TRANSDUCTlON AND ODOR DEACTlVATlON lN MOTH PHEROMONE RECEPTORS

Karl-Ernst KAlSSLlNG
Max-Planck-lnstitut für Verhaltensphysiologie Seewiesen, 82319 Starnberg, FRG


Olfactory transduction involves two groups of processes: Extracellular or perireceptor events comprise the adsorption and transport of odorants towards the receptor cell membrane as well as their removal or deactivation. The subsequent events of cellular excitation start with the activation by the odorant of - still hypothetical - receptor molecules in the receptor cell membrane. Their activation initiates intracellular signal processes leading to an opening of ion channels and a change of membrane potential. This "receptor potential" spreads passively (electrotonically) towards an electrically excitable region of the receptor cell membrane, the nerve impulse generator, and elicits a train of nerve impulses.

All of these perireceptor and cellular events consist of several molecular processes each of which can, in principle, contribute to the kinetics of the bioelectrical responses, the receptor potential and, finally, the nerve impulses. Here we ask which of the transduction processes is the slowest one and hence governs the kinetics of the receptor potential. One possible candidate is the process of rapid odorant deactivation as exemplified by pheromone receptor cells in the moth Antheraea polyphemus. This process was postulated in order to explain the decline of the receptor potential after the end of stimulus exposure (1). The decline cannot be due to the much slower ertzymatic degradation of the pheromone adsorbed on the antenna2. To determine the velocity of the postulated pheromone deactivation we assume that the actual amplitude of the receptor potential depends on the concentration of active pheromone in the sensillum lymph surrounding the receptor cell dendrites and that the relationship between active concentration and receptor potential can be taken from the static dose response curve. From the decline of the receptor potential after weak pheromone stimuli we can now determine a (maximum) half life of the active pheromone of 0.5-2s, a measure for the (minimum) velocity of pheromone deactivation in situ.

Recent in vitro experiments with homogenates of isolated olfactory hairs suggest that the pheromone binding protein (PBP) is involved not only in pheromone solubilization and transport but also in pheromone deactivation (3). PBP occurs in a reduced form with one or two disulfide bridges (PBPred) and in the oxidized form with three bridges (PBPox). From kinetic experiments it was concluded that the pheromone is first bound to the reduced form. This complex activates the receptor molecules and then turns into the oxidized form which - according to our working hypothesis - is unable to activate further receptor molecules. The pheromone bound to the PBP both forms - is protected from enzymatic degradation into non-excitatory metabolites (4). A quantitative kinetic model of pheromone deactivation was developed (in collaboration with J. Thorson, Oxford) in which the receptor molecules are considered to act as enzymes catalyzing the redox shift of the binding protein.

The model is consistent with biochemical and electrophysiological data. It is compatible with a density of receptor molecules equal to the density of rhodopsin molecules in the disc membrane of visual receptor cells (40 O0O units per &3181;m2), a Michaelis constant Km = 100 µM, and a rate constant of the redox shift k3 = 5/s. Furthermore, it is proposed that the duration of the "elementary receptor potential" elicited by single pheromone molecules represents the lifetime of the temary complex PBPred - pheromone - receptor molecule t = 1/(k2 + k3) = 50 ms. With this assumption one finds the rate constants of formation k1 = 0.2/(s x µM) and dissociation of the temary complex k2 = 15/s. The dissociation constant of the ternary complex would then be Kd = k2/k1 = 75 µM which means that the binding affinity of the complex PBPred - pheromone and the receptor molecule is much weaker than the binding affinity of PBPred and the pheromone, for which Kd = 60 nM (5). - The advantage of this model is its one-way character, which guarantees that the odorant is deactivated only after it hits a receptor molecule. This would help to avoid deactivation of pheromone before it had a chance to activate a receptor molecule and, thus, to lower the threshold of odor detection. - Further work is necessary to determine the kinetic constants more exactly and to investigate whether alternative models are also compatible with the experimental data.

For understanding cellular transduction the key question has to be answered how the interaction of the complex PBPred - pheromone with a receptor molecule generates an increase of cell membrane conductance by about 30 pS. According to an analysis of the electrical circuit of the olfactory sensillum, this conductance increase underlies the elementary receptor potential that can be elicited by a single pheromone molecule (6). It corresponds to opening of a single ion channel of the type of acetylcholine receptors. Recent findings in the moth Bombyx mori suggest that a single receptor activation leads to simultaneous opening of several but smaller ion channels (7). Possibly, the receptor activation causes the activation of G-proteins and the formation of second messenger molecules which activate several ion channels in the neighborhood of the receptor site. Rapid formation of the second messenger lP3 upon pheromone stimulation has been shown in homogenates of moth antennae (8). More slowly, cGMP is formed in intact antennae upon pheromone stimulation (9). The latter compound, as well as an analog of diacyl glycerol (both of them in the presence of MgATP), also induced ion channel opening in patch clamp experiments (10). However, a quantitative model of cellular transduction has not yet been developed.

Reterences

  1. Kaissling KE (1972) ln: Schneider D. (ed) lnt Symp Oltaction and Taste lV, Wiss. Verlagsges., Stuttgart, pp 207-213
  2. Kasang G. von Proff L, Nicholls M (1988) Z. Naturforsch. 43c, 257-284. 3) Ziegelberger G (1995) Eur. J. Biochem., 232, 706 -711
  3. Vogt RG and Riddiford LM (1986) ln: Payne TL, Birch MC, Kennedy CEJ (eds) Mechanisms in insect Olfaction, Clarendon Press, Oxtord, pp 201-208
  4. Kaissling KE, Klein U. de Kramer JJ, Keil TA, Kanaujia S. Hemberger J (1985) in: Molec. Basis of Nerve Activrly, Changeux JP et al. (eds), W. de Gruyter & Co., Berlin, New York, 173-183.
  5. Kaissling KE, Thorson, J (1980) In: Sattelle DB, Hall LM, Hildebrand JG (ads) Receptors for Transmitters, Hormones and Pheromones in lnsec~s, Elsevier/North-Holl., Amsterdam, pp 261-282
  6. Redkozubov A and Kaissling KE, unpublished
  7. Kaissling KE and Boekhoff l (1993) fn: Wiese K, Gribakin FG, Popov AV, Renninger G (eds) Sensory Systems of Arthropods, Birkhäuser Verlag Basel, Boston, Berlin, pp 489-502
  8. Ziegelberger G. Van den Berg, MJ, Kaissling KE, Klumpp S. Schulz JE (19g0) J. Neurosci., 10:1217-1225
  9. Zufall F. Hatt H (1991) Proc. Natl. Acad. Sci. USA, 88: 8520-8524

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