Takashi Kurahashi

Activation by odorants of cation-selective conductance in the olfactory receptor cell isolated from the newt.

1. Ionic selectivity of the conductance activated by n‐amyl acetate (odorant‐activated conductance) was analysed in isolated olfactory receptor cells under the whole‐cell voltage clamp condition. 2. Solitary receptor cells had a resting membrane potential of ‐44.7 +/‐ 7.0 mV (mean +/‐ S.D.; n = 70). Application of 10 mM‐n‐amyl acetate caused a depolarizing response in about 30% of the cells. Sensitivity to the odorant was maximum at around the apical dendrite. 3. Odorant induced an inward current to cells voltage clamped at their resting potential and bathed in the standard medium. The response amplitude was voltage dependent, and the polarity reversed at +2.5 +/‐ 2.2 mV (n = 6). The I‐V relation was almost linear at membrane potentials more positive than ‐20 mV, with an average slope of 3.14 +/‐ 1.59 nS (measured at 0 mV), but showed a marked outward rectification at voltages more negative than ‐30 mV. 4. Removal of external Ca2+ increased the amplitude of the odorant‐induced current and prolonged response duration, but did not cause a significant change on the reversal potential. Thus, Ca2+ affected the kinetics of the conductance, but did not seem to be a dominant charge carrier in the physiological condition. 5. Reduction of external Na+ concentration [( Na+]o) (replaced with choline) shifted the reversal potential by about 57 mV per 10‐fold change of [Na+]o. Removal of external Cl‐ (replaced with glutamate ions) did not affect the reversal potential. 6. The odorant‐activated conducting channels were permeable to all alkali metal ions. The permeability ratios were: PLi:PNa:PK:PRb:PCs = 1.25:1:0.98:0.84:0.80. 7. The present study strongly suggests that the olfactory receptor potential is generated by an increase in the membrane conductance to alkali metal ions.

The response induced by intracellular cyclic AMP in isolated olfactory receptor cells of the newt.

1. Responses induced by intracellular cyclic nucleotides were analysed in isolated olfactory receptor cells of the newt under a voltage‐clamp condition by using the patch pipette in a whole‐cell recording configuration. Cyclic nucleotides were applied by diffusion from the patch pipette. 2. Introduction of either cyclic AMP or cyclic GMP caused a transient inward current in cells held at ‐50 mV. The response amplitude was dose‐dependent with the Hill coefficient of 3 and half‐saturating concentration of 300 microM (concentration in the pipette) for both cyclic AMP and cyclic GMP. Cyclic CMP was less effective than those two nucleotides. 3. The response to intracellular cyclic AMP was seen in all cilia‐bearing cells, but not in cells which lost the cilia during dissociation. The response latency was shorter when cyclic AMP was introduced into the ciliated terminal swelling (ca 0.2 s) rather than into the cell body (ca 1.4 s). These results suggest that the sensitivity to intracellular cyclic AMP is confined to the cilia. 4. The cyclic AMP‐induced current was transient (half decay time, ca 2.3s) despite the fact that cyclic AMP was continuously loaded from the patch pipette. The response time course was controlled by Ca2+; the reduction of external Ca2+ concentration (replaced with Mg2+) or loading the cell with 50 mM‐EGTA prolonged the cyclic AMP‐induced responses. The Ca2(+)‐induced suppression was reversible. 5. The reversal potential of the cyclic AMP‐induced transient current was ‐4.8 +/‐ 3.8 mV, and that of the current re‐induced by Ca2+ removal was 1.5 +/‐ 2.1 mV, suggesting that both currents flowed through the same ionic channel. The channel permeates all alkali metal ions with the permeability ratios of PLi:PNa:PK:PRb:PCs = 0.93:1:0.93:0.91:0.72, but not Cl‐ or choline ions. 6. These results demonstrate that the cyclic AMP‐induced response and the odorant‐induced response of the isolated olfactory cell have nearly identical characteristics. The present study supports the notion that cyclic AMP is the internal messenger mediating olfactory transduction.

Adrenaline enhances odorant contrast by modulating signal encoding in olfactory receptor cells

Olfactory perception is influenced by hormones. Here we report that adrenaline can directly affect the signal encoding of olfactory receptor cells. Application of adrenaline suppressed action potentials near threshold and increased their frequency in response to strong stimuli, resulting in a narrower dynamic range. Under voltage–clamp conditions, adrenaline enhanced sodium current and reduced T–type calcium current. Because sodium current is the major component of spike generation and T–type calcium current lowers the threshold in olfactory receptor cells, the effects of adrenaline on these currents are consistent with the results obtained under current–clamp conditions. Both effects involved a common cytoplasmic pathway, cAMP–dependent phosphorylation. We suggest that adrenaline may enhance contrast in olfactory perception by this mechanism.

Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6‐bromo‐7‐hydroxycoumarin‐4‐ylmethyl esters of cNMP (Bhc‐cNMP) and their acetyl derivatives (Bhc‐cNMP/Ac) as new caged second messengers. Irradiation of Bhc‐cNMPs quantitatively produced the parent cNMPs with one‐photon uncaging efficiencies (Φε) of up to one order of magnitude better than those of 2‐nitrophenethyl (NPE) cNMPs. In addition, two‐photon induced photochemical release of cNMP from Bhc‐cNMPs (7 and 8) can be observed with the two‐photon uncaging action cross‐sections (δu) of up to 2.28 GM (1 GM=10−50 cm4 s photon−1), which is the largest value among those of the reported Bhc‐caged compounds. The wavelength dependence of the δu values of 7 revealed that the peak wavelength was twice that of the one‐photon absorption maximum. Bhc‐cNMPs showed practically useful water solubility (nearly 500 μM), whereas 7‐acetylated derivatives (Bhc‐cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP‐mediated transduction channels in newt olfactory receptor cells and cAMP‐mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

High density cAMP-gated channels at the ciliary membrane in the olfactory receptor cell.

Spatial distribution of the cAMP-gated channel was investigated in amphibian olfactory receptor cells. Low doses of cAMP applied to the cytoplasmic side of a membrane patch excised from cilia produced single channel activity of unitary conductance 28pS. Variance analysis showed that the ciliary membrane contained 920 cAMP gated-channels/microns2 in the newt and 2400 channels/microns2 in the toad. In contrast, the membrane of the dendrite and cell body contained only 2 cAMP-gated channels/microns2 (newt) and 6 channels/microns2 (toad). Thus, there is a high density of cAMP-gated channels in the cilia where olfactory transduction is thought to take place.

Olfactory Transduction: Tale of an unusual chloride current

An unusual chloride current seems to play an important role in safeguarding olfactory transduction against an unstable ionic environment, and in nonlinearly amplifying the olfactory signal.

Mechanism of olfactory masking in the sensory cilia

Olfactory masking has been used to erase the unpleasant sensation in human cultures for a long period of history. Here, we show a positive correlation between the human masking and the odorant suppression of the transduction current through the cyclic nucleotide–gated (CNG) and Ca2+-activated Cl− (Cl(Ca)) channels. Channels in the olfactory cilia were activated with the cytoplasmic photolysis of caged compounds, and their sensitiveness to odorant suppression was measured with the whole cell patch clamp. When 16 different types of chemicals were applied to cells, cyclic AMP (cAMP)-induced responses (a mixture of CNG and Cl(Ca) currents) were suppressed widely with these substances, but with different sensitivities. Using the same chemicals, in parallel, we measured human olfactory masking with 6-rate scoring tests and saw a correlation coefficient of 0.81 with the channel block. Ringers solution that was just preexposed to the odorant-containing air affected the cAMP-induced current of the single cell, suggesting that odorant suppression occurs after the evaporation and air/water partition of the odorant chemicals at the olfactory mucus. To investigate the contribution of Cl(Ca), the current was exclusively activated by using the ultraviolet photolysis of caged Ca, DM-nitrophen. With chemical stimuli, it was confirmed that Cl(Ca) channels were less sensitive to the odorant suppression. It is interpreted, however, that in the natural odorant response the Cl(Ca) is affected by the reduction of Ca2+ influx through the CNG channels as a secondary effect. Because the signal transmission between CNG and Cl(Ca) channels includes nonlinear signal-boosting process, CNG channel blockage leads to an amplified reduction in the net current. In addition, we mapped the distribution of the Cl(Ca) channel in living olfactory single cilium using a submicron local [Ca2+]i elevation with the laser photolysis. Cl(Ca) channels are expressed broadly along the cilia. We conclude that odorants regulate CNG level to express masking, and Cl(Ca) in the cilia carries out the signal amplification and reduction evenly spanning the entire cilia. The present findings may serve possible molecular architectures to design effective masking agents, targeting olfactory manipulation at the nano-scale ciliary membrane.

Photolysis of caged cyclic AMP in the ciliary cytoplasm of the newt olfactory receptor cell

The effects of cyclic nucleotide monophosphate (cNMP) in the ciliary cytoplasm of the olfactory receptor cell were examined by using photolysis of caged cNMP loaded from the whole‐cell patch clamp pipette. Illumination of the cilia induced an inward current at −50 mV. The current amplitude was voltage dependent and the polarity was reversed at +10 mV. The amplitude of the light‐induced current was dependent on both light intensity and duration. The intensity‐response relation was fitted well by the Hill equation with a coefficient (nH) of 4.99 ± 2.66 (mean ±s.d., n= 19) and the duration‐response relation with a coefficient of 4.03 ± 1.43 (n= 17). The activation time course of adenylyl cyclase was estimated by comparing the light‐induced response with the odorant‐induced response. Adenylyl cyclase was activated approximately 260 ms later from the onset of the odorant‐stimulation. The light‐induced current developed very sharply. This could be explained by the sequential openings of cAMP‐gated and Ca2+‐activated Cl− channels. At +100 mV, where Ca2+ influx is expected to be very small, the current rising phase became less steep. When the cells were stimulated by long steps of either odour or light, the odorant‐induced current showed stronger decay than the light‐induced response. This observation suggests that the molecular system regulating desensitization is situated upstream of cAMP production.

Mechanism of signal amplification in the olfactory sensory cilia.

Molecular mechanisms underlying olfactory signal amplification were investigated by monitoring cAMP dynamics in the intact sensory cilia. We saw that [cAMP]i increased superlinearly with time during odorant stimuli for >1 s. This time course was remarkably different from that obtained with the rapid quench method previously applied to the in vitro preparation, in which [cAMP]i change has been reported to be transient. The superlinear increase of [cAMP]i was attributable to a gradual increase of cAMP production rate that was consistent with the thermodynamical interaction model between elemental molecules, as has been revealed on the rod photoreceptor cell. It thus seems likely that the fundamental mechanism for molecular interactions between olfactory transduction elements is similar to that of the rod. In olfaction, however, cAMP production was extremely small (∼200,000 molecules/s/cell at the maximum), in contrast to the cGMP hydrolysis in the rod (250,000 molecules/photon). The observed numbers indicate that the olfactory receptor cell has lower amplification at the enzymatic cascade. Seemingly, such low amplification is a disadvantage for the signal transduction, but this unique mechanism would be essential to reduce the loss of ATP that is broadly used for the activities of cells. Apparently, transduction by a smaller number of second-messenger formations would be achieved by the fine ciliary structure that has a high surface-volume ratio. In addition, it is speculated that this low amplification at their enzymatic processes may be the reason why the olfactory receptor cell has acquired high amplification at the final stage of transduction channels, using Ca2+ as a third messenger.

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

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 Ca2+ 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.