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

Abstract

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.

Keywords: channel pore; electrophysiology; ion channel; ionotropic glutamate receptor; kainate receptor; membrane potential; polyamine; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor, AMPAR).

Figure 1.

Ionotropic 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 (189); (ii) four clamshell-like ligand-binding pockets (LBD) (190); (iii) a TMD that forms a central ion channel pore that transports Na⁺ and Ca²⁺ ions in response to neurotransmitter binding (42, 43); and (iv) a cytoplasmic C-terminal domain (not shown) that directs receptor trafficking and synaptic anchoring (191). Image of structure was adapted from Ref. . Right, table of the four classes of iGluR arranged with each of their respective subunits.

Figure 2.

Sequence 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.

Figure 3.

Closed 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. with permission.

Figure 4.

Open 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. with permission.

Figure 5.

AMPAR 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. with permission.

Figure 6.

Auxiliary 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 (k_on), unbinding (k_off), and permeation (k_perm) 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 (k_off and k_perm) 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. .

Figure 7.

Asymmetric 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 (193) 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. .