STOICHIOMETRY OF RECOMBINANT N-METHYL-D-ASPARTATE RECEPTOR CHANNELS INFERRED FROM SINGLE-CHANNEL CURRENT PATTERNS

Single-channel currents were recorded from mouse NR1-NR2B (zeta-epsilo n(2)) receptors containing mixtures of wild-type and mutant subunits e xpressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glut amine (N-to-Q) mutation at the N-0 site of the M2 segment (NR1:598, NR 2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated sin gle-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current Ir ere identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mix ture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subu nits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N recepto rs. The third, novel pattern presumably arose from hybrid receptors ha ving both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel cur rent patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns pr esumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depend ed on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that the re are two NR2 subunits per receptor and is consistent with either thr ee or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforwar d interpretation of the results is that N-methyl-D-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.