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Gating properties of the cAMP-gated channel in toad olfactory receptor cells
1National Institute for Physiological Sciences, Okazaki, Japan.
Abstract:
1. Inside-out membrane patches were excised from different parts of isolated olfactory receptor cells of the toad, and the gating properties of cAMP-gated channels were investigated under low concentrations of divalent cations. 2. At +50 mV, an outward current was observed when 1 mM cAMP was applied to the cytoplasmic side of membrane patches excised from cilia. cGMP had a similar effect. The dose-response relation for the cAMP-induced current could be fitted with the Hill equation with a coefficient of 1.5 for both cAMP and cGMP, and half-maximal concentration (K1/2) values of 19 microM (cAMP) and 16 microM (cGMP). 3. cAMP at low concentration (1 microM) induced step-like currents representing the opening of individual channels with a unitary conductance of about 30 pS. The I-V relation for the unitary events showed a weak outward rectification. 4. The relation between variance and mean current amplitude was well described with a parabola. Even at a saturating ligand concentration (1 mM cAMP) the current fluctuations did not disappear, indicating that fully liganded channels still switched between open and closed states. The maximum open probability was about 80% (+40 to +60 mV). 5. The current fluctuations at 1 mM cAMP were analysed with power spectral analysis. At +50 mV, frequency components lower than 10 Hz were well described with a Lorentzian function with a corner frequency of 1.7 +/- 0.2 Hz (+/- S.D.). In addition, higher flat frequency components were observed. At -50 mV the corner frequency became 0.6 +/- 0.1 Hz. 6. Membrane patches having a single cAMP-gated channel were obtained from the dendro-somatic membrane. These channels were very similar to the ciliary channels in electrophysiological characteristics. 7. The single cAMP-gated channel current showed flickering bursts that were interrupted with gaps. In saturating cAMP condition (1 mM), both burst- and gap-time histograms were fitted with single-exponential functions with time constants of 148 and 141 ms, respectively. 8. The channels were present at a high density of around 1750 microns-2 on the ciliary plasma membrane, as compared to 6 microns-2 on dendro-somatic membrane.
Insights
This study investigated cyclic nucleotide-gated (CNG) channels in toad olfactory receptor cells. These channels, crucial for smell, exhibit unique gating properties and are highly concentrated in cilia.
Area of Science:
- Olfactory receptor cell physiology
- Ion channel biophysics
- Sensory transduction mechanisms
Background:
- Olfactory receptor cells (ORCs) detect odorants via signal transduction cascades.
- Cyclic nucleotide-gated (CNG) channels are key players in ORC depolarization.
- Understanding CNG channel gating is essential for elucidating olfactory mechanisms.
Purpose of the Study:
- To characterize the gating properties of cAMP-gated channels in toad ORCs.
- To investigate the electrophysiological behavior of these channels in different cellular compartments.
- To determine the density and kinetic parameters of CNG channels in ciliary and somatic membranes.
Main Methods:
- Excised inside-out membrane patches from isolated toad ORCs (cilia and dendro-somatic regions).
- Electrophysiological recordings (single-channel and whole-cell currents) under controlled ligand concentrations and voltages.
- Analysis of dose-response relationships, unitary currents, variance-mean analysis, power spectral analysis, and dwell-time distributions.
Main Results:
- cAMP and cGMP activated outward currents with Hill coefficients of 1.5 and EC50 values around 16-19 µM.
- Single channels exhibited a unitary conductance of ~30 pS with weak outward rectification and flicking behavior.
- Channels displayed significant open probability (~80%) even at saturating ligand concentrations, with kinetic analysis revealing distinct burst and gap times.
Conclusions:
- Toad ORC CNG channels exhibit characteristic gating kinetics and ligand sensitivity.
- These channels are highly concentrated in the ciliary membrane (approx. 1750/µm²) compared to the somatic membrane (approx. 6/µm²).
- The findings provide detailed biophysical insights into the function of CNG channels in olfactory transduction.