      • Cull-Candy S.G.
      Cornichons modify channel properties of recombinant and glial AMPA receptors.
      ) suggesting that auxiliary proteins may have a broader structural impact on both the inner electronegative cavity of the pore and the outer extracellular vestibule formed by the M3-S2 linker. The fact that the γ2 TARP enhances Ca2+ permeability of GluA1 (
      • Coombs I.D.
      • Soto D.
      • Zonouzi M.
      • Renzi M.
      • Shelley C.
      • Farrant M.
      • Cull-Candy S.G.
      Cornichons modify channel properties of recombinant and glial AMPA receptors.
      ) but not GluA4 (
      • Soto D.
      • Coombs I.D.
      • Kelly L.
      • Farrant M.
      • Cull-Candy S.G.
      Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors.
      ) homomers is not understood, but it may suggest that the ability of TARPs to enhance divalent transport through the pore is AMPAR subunit-dependent. GSG1L also affects both ion permeation and channel block (
      • Shanks N.F.
      • Savas J.N.
      • Maruo T.
      • Cais O.
      • Hirao A.
      • Oe S.
      • Ghosh A.
      • Noda Y.
      • Greger I.H.
      • Yates 3rd., J.R.
      • Nakagawa T.
      Differences in AMPA and kainate receptor interactomes facilitate identification of AMPA receptor auxiliary subunit GSG1L.
      ,
      • McGee T.P.
      • Bats C.
      • Farrant M.
      • Cull-Candy S.G.
      Auxiliary subunit GSG1L acts to suppress calcium-permeable AMPA receptor function.
      ). In this case, however, GSG1L acts to diminish AMPAR divalent permeability and to enhance polyamine block (albeit at membrane potentials >+50 mV) (
      • McGee T.P.
      • Bats C.
      • Farrant M.
      • Cull-Candy S.G.
      Auxiliary subunit GSG1L acts to suppress calcium-permeable AMPA receptor function.
      ), which, although contrary to the effect of TARPs and CNIHs, is in keeping with auxiliary subunits having a broader impact on the structural integrity of the ion-permeation pathway. Third, and finally, two separate studies have identified that the cytoplasmic tails of NETOs and TARPs can attenuate polyamine block of KARs (
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ) and AMPARs (
      • Soto D.
      • Coombs I.D.
      • Gratacòs-Batlle E.
      • Farrant M.
      • Cull-Candy S.G.
      Molecular mechanisms contributing to TARP regulation of channel conductance and polyamine block of calcium-permeable AMPA receptors.
      ), respectively. Fisher and Mott (
      • Fisher J.L.
      • Mott D.D.
      The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.
      ) observed that neutralization of three positively charged residues, namely Arg–Arg–Lys, which lie close to the transmembrane region of NETOs, eliminated the attenuation of polyamine block of KARs. Given this, the authors speculated that the C-tail of NETOs may act by a charge-screening mechanism to prevent polyamines entering the pore. A charge-screening mechanism, however, is unlikely because auxiliary subunits do not affect the rate of polyamine binding and therefore do not hinder their ability to access the pore (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). Neutralization of the homologous residues in γ2 TARP, namely Arg–His–Lys, had no effect on polyamine block of AMPARs (
      • Soto D.
      • Coombs I.D.
      • Gratacòs-Batlle E.
      • Farrant M.
      • Cull-Candy S.G.
      Molecular mechanisms contributing to TARP regulation of channel conductance and polyamine block of calcium-permeable AMPA receptors.
      ) suggesting that auxiliary proteins probably impact polyamine block differently between AMPARs and KARs and that other mechanisms are likely to be at play.

      Linking neurodevelopmental disorders and cancer to polyamines and their transport

      One of the most unexpected effects of auxiliary proteins is their ability to enhance the transport of polyamines across the plasma membrane (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). This is particularly evident at AMPARs where the permeability of Spm relative to Na+ at GluA2(Q) AMPARs (PSpm/PNa = 0.3) increased about 4- and 10-fold when GluA2 was co-assembled with γ2 TARP (PSpm/PNa = 1) and CNIH-3 (PSpm/PNa = 4), respectively (
      • Brown P.M.G.E.
      • McGuire H.
      • Bowie D.
      Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.
      ). Given that other endogenous polyamines, such as Spd and Put, have fewer amine groups, they would be expected to be transported at a greater rate than Spm, as has already been shown at KARs (
      • Bähring R.
      • Bowie D.
      • Benveniste M.
      • Mayer M.L.
      Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines.
      ,
      • Brown P.M.
      • Aurousseau M.R.
      • Musgaard M.
      • Biggin P.C.
      • Bowie D.
      Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.
      ). Although it is not immediately clear how enhanced polyamine transport might be important to the mammalian CNS, it is worth noting that the developing brain is thought to express a greater abundance of GluA2-lacking AMPARs (
      • Pellegrini-Giampietro D.E.
      • Bennett M.V.
      • Zukin R.S.
      Are Ca2+-permeable kainate/AMPA receptors more abundant in immature brain?.
      ,
      • Talos D.M.
      • Fishman R.E.
      • Park H.
      • Folkerth R.D.
      • Follett P.L.
      • Volpe J.J.
      • Jensen F.E.
      Developmental regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex.
      ), where polyamine transport may play a role. Outside the brain, polyamines are recognized for fulfilling many roles in cells, such as regulating protein and nucleic acid synthesis and structure, cell proliferation, differentiation, and apoptosis (
      • Miller-Fleming L.
      • Olin-Sandoval V.
      • Campbell K.
      • Ralser M.
      Remaining mysteries of molecular biology: the role of polyamines in the cell.
      ,
      • Pegg A.E.
      Functions of polyamines in mammals.
      ). Consequently, it is likely that polyamines contribute to similar roles in the developing CNS. Interestingly, disrupted polyamine levels, due to the inactivation of the Spm synthase gene, give rise to the X-linked neurodevelopmental disorder called Snyder-Robinson syndrome that is associated with mental retardation (
      • Cason A.L.
      • Ikeguchi Y.
      • Skinner C.
      • Wood T.C.
      • Holden K.R.
      • Lubs H.A.
      • Martinez F.
      • Simensen R.J.
      • Stevenson R.E.
      • Pegg A.E.
      • Schwartz C.E.
      X-linked spermine synthase gene (SMS) defect: the first polyamine deficiency syndrome.
      ). Although the potential role of polyamine-sensitive K+ channels and AMPARs has been speculated upon (
      • Cason A.L.
      • Ikeguchi Y.
      • Skinner C.
      • Wood T.C.
      • Holden K.R.
      • Lubs H.A.
      • Martinez F.
      • Simensen R.J.
      • Stevenson R.E.
      • Pegg A.E.
      • Schwartz C.E.
      X-linked spermine synthase gene (SMS) defect: the first polyamine deficiency syndrome.
      ), it is still not clear how deficits in cellular polyamines account for the CNS defects.
      Polyamines and their transport by AMPARs may be more compellingly linked to their proposed roles in cancer (
      • Casero Jr., R.A.
      • Murray Stewart T.
      • Pegg A.E.
      Polyamine metabolism and cancer: treatments, challenges and opportunities.
      ). Cell growth and cancer have long been associated with elevated levels of polyamines (
      • Gerner E.W.
      • Meyskens Jr., F.L.
      Polyamines and cancer: old molecules, new understanding.
      ) and with more recent studies establishing a connection to AMPAR activity. For example, pancreatic (
      • Herner A.
      • Sauliunaite D.
      • Michalski C.W.
      • Erkan M.
      • De Oliveira T.
      • Abiatari I.
      • Kong B.
      • Esposito I.
      • Friess H.
      • Kleeff J.
      Glutamate increases pancreatic cancer cell invasion and migration via AMPA receptor activation and Kras-MAPK signaling.
      ,
      • Ripka S.
      • Riedel J.
      • Neesse A.
      • Griesmann H.
      • Buchholz M.
      • Ellenrieder V.
      • Moeller F.
      • Barth P.
      • Gress T.M.
      • Michl P.
      Glutamate receptor GRIA3–target of CUX1 and mediator of tumor progression in pancreatic cancer.
      ) and kidney (
      • Hu H.
      • Takano N.
      • Xiang L.
      • Gilkes D.M.
      • Luo W.
      • Semenza G.L.
      Hypoxia-inducible factors enhance glutamate signaling in cancer cells.
      ,
      • von Roemeling C.A.
      • Radisky D.C.
      • Marlow L.A.
      • Cooper S.J.
      • Grebe S.K.
      • Anastasiadis P.Z.
      • Tun H.W.
      • Copland J.A.
      Neuronal pentraxin 2 supports clear cell renal cell carcinoma by activating the AMPA-selective glutamate receptor-4.
      ) cancers have each been associated with the activation of AMPARs with tumor growth in breast and lung carcinoma, colon adenocarcinoma, and neuroblastoma cells shown to be diminished by AMPAR antagonists (
      • Rzeski W.
      • Ikonomidou C.
      • Turski L.
      Glutamate antagonists limit tumor growth.
      ,
      • Stepulak A.
      • Sifringer M.
      • Rzeski W.
      • Brocke K.
      • Gratopp A.
      • Pohl E.E.
      • Turski L.
      • Ikonomidou C.
      AMPA antagonists inhibit the extracellular signal regulated kinase pathway and suppress lung cancer growth.
      ). The elevated levels of extracellular polyamine levels reported in many cancers coupled with the prolonged activation of AMPARs (
      • Prickett T.D.
      • Samuels Y.
      Molecular pathways: dysregulated glutamatergic signaling pathways in cancer.
      ) would provide the appropriate environment to permit the slow but steady transport of polyamines into cells. Although the mechanisms regulating polyamine biosynthesis and catabolism are well-understood (
      • Casero R.A.
      • Pegg A.E.
      Polyamine catabolism and disease.
      ,
      • Pegg A.E.
      Mammalian polyamine metabolism and function.
      ), the molecular events that lead to polyamine transport in mammalian cells are less clear (
      • Poulin R.
      • Casero R.A.
      • Soulet D.
      Recent advances in the molecular biology of metazoan polyamine transport.
      ). Consequently, whether the AMPAR in complex with auxiliary proteins, such as CNIHs, could contribute to this function would be an important question to consider in future studies.

      Conclusion

      Much has been learned about polyamine channel block of iGluRs since it was first identified over 2 decades ago. The discovery of auxiliary proteins and their regulation of iGluRs as well as breakthroughs in structural biology open new opportunities for the development of clinically-relevant compounds that might exploit these novel protein–protein interactions. Recent advances in gene-editing techniques, such as CRISPR, also make it possible to pinpoint how distinct families of auxiliary proteins shape iGluR signaling within neuronal circuits and how they may give rise to the aberrant activity that underlines neurological disease. Consequently, there is still much to be uncovered about how polyamine channel block contributes to membrane excitability and its potential role in disease.

      Acknowledgments

      I thank the present and former members of the Bowie lab for their ongoing discussions and careful reading of this manuscript. I also thank Yuhao Yan for providing the pore structure shown in Fig. 2. I am grateful to the two reviewers for their thoughtful critique that has helped improve the overall clarity and readability of the text.

      References

        • Anderson D.
        Still going strong: Leeuwenhoek at eighty.
        Antonie Van Leeuwenhoek. 2014; 106 (24682589): 3-26
        • Lewenhoeck A.
        Observationes D. Anthonii Lewenhoeck, de natis è semine genitali animalculis.
        Philo. Trans. R. Soc. Lond. 1678; 12: 1040-1042
        • Ladenburg A.
        • Abel J.
        Ueber das aethylenimin (Spermin?).
        Ber. Ditsch. Chem. Ges. 1888; 21: 758-766
        • Dudley H.W.
        • Rosenheim M.C.
        • Rosenheim O.
        The chemical constitution of spermine. I. The isolation of spermine from animal tissues, and the preparation of its salts.
        Biochem. J. 1924; 18 (16743399): 1263-1272