Chen TY et al. (2006), Odorant inhibition of the olfactory cyclic nucl...

XB-ART-36156

J Gen Physiol 2006 Sep 01;1283:365-71. doi: 10.1085/jgp.200609577.

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Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly.

Chen TY , Takeuchi H , Kurahashi T .

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Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein-coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca(2+) are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.

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Species referenced: Xenopus
Genes referenced: camp cnga2

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	Figure 1. Comparing the odorant inhibition in the homo-oligomeric and hetero-oligomeric CNG channels. (A) Sensitivity of the homo-oligomeric channel formed by subunit A2 to odorants at a concentration of 0.5 μl/ml. Top panel tests ANI suppressions. The gap in the recording represents the 30-min incubation of the oocyte in the solution with 100 μM 8Br-cGMP. Bottom panel, experiments using IAA, CIN and LIM in another oocyte. Three other oocytes showed similar results. (B) Sensitivity of the hetero-oligomeric channel to odorants. All recordings were from the same oocyte. The channels were generated by injecting RNAs of subunits A2, A4, and B1 (RNA ratio, 2:1:1) into the oocyte. IAA, ANI, LIM, and CIN were all at a concentration of 0.5 μl/ml. Mg2+ was 10 mM. Recording gap on top represents the 8Br-cGMP incubation period. Three other oocytes showed similar results.
	Figure 2. Dose dependence of the odorant inhibition of olfactory CNG channels. (A) Suppression of the olfactory CNG channel (A2 + A4 + B1) by various concentrations of ANI. The labeling of “Mg2+” indicates the application of 10 mM MgCl2. Arrow at the end of the recording indicates a membrane breakdown, which is followed by a sudden increase in leak current. (B) Low sensitivity of the triple-subunit channel to LIM. Note that even at the highest concentration, 5 μl/ml, LIM does not have an appreciable suppression on the channel. The oocyte is also intact at such a high concentration of LIM. (C) Suppression of the homo-oligomeric channel (CNGA2) by IAA. The current is not inhibited by IAA at concentrations ≤0.5 μl/ml. At 2 μl/ml, a slight and slow suppression can be detected although the oocyte becomes leaky at this concentration of IAA.
	Figure 3. Dose–suppression curves of various types of olfactory CNG channels. (A) Homo-oligomeric CNG channels formed entirely by subunit CNGA2. The number of oocytes (n) tested are 4, 3, 4, and 5 for ANI, IAA, CIN, and LIM, respectively. (B) Hetero-oligomeric channels formed by subunits A2 and A4 (A2 + A4); n = 3–5. (C) Hetero-oligomeric channels (A2 + B1); n = 4–5. (D) Hetero-oligomeric channels (A2 + A4 + B1); n = 3–4. In all four panels, symbols are as follows: solid squares, ANI; open squares, IAA; open triangles, CIN; open circles, LIM. Data represented as mean ± SEM.
	Figure 4. Sensitivity of odorant-induced current in the olfactory neuron clamped at two voltages. Cells that did not respond to the applied odorant were selected for experiments. (A) Suppression of the cAMP-induced current by LIL. Cytoplasmic cAMP was jumped by a photolysis of caged cAMP. The concentration of LIL used to suppress the current was 0.2 μl/ml. Top, Vm = −50 mV; bottom, Vm = +120 mV. The suppression on the basal current was subtracted from the data. (B) Suppression percentage versus the pressure applied to the puff pipette. Odorant, AA (0.1 μl/ml). Top, Vm = −50 mV. Solid curve was Hill fitting of the data points with K1/2 = 18 kPa. Bottom, Vm = +120 mV. Hill fitting shows K1/2 = 25 kPa. Error bars represent SD, and numbers in parentheses indicate the numbers of cells tested.

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