Olfactory receptor neurons (ORNs) are ideally suited as a model to study the kinetics of a second messenger system since the second messenger cascade is coupled directly to ion channels gated by either cAMP or cGMP (cN channels). Because these channels occupy a critical role in the odor transduction cascade, information about the underlying biochemical events can be gathered by measuring the electrical activity of cN channels. The kinetics of receptor-effector coupling in G-protein signal transduction systems in general is incompletely understood. Previous studies of second messenger kinetics conducted under equilibrium conditions have given rise to a model in which the output is presumed to be proportional to the accumulating concentration of a second messenger product. However, more recent biochemical studies utilizing rapid kinetic measurements suggest that activation of the olfactory G-protein cascade is transient rather than cumulative. The overall goal of this project is to determine whether a pulsatile model of second messenger signalling can explain the known characteristics of the response of olfactory receptor cells to odor application thus solving a long-standing controversy in olfactory transduction. The cN channels under study will be either a native one from salamander ORNs or a recombinant channel from rat olfactory epithelium expressed in a kidney cell line. The novel strategy in these studies will be to mimic a transient second messenger production by using a fast perfusion system and measuring the response of isolated membrane patches containing the cN channel to rapid application of cAMP/cGMP. These responses obtained under non-steady state conditions will be compared to single-channel kinetics obtained in the constant presence of second messenger. Further, we will compare the kinetics of isolated cN channels to the kinetics of the odor response in single ORNs obtained from odor pulses with well-controlled timing and concentration. Our data will lead to a biophysical state model for gating of cN channels providing estimates for the rates constants of association and dissociation of cyclic nucleotides to their target ion channel and eventually these numbers will be included into a comprehensive model for the olfactory second messenger cascade. As our pilot data show, activation of the cN channel could be a rate limiting step in the second messenger cascade. Therefore, the characteristic time course of the odor response may result, not from the biochemical steps of the second messenger system, but from inherent channel gating characteristics. Recent evidence suggested that cN channels could be widely expressed in the brain. Thus it appears possible that cN channels have important roles in modulation of neuronal excitability in the CNS as well and it is therefore anticipated that the results obtained from olfactory cells will provide fundamental information about kinetics of second messenger signalling systems in general.
