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J Physiol. May 1, 2009; 587(Pt 9): 1861–1862.
PMCID: PMC2689325

Ca2+-permeable AMPA receptors in central neurons

Ca2+ entry through ionotropic glutamate receptors plays a pivotal role in the induction of long-term changes in the efficacy of synaptic transmission, in excitotoxicity and in other processes such as the development of the central nervous system (CNS). It has been demonstrated that NMDA-type glutamate receptors are highly permeable to Ca2+, whereas the Ca2+ permeability of non-NMDA receptors (AMPA/kainite-type receptors) is about 70-fold lower than that of NMDA receptors (Mayer & Westbrook, 1987).

However, in the course of the experiments conducted in 1988–1989 in our laboratory, we reached the conclusion that the view that the non-NMDA receptor channel is almost impermeable to Ca2+ should be partly modified (Iino et al. 1990). We estimated the Ca2+ permeability of receptor channels activated by NMDA, quisqualate and kainate in cultured rat hippocampal neurons using the whole-cell voltage-clamp technique, and found that there are two distinct types of current responses to kainate. In the type 1 response, which was seen in most neurons tested (type 1 neurons), the receptor channels activated by kainate displayed a slight outward rectification and little permeability to Ca2+ (PCa/Palkali-metal < 0.18). In contrast, in the type 2 response, which was only seen in a small population of neurons (type 2 neurons), they displayed a substantial permeability to Ca2+ and a strong inward rectification (Iino et al. 1990). Later, this inward rectification turned out to be attributed to voltage-dependent blocking by intracellular polyamines (Isa et al. 1995, also see Cull-Candy et al. 2006 for review). To define the type 2 response more quantitatively, we introduced the following value as a rectification index (RI):

equation image

where I+40 and I–60 are the peak amplitudes of the kainate-induced currents at +40 mV and −60 mV, respectively, and Erev is the reversal potential. In neurons with this value <0.25 (type 2 neurons), the kainate-activated receptors displayed a significant Ca2+ permeability (PCa/Palkali-metal= 2.3–2.7), although they were less permeable than NMDA receptors (PCa/Palkali-metal= 6.2). The relative permeability of these kainate-activated receptors for divalent cations was Ba2+ (1.27) > Ca2+ (1.00) > Sr2+ (0.90) > Mg2+ (0.79) > Mn2+ (0.71), indicating that these receptors have a much weaker selectivity among the divalent cations than NMDA receptors (Iino et al. 1990; Ozawa et al. 1991). The mean single channel conductance of the type 2 kainate response was estimated to be 8.7 pS, being much smaller than that of the NMDA response (~50 pS) but fourfold larger than that of the type 1 response (2.2 pS) (Ozawa et al. 1991).

Molecular cloning has identified four cDNAs in the rodent brain that encode AMPA receptor subunits, GluR1 (GluR-A)–GluR4 (GluR-D) (see Seeburg, 1993; Hollmann & Heinemann, 1994 for reviews). Expression studies have demonstrated that the presence of the GluR2 subunit determines both the rectification properties and the Ca2+ permeability of the AMPA receptor channels. The AMPA receptors that contain GluR2 display a linear or outwardly rectifying current–voltage (IV) relationship and little Ca2+ permeability. In contrast, the receptors that lack this subunit exhibit an inwardly rectifying IV relationship and substantial Ca2+ permeability (Hollmann et al. 1991; Verdoorn et al. 1991). The unique properties of GluR2 can be traced to a single amino acid residue within its re-entrant M2 membrane loop region. This residue is a positively charged arginine (R) in GluR2, whereas it is a glutamine (Q) with a neutral charge in the other AMPA receptor subunits. When the arginine at this site (referred to as the Q/R site) is replaced with glutamine using site-directed mutagenesis, the mutant GluR2(Q) shows similar Ca2+ permeability and rectification properties to those of the wild-type GluR1, GluR3 and GluR4 subunits (Hume et al. 1991; Mishina et al. 1991; Burnashev et al. 1992). Interestingly, the arginine at the Q/R site is not encoded in the GluR2 genomic DNA, but is introduced by RNA editing to replace the gene-encoded glutamine. In the adult CNS, almost all GluR2 subunits are expressed in the edited form (Sommer et al. 1991).

The above results, which were obtained by molecular biological studies, strongly suggest that both the type 1 and type 2 responses induced by kainate in the cultured hippocampal neurons are due to the activation of AMPA receptors with and without the GluR2 subunit, respectively. We confirmed that these two types of responses are mimicked by AMPA, which cross-desensitizes both types of kainate responses (Ozawa & Iino, 1993). We next attempted to determine the subunit composition of naturally occurring AMPA receptors in type 1 and 2 neurons at the single-cell level using whole-cell patch-clamp recordings coupled with reverse transcription followed by PCR amplification (patch-clamp RT-PCR). The mean numbers of mRNA molecules harvested from a single type 1 pyramidal-like neuron of the rat hippocampus in culture were 1150 ± 324 molecules of GluR1, 1080 ± 273 molecules of GluR2, 100 ± 20 molecules of GluR3 and 50 ± 10 molecules of GluR4 (mean ±s.e.m., n= 12). In the type 2 non-pyramidal neuron, the numbers of GluR1, GluR3 and GluR4 mRNA molecules harvested per cell were 354 ± 64, 25 ± 17 and 168 ± 36 (mean ±s.e.m., n= 8). GluR2 was not detected in type 2 neurons. These results demonstrate that the substantial Ca2+ permeability and inward rectification displayed by the AMPA receptors in type 2 neurons are due to the absence of GluR2 (Bochet et al. 1994; Tsuzuki et al. 2001).

In cultured rat hippocampal neurons, Ca2+-permeable AMPA receptors were detected exclusively in small cells with elliptical somata. These type 2 neurons expressed glutamic acid decarboxylase (GAD), suggesting that they are derived from inhibitory GABAergic interneurons (Bochet et al. 1994). In rat hippocampal slices, a population of interneurons with inwardly rectifying IV relationship of kainate-activated current was observed in the stratum radiatum of the CA3 region (McBain & Dingledine 1993). We found type 2 responses in neurons with round or elliptical somata in various areas in hippocampal slices of young rats, most frequently in the stratum moleculare of the dentate gyrus and in the stratum radiatum and the stratum lacunosum-moleculare of both the CA1 and CA3 regions. In these neurons, inwardly rectifying AMPA receptors were involved in excitatory synaptic transmission (Isa et al. 1996).

In addition to hippocampal interneurons, Ca2+-permeable AMPA receptors are found to occur in various neurons and glia in the CNS, including neocortical and amygdaloid GABAergic interneurons, cerebellar sellate cells, spinal dorsal horn interneurons, large striatal cholinergic interneurons, relay neurons of the auditory pathway, Bergmann glia, oligodendrocyte precursor cells, etc. (see Liu & Zukin, 2007 for review). Using the patch-clamp RT-PCR method, the relationship between functional and molecular properties of native AMPA receptors was investigated in principal neurons and interneurons of the hippocampus and neocortex, auditory relay neurons and Bergmann glia in acute brain slices of rats (Jonas et al. 1994; Geiger et al. 1995; also see Jonas & Burnashev, 1995 for review). These studies have shown that the range of Ca2+ permeability (PCa/PNa) in native AMPA receptors varies from 0.07 to 2.8, and this value is inversely correlated with the relative abundance of GluR2 mRNA.

Ca2+-permeable AMPA receptors are now known to occur in a wider variety of neurons in a cell- and synapse-specific manner during development and in response to neuronal activity. Compelling evidence indicates that they impart a novel form of short- and long-term synaptic plasticity independent of NMDA receptors in these neurons. Furthermore, there is accumulating evidence that downregulation of GluR2, an impairment of GluR2 Q/R site editing or an increase in GluR1 (and/or GluR3, GluR4) expression is related to various neurological disorders such as post-ischaemic neuronal death, amyotrophic lateral sclerosis (ALS), inflammatory and neuropathic pain, epilepsy and drug addiction (see Cull-Candy et al. 2006; Isaac et al. 2007; Liu & Zukin, 2007 for reviews). Thus, Ca2+-permeable AMPA receptors are spread more widely and play more important roles than originally thought. Further studies are expected to reveal the involvement of these receptors in an even wider range of physiological and pathological events in the CNS.

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