Hippocalcin functions as a calcium sensor in hippocampal LTD.

It is not fully understood how NMDAR-dependent LTD causes Ca(2+)-dependent endocytosis of AMPARs. Here we show that the neuronal Ca(2+) sensor hippocalcin binds the beta2-adaptin subunit of the AP2 adaptor complex and that along with GluR2 these coimmunoprecipitate in a Ca(2+)-sensitive manner. Infusion of a truncated mutant of hippocalcin (HIP(2-72)) that lacks the Ca(2+) binding domains prevents synaptically evoked LTD but has no effect on LTP. These data indicate that the AP2-hippocalcin complex acts as a Ca(2+) sensor that couples NMDAR-dependent activation to regulated endocytosis of AMPARs during LTD.

ATP hydrolysis is required for the rapid regulation of AMPA receptors during basal synaptic transmission and long-term synaptic plasticity.

ATP hydrolysis is critical for many cellular processes; however, the acute requirement for ATP hydrolysis in synaptic transmission and plasticity in neurons is unknown. Here we studied the effects of postsynaptically applying the non-hydrolyzable ATP analogue adenosine 5'-[beta,gamma-methylene]triphosphate (AMP-PCP) into hippocampal CA1 pyramidal cells in hippocampal slices. The effects of this manipulation were investigated on basal transmission and on two forms of long-term synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). AMP-PCP caused an increase in basal AMPA receptor (AMPAR)-mediated transmission, which occurred rapidly within minutes of infusing the drug. This effect was selective for AMPARs, since pharmacologically isolated NMDAR-mediated synaptic currents did not exhibit this run up. In two-pathway experiments infusion of AMP-PCP blocked the induction of both LTD and LTP. These findings show an acute and selective role for ATP hydrolysis in regulating AMPAR function both during basal transmission and long-term synaptic plasticity. Recent evidence indicates that AMPARs are selectively and acutely regulated by the ATPase N-ethylmaleimide-sensitive factor (NSF), which forms part of a multi-protein complex with AMPARs. Our data are consistent with the idea that such a mechanism that can acutely bi-directionally regulate AMPAR function at synapses and requires ATP hydrolysis is necessary for rapid activity-dependent changes in synaptic strength.

Receptor-specific inhibition of GABAB-activated K+ currents by muscarinic and metabotropic glutamate receptors in immature rat hippocampus.

It has been shown that the activation of G(q)-coupled receptors (G(q)PCRs) in cardiac myocytes inhibits the G protein-gated inwardly rectifying K(+) current (I(GIRK)) via receptor-specific depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)). In this study, we investigated the mechanism of the receptor-mediated regulation of I(GIRK) in acutely isolated hippocampal CA1 neurons by the muscarinic receptor agonist, carbachol (CCh), and the group I metabotropic glutamate receptor (mGluR) agonist, 3,5-dihydroxyphenylglycine (DHPG). I(GIRK) was activated by the GABA(B) receptor agonist, baclofen. When baclofen was repetitively applied at intervals of 2-3 min, the amplitude of the second I(GIRK) was 92.3 +/- 1.7% of the first I(GIRK) in control. Pretreatment of neurons with CCh or DHPG prior to the second application of baclofen caused a reduction in the amplitude of the second I(GIRK) to 54.8 +/- 1.3% and 51.4 +/- 0.6%, respectively. In PLCbeta1 knockout mice, the effect of CCh on I(GIRK) was significantly reduced, whereas the effect of DHPG remained unchanged. The CCh-mediated inhibition of I(GIRK) was almost completely abolished by PKC inhibitors and pipette solutions containing BAPTA. The DHPG-mediated inhibition of I(GIRK) was attenuated by the inhibition of phospholipase A(2) (PLA(2)), or the sequestration of arachidonic acid. We confirmed that DHPG eliminated the inhibition of I(GIRK) by arachidonic acid. These results indicate that muscarinic inhibition of I(GIRK) is mediated by the PLC/PKC signalling pathway, while group I mGluR inhibition of I(GIRK) occurs via the PLA(2)-dependent production of arachidonic acid. These results present a novel receptor-specific mechanism for crosstalk between G(q)PCRs and GABA(B) receptors.

Ca2+ enhances U-type inactivation of N-type (CaV2.2) calcium current in rat sympathetic neurons.

Ca2+ -dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (tau approximately 150 ms) and slow (tau approximately 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5-30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.

GluR2 protein-protein interactions and the regulation of AMPA receptors during synaptic plasticity.

AMPA-type glutamate receptors mediate most fast excitatory synaptic transmissions in the mammalian brain. They are critically involved in the expression of long-term potentiation and long-term depression, forms of synaptic plasticity that are thought to underlie learning and memory. A number of synaptic proteins have been identified that interact with the intracellular C-termini of AMPA receptor subunits. Here, we review recent studies and present new experimental data on the roles of these interacting proteins in regulating the AMPA receptor function during basal synaptic transmission and plasticity.

Effect of acute hypoxia on ATP-sensitive potassium currents in substantia gelatinosa neurons of juvenile rats.

Although hypoxia is known to affect membrane excitability of various neurons by various mechanisms, the effects of hypoxia on substantia gelatinosa (SG) neurons have not yet been elucidated. In whole-cell or perforated patch-clamp recordings from SG neurons, we showed that acute hypoxia induces a reversible hyperpolarization of -6.1+/-1.3 mV of the resting membrane potential and an outwards current of 9.48+/-1.71 pA at a holding potential of -60 mV. The reversal potentials of the hypoxia-induced current depended on [K(+)](o). The hypoxia-induced hyperpolarization and outwards current were abolished completely by BaCl(2), but not by CsCl. Glibenclamide, a blocker of K(ATP) channels, blocked the hypoxia-induced hyperpolarization. Pretreatment with cromakalim, an opener of K(ATP) channels, occluded the hypoxia-induced hyperpolarization. Any alteration by hypoxia was not observed in the presence of an internal solution with a high [ATP] (10 mM). The above results suggest that hypoxia-induced hyperpolarization in SG neurons is mediated by activation of K(ATP) channels.

Role of K(+) channels in frequency regulation of spontaneous action potentials in rat pituitary GH(3) cells.

The frequency of spontaneous action potentials (SAP) is important in the regulation of hormone secretion. The decrease in K(+) conductance is known as a primary mechanism for increasing SAP frequency. To investigate the nature of K(+) channels that contribute to the frequency regulation of the SAP in rat clonal pituitary GH(3) cells, the effect of various K(+) channel blockers on the SAP and membrane currents were recorded using the patch-clamp technique. A classical inward rectifying K(+) channel blocker, Cs(+) (5 mM), caused an increase in firing frequency and depolarization in after-hyperpolarization (AHP) voltage. An ETHER-A-GO-GO(ERG) type K(+) channel blocker, E-4031 (5 microM), caused no significant effect on the SAP. Tetraethylammonium (TEA, 10 mM) decreased firing frequency and increased the duration of SAP. These effects were not changed by the presence of high concentration of Ca(2+) buffer (10 mM EGTA or BAPTA) in pipette solutions. In voltage-clamp experiments, Cs(+) and E-4031 did not affect outwardly rectifying K(+) currents, but significantly inhibited inwardly rectifying K(+) currents recorded in isotonic K(+) solution. However, the kinetics of Cs(+)-sensitive current and E-4031-sensitive current were distinctive: the time to peak was more immediate and the decay rate was slower in Cs(+)-sensitive current than in E-4031-sensitive current. These results imply that Cs(+) and E-4031 inhibit the distinct components of inwardly rectifying K(+) currents, and that the contribution of the Cs(+)-sensitive current can be immediate on repolarization and can last more effectively over pacemaking potential range than E-4031-sensitive current.