Subconductance states of a mutant NMDA receptor channel kinetics, calcium, and voltage dependence

The kinetic properties of main and subconductance states of a mutant mouse N-methyl-D-aspartate (NMDA) receptor channel were examined. Recombinant receptors made of zeta-epsilon 2 (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (zeta N598Q/epsilon 2N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In Ca(2+)-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62 pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single main level state. At -80 mV, the lifetime of the main and the longer-lived sub level are both approximately 3.3 ms. The gating of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (approximately 120 mV/e-fold change), but does not alter the equilibrium occupancies. Extracellular Ca2+ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore.