SINGLE-CHANNEL ACTIVATIONS AND CONCENTRATION JUMPS - COMPARISON OF RECOMBINANT NR1A NR2A AND NR1A/NR2D NMDA RECEPTORS/

1. We have expressed recombinant NR1a/NR2A and NR1a/NR2D N-methyl-D-as partate (NMDA) receptor channels in Xenopus oocytes and made recording s of single-channel and macroscopic currents in outside-out membrane p atches. For each receptor type we measured (a) the individual single-c hannel activations evoked by lour glutamate concentrations in steady-s tate recordings, and (b) the macroscopic responses elicited by brief c oncentration jumps with high agonist concentrations, and we explore th e relationship between these two sorts of observation. 2. Low concentr ation (5-100 nM) steady-state recordings of NR1a/NR2A and NR1a/NR2D si ngle-channel activity generated shut-time distributions that were best fitted with a mixture of five and six exponential components, respect ively. Individual activations of either receptor type were resolved as bursts of openings, which we refer to as 'super-clusters'. 3. During a single activation, NR1a/NR2A receptors were open for 36% of the time , but NR1a/NR2D receptors were open for only 4% of the time. For both, distributions of supercluster durations were best fitted with a mixtu re of six exponential components. Their overall mean durations were 35 .8 and 1602 ms, respectively. 4. Steady-state super-clusters were alig ned on their first openings and averaged. The average was well fitted by a sum of exponentials with time constants taken from fits to super- cluster length distributions. It is shown that this is what would be e xpected for a channel that shows simple Markovian behaviour. 5. The cu rrent through NR1a/NR2A channels following a concentration jump from z ero to 1 mM glutamate for 1 ms was well fitted by three exponential co mponents with time constants of 13 ms (rising phase), 70 ms and 350 ms (decaying phase). Similar concentration jumps on NR1a/NR2D channels w ere well fitted by two exponentials with means of 45 ms (rising phase) and 4408 ms (decaying phase) components. During prolonged exposure to glutamate, NR1a/NR2A channels desensitized with a time constant of 64 9 ms, while NR1a/NR2D channels exhibited no apparent desensitization. 6. We show that under certain conditions, the time constants for the m acroscopic gump response should be the same as those for the distribut ion of super-cluster lengths, though the resolution of the latter is s o much greater that it cannot be expected that all the components will be resolvable in a macroscopic current. Good agreement was found for jumps on NRla/NR2D receptors, and for some jump experiments on NR1a/NR 2A. However, the latter were rather variable and some were slower than predicted. Slow decays were associated with patches that had large cu rrents.