Because the structures of the open and deep desensitized states are likely to differ appreciably, the connection between open and desensitized states may consist of multiple transitions. Such a correlation could also result without desensitization from the open state, but other features of our data are not described in this EX527 case. Simple changes in affinity do not predict the existence of mutants (or wild-type receptors) where apparent affinities do not differ much but which have dramatically different recovery. In NMDA and GABA receptors, agonist unbinding is slow. Thus long shut sojourns (which may involve desensitized states) contribute

considerably to the synaptic decay for both receptor classes. Reopening of NMDA and GABA receptors following a long shut state

occurs because the channel opening rate is similar to the unbinding rate ( Jones and Westbrook, 1995 and Popescu et al., 2004). If AMPA channels are functioning in a similar way, only accelerated about 100-fold, faster recovery of receptors from the desensitized state and speeding of channel closure might be a way of sharpening the synaptic current and limiting noise by minimizing reopening, as well as ensuring maximum availability of receptors over a wide input bandwidth. To construct S1S2 chimeras, 3-deazaneplanocin A clinical trial we amplified inserts containing the GluA2 or GluK2 ligand binding domains with splice sites to the parent backbone via overlap PCR. Domain boundaries, which were sequence neutral, were as follows: B2P6 – K2 (T1-N399) A2 (N382-P507) K2 (P513-S635) A2 (S631-K781) K2 (K779-A877); B6P2: A2 (V1-N382) K2 (N399- P513) A2

(P507-S631) K2 (S635-K779) A2 (K781-I862). Point mutations were introduced by overlap PCR and confirmed by double-stranded sequencing. Numbering nearly refers to the mature polypeptide chain. Wild-type and mutant glutamate receptors were overexpressed in HEK293 cells as described (Chen et al., 1999). For most experiments, the external solution contained (in mM): 150 NaCl, 0.1 MgCl2, 0.1 CaCl2, and 5 HEPES, titrated to pH 7.3 with NaOH, to which we added drugs as required. In experiments to assess the ion sensitivity of chimeras, we replaced NaCl with NaNO3 or CsCl. Drugs were obtained from Ascent Scientific (Weston-Super-Mare, UK). The pipette solution contained (in mM): 115 NaCl, 10 NaF, 0.5 CaCl2, 1 MgCl2, 5 Na4BAPTA, 5 HEPES and 10 Na2ATP (pH 7.3). We applied ligands to outside out patches via a piezo driven fast perfusion system. Typical 10%–90% solution exchange times were faster than 300 μs, as measured from junction potentials at the open tip of the patch pipette. For single-channel recording, outside-out patches were clamped at –80mV during long applications (8 s) of 10 mM glutamate. Records were filtered at 1–2 kHz and idealized using time course fitting (SCAN, available from onemol.org.uk). To measure recovery from desensitization, we used a two-pulse protocol with a variable interpulse interval.