3and and Fig. fast activation and desensitization kinetics (mean = 9 patches; Fig. 2 and 9 patches; bars indicate mean values. Statistical testing was performed with paired tests: *** 0.001. See Rabbit Polyclonal to SEPT6 also = 9 patches; Fig. 2 and = 9 patches; Fig. 2= 9 patches; Fig. 2= 6 patches). Fitting the kinetic scheme in with = 6 patches; Fig. 3= 6 patches; Fig. 3= 3 patches; Fig. 3and and Fig. S9). Since iGluR tetramers assemble with pseudo-twofold symmetry (36; but see ref. 60 for a recent exception) we assumed that UBP-310 dissociation starts from a symmetric configuration. YC-1 (Lificiguat) Different configurations can be distinguished during antagonist dissociation, however. Most obviously, two antagonists (agonists) can be bound at different LBD dimers (path I) or at the same LBD dimer (path II). The situation can be summarized in a branched kinetic scheme (Fig. 3and Fig. S8and and Figs. S4and S10and = 9 patches; Fig. 4and = 6 patches; Fig. 4and indicates the number of different patches. Statistical testing was performed with Welch’s tests: * 0.05, *** 0.001. More details on the experiment and the controls are provided in = 10 patches; Fig. 4= 8 patches; Fig. 1). Taken together, these experiments confirm that partial agonist occupancies can result in incomplete desensitization of various kainate receptor types. Partial Agonist Occupancy Also Reduces Desensitization of Heteromeric AMPA Receptors. We next tested whether subunit-specific manipulations would also affect AMPA receptor desensitization. For this, we artificially modified the GluA2 ligand selectivity by substituting an amino acid side chain in the binding pocket (leucine 671 to threonine; L671T). The L671T substitution is known to increase kainate binding and efficacy at GluA2 receptors (62), and it has been suggested that leucine 671 obstructs binding of the kainate receptor agonist (2= 11 patches), significantly higher than those seen on application of glutamate (mean = 6; = 10 patches; Fig. 4= 8 patches) compared with GluA2(wt) homotetramers (mean = 26 patches; Fig. 4and and and and and and em C /em ). These observations can be readily explained if we assume that antagonist dissociation is YC-1 (Lificiguat) not equally fast at all four receptor subunits. These differences could be due to binding cooperativity and/or positional differences within the tetramers ( em SI Appendix /em , em Note 2 /em ). Our data also show heterogeneity in the receptor configurations with two bound agonists: some configurations produce nondesensitizing currents, while other configurations with two bound agonists produce either no or fully desensitizing responses. This situation can be illustrated by a branched kinetic scheme (Fig. 3 em A /em ). More generally, our experiments show that ligand occupancy is an important determinant of AMPA and kainate receptor desensitization, in addition to ligand efficacy (67, 68) and structural rearrangements of the LBD dimer interface (42C44, 69). The prevailing view that one agonist-occupied subunit is sufficient to induce efficient desensitization of YC-1 (Lificiguat) the tetrameric receptors (26, 47) had already been challenged by work on kainate receptors incorporating the high-affinity subunits GluK4 and GluK5 (48C50) and here is also disproven for other types of kainate receptors, as well as GluA2 AMPA receptor pseudoheteromers. Further experimental work is needed to address the role of binding cooperativity and interdimer coupling (34), the behavior of different types of AMPA receptor heteromers, and the contribution of auxiliary subunits. Besides the mechanistic implications of our findings, we show that partial receptor occupancies can have significant practical consequences, for instance, when subunit-selective antagonists are used to treat heteromeric receptor populations. The desensitization block that can arise from using subunit-selective antagonists may be small and variable, but the physiological effects could be large. Heteromeric AMPA receptors play an important part in the nervous system (4, 21C23), and YC-1 (Lificiguat) recent work suggests that their assembly occurs inside a biased, nonrandom fashion (22, 25, 70). In this case, the formation of nondesensitizing configurations may be favored, much like GluK5-comprising kainate receptor heteromers in which preferential assembly (58, 61, 66) yields nondesensitizing configurations while high-affinity glutamate binding is definitely maintained (Fig. 2). The impact on synaptic function may be serious if iGluRs maintain nondesensitizing currents in the presence of falling or low basal glutamate concentrations. The use of subunit-specific antagonists may prolong synaptic activations or cause undesirable tonic activations. Moreover,.