The purpose of this proposal is to study the kinetic parameters that determine the probability of opening (Po) of NMDA channels at synaptic and non-synaptic sites in conditions that favor phosphorylation or dephosphorylation to determine the rage over which modulation is possible. Because the Po is generally found to be the channel property affected by modulatory events (changes in single channel conductance and channel number are only rarely reported), the means of altering Po and the range over which Po can change are vital pieces of information in understanding the consequences of activating modulatory mechanisms. Estimates in the literature of NMDA channel Po range from 0.003 to 0.3; that is, if an NMDA channel were to remain in a state from which it could open (bound by glutamate and glycine) for 1 second, it would spend a total of either 3 milliseconds or 300 milliseconds in the open state, respectively. Recent reports suggest that the Po is increased about 10- fold by excising an outside-out patch and that both non-synaptic and synaptic receptors in undisturbed cells have similar Po's. The evidence for altering Po by patch detachment appears good, but the estimates of synaptic receptor Po are not straightforward and, again, estimates vary 10-fold. If the variability is real but due to different experimental conditions, it implies that alterations in synaptic strength as high as 10-fold can be achieved by normal physiological mechanisms. The proposed experiments will assess the Po of NMDA channels in a variety of experimental conditions. NMDA receptor responses will be evoked by synaptic stimulation and rapid agonist applications in hippocampal neurons in culture and in slice preparations. Responses will be recorded with whole cell, cell-attached and detached patch clamp techniques.
