The four subunits of the AMPA-type glutamate receptor (GluR1-GluR4 or GluR-A-GluR-D) exist in two distinct forms, flip and flop, generated by alternative splicing of a 115 bp region. The GluR2 subunit plays a key role in determining the functional properties of the AMPA receptor channel. In this study, we examined the differences in kinetic properties between the flip and flop splice variants of the GluR2 subunit expressed in Xenopus oocytes using fast agonist application techniques. Glutamate was applied to outside-out patches from oocytes with piezo-driven double-barreled application pipettes. Because homomeric receptor channels composed of the edited form of GluR2 (GluR2R) produce no appreciable current responses, we expressed the unedited form of GluR2 (GluR2Q) in oocytes, which produced large current responses sufficient for analysis of the kinetic properties. The time constant for desensitization during application of 1 mM glutamate was 5.89 +/- 0. 17 msec (n = 50) in flip and 1.18 +/- 0.05 msec (n = 37) in flop. The deactivation time constant was 0.62 +/- 0.06 msec (n = 10) in flip and 0.54 +/- 0.05 msec (n = 10) in flop. The steady-state nondesensitizing current was 6.8 +/- 0.4% (n = 53) of the peak current in flip, whereas it was almost negligible in flop, being only 1.1 +/- 0.1% (n = 36). The slower desensitization kinetics and larger steady-state current responses in the flip variant were also observed in heteromeric receptors assembled from GluR2Q/GluR2R. Thus, desensitization occurred much more prominently in the flop variant in the recombinant GluR2 receptor channels.

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1. The effect of synthetic joro spider toxin (JSTX-3) on a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels in cultured rat hippocampal neurones was investigated using the whole-cell patch-clamp technique. 2. A population of cultured neurones had AMPA receptors with strong inward rectification and substantial Ca2+ permeability (type II neurones), whereas most neurones (type I neurones)had slight outward rectification and little Ca2P permeability. JSTX-3 selectively suppressed the inwardly rectifying and Ca2+-permeable AMPA receptors expressed in type II neurones without affecting AMPA receptors in type I neurones.3. The effect of JSTX-3 on the Ca2P-permeable AMPA receptors was use and voltage dependent. In the steady state, current responses induced by ionophoretic applications of kainate (a non-desensitizing agonist of AMPA receptors) were suppressed by the toxin in a dose-dependent manner at negative potentials (IC50 = 56 nm at -60 mV). 4. At the standard membrane potential (-60 mV), recovery from the blockage by JSTX-3 was very slow. Even after washout for more than 7 min, the recovery was only partial. However, the blockage was completely removed immediately after application of a +60 mV voltage pulse for 5 s in conjunction with a single ionophoretic application of kainate.

Substance P (SP) excites large neurons of the nucleus basalis (NB) by inhibiting an inward rectifier K ؉ channel (Kir). The properties of the Kir in NB (KirNB) in comparison with the G protein-coupled Kir (GIRK) were investigated. Single-channel recordings with the cellattached mode showed constitutively active KirNB channels, which were inhibited by SP. When the recording method was changed from the on-cell to the inside-out mode, the channel activity of KirNB remained intact with its constitutive activity unaltered. Application of G␤ 1␥2 to inside-out patches induced activity of a second type of Kir (GIRK). Application of G␤ 1␥2, however, did not change the KirNB activity. Sequestering G␤ 1␥2 with G␣i2 abolished the GIRK activity, whereas the KirNB activity was not affected. The mean open time of KirNB channels (1.1 ms) was almost the same as that of GIRKs. The unitary conductance of KirNB was 23 pS (155 mM [K ؉ ]o), whereas that of the GIRK was larger (32-39 pS). The results indicate that KirNB is different from GIRKs and from any of the classical Kirs (IRKs). Whole-cell current recordings revealed that application of muscarine to NB neurons induced a GIRK current, and this GIRK current was also inhibited by SP. Thus, SP inhibits both KirNB and GIRKs. We conclude that the excitatory transmitter SP has two types of Kirs as its effectors: the constitutively active, G␤␥-independent KirNB channel and the G␤␥-dependent GIRK.T he nucleus basalis (NB) is located in the basal forebrain and contains a population of large cholinergic neurons that have axon projections to a wide area of brain regions including the cerebral cortex and the hippocampus (1). These cholinergic neurons play a crucial role in executing vital brain functions such as cognition and arousal (2), and degeneration of these neurons could be a major cause for the memory loss in Alzheimer's disease (3). Our group has been using these large neurons from the NB in culture together with neurons from the locus coeruleus (LC) as model neurons in the brainstem and the basal forebrain. One of the main objectives has been to elucidate the mechanism by which ''slow transmitters'' produce neuronal excitation through the modulation of the inward rectifier K ϩ channels (Kirs).Substance P (SP) is a peptide transmitter (4) that induces slow excitation in peripheral and central neurons (5-8). Our initial studies revealed that the application of SP causes depolarization by inhibiting Kir current in NB neurons (will be referred to as the KirNB channel; refs. 9 and 10). Subsequent studies have shown that a considerable portion of this SP-induced inhibition of the KirNB is mediated through a transduction cascade consisting of G␣q͞11, phospholipase C␤1, and PKC (11,12).In parallel with these studies on KirNB channels, we have been investigating the characteristics of G protein-coupled Kirs (GIRKs) in noradrenergic neurons of the LC (13, 14). A large part of the GIRK mRNAs in LC neurons consists of GIRK1 and -2, § and the channel properties of the GIRKs in LC are very simi...