A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures.

Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.

Purinoceptors evoke different electrophysiological responses in pancreatic ducts. P2Y inhibits K(+) conductance, and P2X stimulates cation conductance.

In epithelia, extracellular nucleotides are often associated with regulation of ion transporters, especially Cl(-) channels. In this study, we investigated which purinoceptors are present in native pancreatic ducts and how they regulate ion transport. We applied whole-cell patch-clamp recordings, intracellular Ca(2+) and pH measurements, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. The data show two types of purinoceptors and cellular responses. UTP and ATP produced large Ca(2+) transients, a decrease in intracellular pH, 8-10-mV depolarization of the membrane voltage, and a decrease in the whole-cell conductance. The membrane effects were due to closure of K(+) channels, as confirmed by dependence on extracellular K(+). UTP/ATP effects could be associated with P2Y(2) purinoceptors, and RT-PCR revealed mRNAs for P2Y(2) and P2Y(4) receptors. On the other hand, 2', 3'-O-4-benzoylbenzoyl-ATP induced Ca(2+) influx and approximately 20-mV depolarization of the membrane voltage with a concomitant increase in the whole-cell conductance. These effects were dependent on extracellular Na(+), not Cl(-), indicating opening of cation channels associated with P2X(7) purinoceptors. RT-PCR showed mRNAs for P2X(7) and P2X(4) receptors. In microperfused ducts, luminal (but not basolateral) ATP caused large depolarizations of membrane voltages recorded with microelectrodes, consistent with luminal localization of P2X(7) receptors. Thus, P2Y(2) (and possibly P2Y(4)) purinoceptors inhibit K(+) channels and may not support secretion in native ducts. P2X(7) (and possibly P2X(4)) receptors are associated with cation channels and may contribute to regulation of secretion.