Of the two quadruple

mutant combinations we made, one was nonfunctional (GluK2 D656A S675R M706L T715E) and the other typically gave patch currents in the 10 pA range. This quadruple mutant (GluK2 E650A S675R M706L T715E; ARLE) recovered about 20-fold faster than wild-type GluK2 (Figures 6C and 6D; 8.6 ± 0.7 s−1, n = 6 patches), with a halftime of recovery (t50) of 80 ms, only about 3-fold longer than wild-type GluA2 (26 ms). Quintuple and sextuple combinations (e.g., GluK2 L511M Y512I E650A S675R M706L T715E) also failed to give expression of membrane currents. Selleck Bcl 2 inhibitor We were not able to measure glutamate apparent affinity for the quadruple ARLE mutant because currents were small and exhibited strong rundown. The triple S675R M706L T715E mutant (GluK2 RLE) had glutamate potency slightly lower than that at wild-type GluA2 (EC50 = 2.8 ± 0.1 mM, n = 5 patches). The potency of glutamate at the M706L single mutant was indistinguishable (EC50 = 3.1 ± 0.3 mM, n = 10 patches), even though the RLE mutant recovers about twice as fast as M706L alone ( Table 1). Notably, a similar 20-fold selleck screening library shift in potency due to point mutations in the jaws of GluK2 ( Weston et al., 2006b) such as K456A, only speeds recovery 4-fold (compared to the more than 10-fold speeding for GluK2 RLE). Mutations that sped K2 recovery also sped up the deactivation

decay (Figure 6E), mirroring the situation in AMPA receptors, and we obtained a similar correlation across the mutant series (Figure 6F, Pearson r = 0.64 for the correlation between krec and kdeact). Only one

mutation (GluK2 L511M Y512I) altered the desensitization rate outside a 2-fold range across the entire series, closely matching the situation in AMPA receptors ( Table S1; Pearson r = –0.01 for the correlation between krec and kdes). A positive correlation between recovery and deactivation rates is expected if glutamate affinity changes for all states. Such a change should also strongly alter glutamate potency for channel activation, a phenomenon that we detected only in GluK2 constructs harboring the M706L mutation. The behavior of other mutants, such as A2 TR, for which deactivation decays and recovery both changed with only limited shifts in EC50, are not predicted by the mechanism in Figure 2A (data not shown). We reasoned that the correlation could be recapitulated by linking the open state to all the deep desensitized state. Schemes with long lived desensitized states connected to open states were previously proposed to describe the activation of native glutamate receptors ( Häusser and Roth, 1997 and Jonas et al., 1993). However, in other studies, desensitization was taken to proceed only from shut states ( Vyklicky et al., 1991 and Robert and Howe, 2003) or from either shut or open states ( Lin and Stevens, 1994 and Raman and Trussell, 1995), and the concept of desensitization from open states has remained controversial ( Colquhoun and Hawkes, 1995a).