title: Illustrating the steady-state condition and the single-molecule kinetic method with the NMDA receptor.
abstract: The steady-state is a fundamental aspect of biochemical pathways in cells; indeed, the concept of steady-state is a definition of life itself. In a simple enzyme kinetic scheme, the steady-state condition is easy to define analytically but experimentally often difficult to capture because of its evanescent quality; the initial, constant velocity condition that signifies the steady-state of the E(f) and ES system is often short-lived. The recording of electrophysiologic events through a receptor channel is used here to illustrate the steady-state and to introduce the single-molecule approach to the quantification of biologic kinetic schemes. This article first briefly reviews the Michaelis-Menten and Briggs-Haldane formulations for the simple enzyme kinetic scheme. The salient structural features of the NMDA (N-methyl-D-aspartic acid) receptor are introduced as is the single NMDA molecule patch-clamp method; this is a high-resolution method for recording charge (ion) transfer through this and other ionotropic (ion-conducting) receptors. The concept of a time constant is related directly to a rate constant in showing that durations of channel opening and closing directly provide values for the rate constants that link the various conformation states adopted by the receptor when it is at a steady state. These rate constants provide the basis for illustrating the energy relationships between the multiple protein conformation states that the receptor populates during steady-state ion conduction across the cell membrane. The article emphasizes the advantages of collecting the mean behavior of a single molecule over time in comparison to the mean behavior of a large collection of independent molecules at a single time point.
authors.affiliation: Department of Biochemistry, The University at Buffalo, Buffalo, New York 14214. camkos@buffalo.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Kosman