Slow deactivation kinetics of NMDA receptors containing NR1 and NR2D subunits in rat cerebellar Purkinje cells.
2000
We have examined the deactivation kinetics of native N-methyl-D-aspartate receptors (NMDARs) containing NR1 and NR2D subunits by patch-clamp recording from Purkinje cells in cerebellar slices from young rats.
NMDAR-mediated whole-cell currents were elicited in response to bath application of 20 μm NMDA and 50 μm glycine. The NMDAR-mediated currents were small, with an average whole-cell conductance of approximately 750 pS.
Following the rapid application of brief pulses (1–10 ms) of 1 mM glutamate to outside-out membrane patches, we observed a low-conductance type of single-channel activity which lasted up to 30 s after the removal of agonist.
Analysis of individual channel openings revealed asymmetry of transitions between the main- and subconductance states – a characteristic of NR1/NR2D-containing NMDARs. The averaged macroscopic current exhibited a decay time course which was well described by a single exponential function with a time constant of ∼3 s.
We conclude that native NR1/NR2D-containing NMDARs, like their recombinant counterparts, display very slow deactivation kinetics. This feature should provide a means for identification of these receptors at synapses, and indicates that they do not contribute to the synaptic NMDAR currents so far described.
Fast excitatory synaptic transmission in the mammalian central nervous system (CNS) usually involves the activation of postsynaptic NMDARs by the neurotransmitter L-glutamate. NMDARs are heteromultimeric assemblies formed from NR1 subunits in combination with at least one type of NR2 subunit. The identity of the NR2 subunit is important in determining both pharmacological and biophysical properties of the receptor (for reviews see Feldmeyer & Cull-Candy, 1996; Dingledine et al. 1999).
Studies of both recombinant and native systems suggest that NMDARs containing NR1/NR2D subunits give rise to receptors with unique properties (Monyer et al. 1994; Momiyama et al. 1996; Wyllie et al. 1996, 1998; Vicini et al. 1998). These include low single-channel conductance, low sensitivity to block by Mg2+ and a low EC50 for glutamate (Momiyama et al. 1996; Wyllie et al. 1996; Kuner & Schoepfer, 1996). Additionally, recombinant NR1/NR2D-containing NMDARs possess unusually slow deactivation kinetics (Monyer et al. 1994; Vicini et al. 1998; Wyllie et al. 1998). If present at the synapse, receptors with these properties would give rise to long-lasting NMDAR activation, and hence prolonged Ca2+ entry during synaptic transmission. However, to date, even those neurones that have been shown to express functional NR1/NR2D-containing NMDARs in their extrasynaptic membrane do not display slow deactivation kinetics in their synaptic responses (Momiyama et al. 1996; Bardoni et al. 1998; Clark & Cull-Candy, 1999; Misra et al. 2000; Momiyama, 2000). There are two possible explanations for this observation. First, native NR1/NR2D-containing NMDARs do not exhibit slow deactivation, and this behaviour is peculiar to recombinant receptors. Alternatively, native and recombinant NR1/NR2D-containing NMDARs exhibit similar properties, but this NMDAR subtype is absent from those central synapses that have been examined so far.
To distinguish between these possibilities we have made recordings from rat cerebellar Purkinje cells. These are known to contain mRNA for the NR1 and NR2D subunits during the early postnatal period (postnatal days (P) 0–8; Akazawa et al. 1994), and express a homogeneous population of low-conductance NMDARs (Momiyama et al. 1996). These appear to be restricted to the extrasynaptic membrane as NMDAR-mediated synaptic currents have not been detected in Purkinje cells (Perkel et al. 1990; Llano et al. 1991). We have characterized, for the first time, the deactivation kinetics of this pure population of native NR1/NR2D-containing NMDARs. These receptors, in outside-out patches from P6 Purkinje cells, do indeed exhibit unusually slow deactivation kinetics following a brief application of glutamate.
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