Premkumar LS et al. (1997), Subconductance states of a mutant NMDA receptor...

XB-ART-16977

J Gen Physiol 1997 Feb 01;1092:181-9. doi: 10.1085/jgp.109.2.181.

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

Premkumar LS , Qin F , Auerbach A .

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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.

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Species referenced: Xenopus
Genes referenced: grin1 grin2b

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	Figure 2. Kinetic characteristics of NMDA receptor multiple conductance levels in Ca2+-free solutions. (A) Example currents at −80 mV. The top trace is a low time resolution view. The underlined region has been expanded in the lower trace. (B) Interval duration histograms and a kinetic model. The solid lines in the histograms were calculated from the optimal rate constants, estimated using the MIL algorithm. The main level histogram was described by a single exponential with a time constant (τ) equal to 2.27 ms, whereas the sub level histogram was described by the sum of two exponentials (τ1 = 3.67 ms, 75% of all intervals; τ2 = 0.25 ms). In this patch, the rate constant for transition from the main to the longer-lived sublevel (kms) was 328 s−1, and 236 s−1 for the reverse transition (ksm). The idealized currents were fitted by a kinetic model that had four closed, two sub, and one main level state. The solid arrows of the model represent transitions that were included in the fitting process for all patches. In some patches, additional states and transitions (indicated by dashed lines) were included (see Table I). M is the main state, S is a long-lived sublevel state, S1 is a brief sublevel state, C1 is a brief closed state, C0 is a long-lived closed state; additional closed states were added in series with C0 to describe the closed interval distribution. (C) Signal-averaged opening transitions of the current shown in A. 62 opening transitions, each following a sojourn in a closed level of at least 1 ms in duration, were aligned at the point of leaving the closed level (i.e., the first sample to cross the −3 pA threshold), and were averaged. The dashed lines indicate the amplitudes obtained from the SKM analysis for each conductance level. The dotted lines are the predicted amplitudes from the rate constants (upper line is the average of the sub and main level amplitudes, weighted by the probability of opening to either from the C0 state; Table I), and from an all-points amplitude histogram of this patch (lower line is the average of the sub and main level amplitudes, weighted by the relative equilibrium occupancy of each level). The channel opens either the main or the subconductance level, indicating that gating and conductance fluctuations are not strongly coupled processes.
	Figure 3. Voltage dependence of NMDA receptor conductance transitions in Ca2+-free solutions. (A) Example currents at the indicated membrane potentials. The lifetimes of both the main and subconductance level sojourns appear to become shorter with hyperpolarization. (B) Rate constant estimates obtained from kinetic analysis of a single patch. The main-to-sub (kms) and sub-to-main (ksm) rate constants both increase with hyperpolarization, suggesting that voltage influences the height of a barrier separating the conformations that generate the main and sub conductances.
	Figure 4. Effects of Ca2+ on the rate constants of conductance transitions. (A) Example currents obtained in the presence of 4.8 μM extracellular Ca2+ (−80 mV). The top trace was filtered at 10 kHz, the middle trace is the idealization of the top trace using the SKM algorithm, and the lower trace is the same current filtered at 1 kHz. The all-points amplitude histogram (1 kHz filtering) is shown at the right. Ca2+ concentration only modestly affects the amplitude of the main level, but lowers that of the sub level to about half of the amplitude in pure Na+ solutions because of unresolved channel block. (B) Main and sub level interval duration histograms for the patch shown in A. Even though the sublevel was occupied by Ca2+ about half of the time, the main-sub level transition rate constants (kms = 239 s−1, and ksm = 213 s−1) were similar to those obtained in Ca2+-free solutions (see Table I). (C) Rate constants as a function of extracellular Ca2+. At each concentration, each symbol is from a different patch. The rate constants show little, if any, dependence on the external Ca2+.

References [+] :

Auerbach, Single-channel currents from acetylcholine receptors in embryonic chick muscle. Kinetic and conductance properties of gaps within bursts. 1984, Pubmed