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Direct suppression by odorants of cyclic nucleotide-gated currents in the newt photoreceptors
Brain Research 876 (2000) 180–184 www.elsevier.com / locate / bres
Short communication
Direct suppression by odorants of cyclic nucleotide-gated currents in the newt photoreceptors a, a Fusao Kawai *, Ei-ichi Miyachi a
Department of Physiology, School of Medicine, Fujita Health University, 1 -98 Dengakugakubo, Kutsukakechou, Toyoake, Aichi, 470 -1192, Japan Accepted 13 June 2000
Abstract Odorants are known to suppress the cyclic nucleotide-gated (CNG) current in olfactory receptor cells. It is unclear, however, whether odorants suppress the olfactory CNG current directly or whether they suppress the current by decreasing the second messenger (cAMP) through the activation of phosphodiesterase. We found that odorants also suppress CNG currents in photoreceptor cells. Under voltage clamp, an odorant puff immediately suppressed the currents induced by the intracellular cGMP in isolated newt rods and cones. Odorants also suppressed the currents induced by another cGMP analog (8-p-chlorophenylthio-cGMP, which strongly resists hydrolysis by phosphodiesterase), suggesting that the second messenger metabolism via phosphodiesterase is not involved in the suppression by odorants. This suggests that odorants suppress the CNG currents directly rather than via the second messenger system in photoreceptors, and also likely in olfactory receptor cells. 2000 Elsevier Science B.V. All rights reserved. Theme: Sensory Systems Topic: Olfactory senses Keywords: Olfactory receptor cell; Rod; Cone; Odorant; Patch clamp; Newt
The initial step in olfactory sensation involves the binding of odorant molecules to specific receptor proteins on the ciliary surface of olfactory receptor cells. Odorant receptors coupled to G-proteins activate adenylyl cyclase leading to the generation of cAMP, which directly gates a cyclic nucleotide-gated (CNG) cationic channel in the ciliary membrane [1,3,4,12,14]. In contrast, odorants such as amyl acetate, limonene, and acetophenone (banana-, lemon-, and orange-blossom-like-odor) are also known to suppress the CNG current in olfactory receptor cells [11]. Similar suppression was observed in various voltage-gated currents of olfactory cells [6–9] and the currents of retinal horizontal cells [10]. It is unclear, however, whether odorants suppress the CNG current in olfactory receptor cells directly or whether they suppress the currents via the second messenger (i.e., cAMP) metabolism activated by odorant binding to odorant receptors. To investigate these possibilities, we ex*Corresponding author. Tel.: 181-562-93-2466; fax: 181-562-932649. E-mail address: [email protected] (F. Kawai).
amined the CNG currents in the newt rod and cone photoreceptor cells, because the photoreceptors do not have the second messenger systems activated by odorants. Thus, by using the photoreceptors, one can examine direct effects of odorants on the CNG channels. Although odorants are not stimulants or endogenous molecules in the retina, it would be interesting to elucidate biophysical and pharmacological properties of odorants against the photoreceptor CNG channels. In the present study, we investigated mechanisms underlying the suppression by odorants of the CNG currents by using the whole-cell version of the patch-clamp technique. Rod and cone photoreceptor cells were dissociated mechanically from the newt retina. Dissociation protocols were similar to those reported previously [2,13]. In short, individual photoreceptors were obtained by chopping the retina with a razor blade in Ringer solution containing (in mM): 110 Na 1 , 3.7 K 1 , 1 Ca 21 , 2 HEPES, 15 glucose, 10 ppm phenol red (pH adjusted to 7.4 with NaOH). Isolated cells were plated on the concanavalin A-coated glass cover-slip. Cells were maintained at 48C (up to 10 h) before use.
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02613-5
F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
The recording pipette was filled with Cs 1 solution: 124 CsCl, 1 CaCl 2 , 5 EGTA, 10 HEPES (pH adjusted to 7.4 with CsOH). To block K 1 currents high internal Cs 1 was used and 20 mM tetraethylammonium chloride was substituted for NaCl in the bath solution. The resistance of the pipette was about 6 MV. Membrane current was recorded under whole-cell patch clamp configuration [5]. For recording, the culture dish was mounted on the stage of an Olympus upright microscope with differential interference contrast optics (BX50WI, Tokyo, Japan). A patch-clamp amplifier (Axon Instruments Inc., Axopatch-200B, Burlingame, CA, USA), linked to an IBM-compatible PC, was used to measure membrane current. The voltage-clamp procedures were controlled by using the software pCLAMP (Axon Instruments Inc., Burlingame, CA, USA). Data were low-pass filtered (4-pole Bessel type) with a cut-off frequency of 5 kHz and then digitized at 10 kHz by an analog-to-digital interface. All experiments were performed at room temperature (23–258C). Test substances were applied via the patch pipette (cGMP, 8-p-chlorophenylthio-cGMP, or 8-bromo-cGMP) or through the bath (8-p-chlorophenylthio-cGMP). Odorants (amyl acetate, acetophenone or limonene) were dissolved in solutions also containing 5 mM dimethylsulfoxide (DMSO) and were applied to the cell from a U-tube system or by pressure ejection. The chemical structures of the odorants used are shown in Table 1. The stream of superfusate was directed at the cell by placing the tip of the U-tube outlet approximately 1 mm from the cell. The puffer pipettes (tip diameter about 5 mm) were placed approximately 30 mm from the cell. Surprisingly, odorants suppressed the CNG currents in the newt rod photoreceptors. At the holding potential (Vh ) of 250 mV, intracellular application of 1 mM cGMP from the recording pipette induced the sustained inward current, which gradually increased with time (Fig. 1A). This current was rapidly (time constant of approximately 10 ms) suppressed by puff application of 1 mM amyl acetate (Fig. 1A). The CNG current was not significantly changed by puff application of the odorant-free solution containing 5
Table 1 Chemical structures of the odorants Molecules
Structure
181
mM DMSO, suggest that the current was suppressed not by DMSO but by amyl acetate. This result raises two possible mechanisms. First, amyl acetate may reduce the rod CNG current by blocking its channel directly. Secondly, amyl acetate might reduce the CNG current by decreasing the intracellular cGMP concentration through the activation of a cGMP-hydrolyzing phosphodiesterase (PDE). To rule out the latter, we used a non-hydrolyzable cGMP analog, 8-p-chlorophenylthiocGMP (8-CPT-cGMP). If the latter were a case, substitution of cGMP with 8-CPT-cGMP would be expected to eliminate the suppression by amyl acetate of the CNG inward current. Application of 0.1 mM 8-CPT-cGMP from the recording pipette induced the sustained inward current, however, 8-CPT-cGMP did not prevent the suppression by amyl acetate (Fig. 1B). Similar suppression was obtained by intracellular application of a poorly hydrolyzable cGMP analog, 8-bromo-cGMP (8-Br-cGMP, 0.2 mM) and also by bath application of 0.1 mM 8-CPT-cGMP. These results exclude the latter possibility. Thus, it is likely that amyl acetate reduces the rod CNG current by blocking its channel directly. These data are summarized in Fig. 1C. Puff application of 1 mM amyl acetate reduced the inward currents induced by cGMP, 8-CPT-cGMP, and 8-Br-cGMP by more than 90%. Similarity of suppression ratio for each cGMP analog suggests that the second messenger metabolism via PDE is not involved in the suppression by amyl acetate. We also examined effects of other odorants on the rod CNG current. Puff application of 1 mM limonene and acetophenone also suppressed the CNG current induced by 0.1 mM 8-CPT-cGMP by approximately 90%, which was similar to the ratio of 1 mM amyl acetate (Fig. 1D). These results suggest that odorants block the rod CNG channel directly. Odorants also suppressed the CNG current in cone photoreceptors. Application of 0.1 mM 8-CPT-cGMP from the recording pipette induced the sustained inward current, which was also suppressed by 10 ms puff application of amyl acetate (Fig. 2A). Amyl acetate puff also suppressed the current induced by internal application of 1 mM cGMP and 0.1 mM 8-Br-cGMP and by bath application of 0.1 mM 8-CPT-cGMP (Fig. 2B). These results suggest that amyl acetate directly blocks the cone CNG channel as well. Dose-response relationships of amyl acetate against the photoreceptor CNG currents were examined by measuring the current-voltage (I–V) relationships. In the control solution without odorant, the rod CNG current induced by 0.1 mM 8-CPT-cGMP showed a linear relationship between 250 mV and 150 mV, which reversed at 14 mV (Fig. 3A). Similar reversal potentials were obtained from five cells (563 mV). Bath application of amyl acetate of several concentrations (0.1, 1 mM) made the slope of I–V curves less steep, and the I–V curves reversed at 14 mV, which was identical with the reversal potential in the
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F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
Fig. 1. Amyl acetate suppresses the cyclic nucleotide-gated (CNG) current of an isolated newt rod photoreceptor cell. (A) The CNG inward current recorded at 2 and 5 min after rupture of the patch membrane at the holding potential (Vh ) of 250 mV. The recording pipette was filled with Cs 1 solution containing 1 mM cGMP. Puffer application of 1 mM amyl acetate for 10 ms (bottom bar) rapidly reduced the amplitude of the inward current. (B) Puffer application of 1 mM amyl acetate for 75 ms (bottom bar) also reduced the amplitude of the inward current induced by 0.1 mM 8-p-chlorophenylthio-cGMP (8-CPT-cGMP) from the recording pipette. (C) Summary of the effects of amyl acetate on the rod CNG current induced by various cGMP analogs in the recording pipette (1 mM [cGMP] p , 0.1 mM [8-CPT-cGMP] p or 0.2 mM [8-Br-cGMP] p ) and the bath solution (0.1 mM 8-CPT-cGMP). Each bin shows the mean ratio of the amplitude of the suppressed inward current to that of the sustained inward current before odor application, and error bar S.E.M. (n55). 1 mM amyl acetate was applied to a cell through the puffer pipette for 100 ms. Five min after rupture of the patch membrane, amplitude of the suppressed current was measured at 50 ms after the onset of puff application. (D) Summary of the effects of various odorants on the rod CNG current induced by 0.1 mM 8-CPT-cGMP in the recording pipette. Each bin shows the mean ratio of the suppression and error bar S.E.M. (n55). 1 mM amyl acetate, limonene or acetophenone was applied to a cell through the puffer pipette for 100 ms.
control condition (Fig. 3A). This observation suggests that amyl acetate decreases the conductance of the rod CNG channel.
Fig. 3B shows the dose-response curve of amyl acetate against the rod CNG current. The dose-response curve of 0.1 mM 8-CPT-cGMP induced current was fitted by the
F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
183
Fig. 2. Amyl acetate suppresses the CNG current of a cone photoreceptor. (A) The CNG inward current recorded at 2 and 5 min after rupture of the patch membrane at the Vh of 250 mV. The recording pipette was filled with Cs 1 solution containing 0.1 mM 8-CPT-GMP. Puffer application of 1 mM amyl acetate for 10 ms (bottom bar) rapidly reduced the amplitude of the inward current. (B) Summary of the effects of amyl acetate (1 mM) on the cone CNG currents induced by various cGMP analogs. Each bin was recorded similarly as Fig. 1C.
Hill equation with a half-blocking concentration (IC 50 ) of 72 mM and a Hill coefficient of 1.3 (Fig. 3B, n55 cells). Similar values were obtained from the cone CNG current. IC 50 of amyl acetate was 87 mM, and the Hill coefficient 1.2 (n54 cells, data not shown).
In the present study, we found that odorants suppress the photoreceptor CNG currents in a similar manner as in olfactory receptor cells [11]. The suppressive action of amyl acetate on the photoreceptor CNG currents was very rapid (|10 ms; Fig. 1, 2), which is consistent with the
Fig. 3. Concentration dependence of amyl acetate against the rod CNG current induced by 0.1 mM 8-CPT-cGMP in the recording pipette. (A) Effects of amyl acetate (concentration indicated to the right of each trace) on I–V curves. The cell was voltage clamped at 250 mV and a ramp depolarization (between –50 mV and 150 mV at a speed of 100 mV/ s) was applied in the presence and absence of amyl acetate (0.1, 1 mM). Note that the slope of I–V curves became less steep during bath application of amyl acetate, suggesting a conductance decrease. (B) Current amplitude at the command voltage of 250 mV in (A) was normalized and plotted as a function of amyl acetate concentration. Data points are mean ratios across cells (6S.E.M., n55). The continuous line shows a least squares fit of the data to the Hill equation.
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F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
observation of the olfactory CNG current [11]. In addition, the suppression was also observed even when hydrolysis of cGMP was inhibited. These results suggest that the second messenger system via PDE is not involved in the suppression. Chemical structures of the odorants used in the present experiment (Table 1) show the high lipid solubility and neutral charge. Therefore, it is likely that these molecules can readily cross the cell membranes and can bind to hydrophobic sites of CNG channels. Furthermore, it is known that odorants non-selectively suppress the voltage-gated currents in the olfactory cells [6–9] and the retinal horizontal cells [10]. Thus, we suggest that odorants directly block both olfactory and photoreceptor CNG channels as well as the voltage-gated channels.
Acknowledgements This work was supported by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, and Grant-in-Aid for Science Research from the Ministry of Education, Science and Culture (No. 12780620 to F.K.; No. 11680794 to E.M.).
References [1] H.A. Bakalyar, R.R. Reed, The second messenger cascade in olfactory receptor neurons, Curr. Biol. 1 (1991) 204–208.
[2] D.A. Baylor, T.D. Lamb, K.-W. Yau, The membrane current of single rod outer segments, J. Physiol. (Lond) 288 (1979) 589–611. [3] S. Firestein, F.S. Werblin, Gating currents in isolated olfactory receptor neurons of the larval tiger salamander, Proc. Natl. Acad. Sci. USA 88 (1987) 6292–6296. [4] G.H. Gold, T. Nakamura, Cyclic nucleotide-gated conductances: a new class of ionic channels mediates visual and olfactory transduction, Trends. Pharmacol. 8 (1987) 312–316. [5] O.P. Hamill, A. Marty, E. Neher, F.J. Sakmann, Sigworth, Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pflugers Arch. 391 (1981) 85–100. [6] F. Kawai, T. Kurahashi, A. Kaneko, Nonselective suppression of voltage-gated currents by odorants in the newt olfactory receptor cells, J. Gen. Physiol. 109 (1997) 265–272. [7] F. Kawai, T. Kurahashi, A. Kaneko, T-type Ca 21 channel lowers the threshold of spike generation in the newt olfactory receptor cell, J. Gen. Physiol. 108 (1996) 525–535. [8] F. Kawai, Odorant suppression of delayed rectifier potassium current in newt olfactory receptor cells, Neurosci. Lett. 269 (1999) 45–48. [9] F. Kawai, Odorants suppress T- and L-type Ca 21 currents in olfactory receptor cells by shifting their inactivation curves to a negative voltage, Neurosci. Res. 35 (1999) 253–263. [10] F. Kawai, E.-I. Miyachi, Odorants suppress voltage-gated currents in retinal horizontal cells, Neurosci. Lett. 281 (2000) 151–154. [11] T. Kurahashi, G. Lowe, G.H. Gold, Suppression of odorant responses by odorants in olfactory receptor cells, Science 265 (1994) 118–120. [12] T. Kurahashi, K.-W. Yau, Tale of an unusual chloride current, Curr. Biol. 4 (1994) 256–258. [13] K. Nakatani, Y. Koutalos, K.-W. Yau, Ca 21 modulation of the cGMP-gated channel of bullfrog retinal rod photoreceptors, J. Physiol. (Lond) 484 (1995) 69–76. [14] D. Restrepo, J.H. Teeter, D. Schild, Second messenger signaling in olfactory transduction, J. Neurobiol. 30 (1996) 37–48.
Short communication
Direct suppression by odorants of cyclic nucleotide-gated currents in the newt photoreceptors a, a Fusao Kawai *, Ei-ichi Miyachi a
Department of Physiology, School of Medicine, Fujita Health University, 1 -98 Dengakugakubo, Kutsukakechou, Toyoake, Aichi, 470 -1192, Japan Accepted 13 June 2000
Abstract Odorants are known to suppress the cyclic nucleotide-gated (CNG) current in olfactory receptor cells. It is unclear, however, whether odorants suppress the olfactory CNG current directly or whether they suppress the current by decreasing the second messenger (cAMP) through the activation of phosphodiesterase. We found that odorants also suppress CNG currents in photoreceptor cells. Under voltage clamp, an odorant puff immediately suppressed the currents induced by the intracellular cGMP in isolated newt rods and cones. Odorants also suppressed the currents induced by another cGMP analog (8-p-chlorophenylthio-cGMP, which strongly resists hydrolysis by phosphodiesterase), suggesting that the second messenger metabolism via phosphodiesterase is not involved in the suppression by odorants. This suggests that odorants suppress the CNG currents directly rather than via the second messenger system in photoreceptors, and also likely in olfactory receptor cells. 2000 Elsevier Science B.V. All rights reserved. Theme: Sensory Systems Topic: Olfactory senses Keywords: Olfactory receptor cell; Rod; Cone; Odorant; Patch clamp; Newt
The initial step in olfactory sensation involves the binding of odorant molecules to specific receptor proteins on the ciliary surface of olfactory receptor cells. Odorant receptors coupled to G-proteins activate adenylyl cyclase leading to the generation of cAMP, which directly gates a cyclic nucleotide-gated (CNG) cationic channel in the ciliary membrane [1,3,4,12,14]. In contrast, odorants such as amyl acetate, limonene, and acetophenone (banana-, lemon-, and orange-blossom-like-odor) are also known to suppress the CNG current in olfactory receptor cells [11]. Similar suppression was observed in various voltage-gated currents of olfactory cells [6–9] and the currents of retinal horizontal cells [10]. It is unclear, however, whether odorants suppress the CNG current in olfactory receptor cells directly or whether they suppress the currents via the second messenger (i.e., cAMP) metabolism activated by odorant binding to odorant receptors. To investigate these possibilities, we ex*Corresponding author. Tel.: 181-562-93-2466; fax: 181-562-932649. E-mail address: [email protected] (F. Kawai).
amined the CNG currents in the newt rod and cone photoreceptor cells, because the photoreceptors do not have the second messenger systems activated by odorants. Thus, by using the photoreceptors, one can examine direct effects of odorants on the CNG channels. Although odorants are not stimulants or endogenous molecules in the retina, it would be interesting to elucidate biophysical and pharmacological properties of odorants against the photoreceptor CNG channels. In the present study, we investigated mechanisms underlying the suppression by odorants of the CNG currents by using the whole-cell version of the patch-clamp technique. Rod and cone photoreceptor cells were dissociated mechanically from the newt retina. Dissociation protocols were similar to those reported previously [2,13]. In short, individual photoreceptors were obtained by chopping the retina with a razor blade in Ringer solution containing (in mM): 110 Na 1 , 3.7 K 1 , 1 Ca 21 , 2 HEPES, 15 glucose, 10 ppm phenol red (pH adjusted to 7.4 with NaOH). Isolated cells were plated on the concanavalin A-coated glass cover-slip. Cells were maintained at 48C (up to 10 h) before use.
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02613-5
F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
The recording pipette was filled with Cs 1 solution: 124 CsCl, 1 CaCl 2 , 5 EGTA, 10 HEPES (pH adjusted to 7.4 with CsOH). To block K 1 currents high internal Cs 1 was used and 20 mM tetraethylammonium chloride was substituted for NaCl in the bath solution. The resistance of the pipette was about 6 MV. Membrane current was recorded under whole-cell patch clamp configuration [5]. For recording, the culture dish was mounted on the stage of an Olympus upright microscope with differential interference contrast optics (BX50WI, Tokyo, Japan). A patch-clamp amplifier (Axon Instruments Inc., Axopatch-200B, Burlingame, CA, USA), linked to an IBM-compatible PC, was used to measure membrane current. The voltage-clamp procedures were controlled by using the software pCLAMP (Axon Instruments Inc., Burlingame, CA, USA). Data were low-pass filtered (4-pole Bessel type) with a cut-off frequency of 5 kHz and then digitized at 10 kHz by an analog-to-digital interface. All experiments were performed at room temperature (23–258C). Test substances were applied via the patch pipette (cGMP, 8-p-chlorophenylthio-cGMP, or 8-bromo-cGMP) or through the bath (8-p-chlorophenylthio-cGMP). Odorants (amyl acetate, acetophenone or limonene) were dissolved in solutions also containing 5 mM dimethylsulfoxide (DMSO) and were applied to the cell from a U-tube system or by pressure ejection. The chemical structures of the odorants used are shown in Table 1. The stream of superfusate was directed at the cell by placing the tip of the U-tube outlet approximately 1 mm from the cell. The puffer pipettes (tip diameter about 5 mm) were placed approximately 30 mm from the cell. Surprisingly, odorants suppressed the CNG currents in the newt rod photoreceptors. At the holding potential (Vh ) of 250 mV, intracellular application of 1 mM cGMP from the recording pipette induced the sustained inward current, which gradually increased with time (Fig. 1A). This current was rapidly (time constant of approximately 10 ms) suppressed by puff application of 1 mM amyl acetate (Fig. 1A). The CNG current was not significantly changed by puff application of the odorant-free solution containing 5
Table 1 Chemical structures of the odorants Molecules
Structure
181
mM DMSO, suggest that the current was suppressed not by DMSO but by amyl acetate. This result raises two possible mechanisms. First, amyl acetate may reduce the rod CNG current by blocking its channel directly. Secondly, amyl acetate might reduce the CNG current by decreasing the intracellular cGMP concentration through the activation of a cGMP-hydrolyzing phosphodiesterase (PDE). To rule out the latter, we used a non-hydrolyzable cGMP analog, 8-p-chlorophenylthiocGMP (8-CPT-cGMP). If the latter were a case, substitution of cGMP with 8-CPT-cGMP would be expected to eliminate the suppression by amyl acetate of the CNG inward current. Application of 0.1 mM 8-CPT-cGMP from the recording pipette induced the sustained inward current, however, 8-CPT-cGMP did not prevent the suppression by amyl acetate (Fig. 1B). Similar suppression was obtained by intracellular application of a poorly hydrolyzable cGMP analog, 8-bromo-cGMP (8-Br-cGMP, 0.2 mM) and also by bath application of 0.1 mM 8-CPT-cGMP. These results exclude the latter possibility. Thus, it is likely that amyl acetate reduces the rod CNG current by blocking its channel directly. These data are summarized in Fig. 1C. Puff application of 1 mM amyl acetate reduced the inward currents induced by cGMP, 8-CPT-cGMP, and 8-Br-cGMP by more than 90%. Similarity of suppression ratio for each cGMP analog suggests that the second messenger metabolism via PDE is not involved in the suppression by amyl acetate. We also examined effects of other odorants on the rod CNG current. Puff application of 1 mM limonene and acetophenone also suppressed the CNG current induced by 0.1 mM 8-CPT-cGMP by approximately 90%, which was similar to the ratio of 1 mM amyl acetate (Fig. 1D). These results suggest that odorants block the rod CNG channel directly. Odorants also suppressed the CNG current in cone photoreceptors. Application of 0.1 mM 8-CPT-cGMP from the recording pipette induced the sustained inward current, which was also suppressed by 10 ms puff application of amyl acetate (Fig. 2A). Amyl acetate puff also suppressed the current induced by internal application of 1 mM cGMP and 0.1 mM 8-Br-cGMP and by bath application of 0.1 mM 8-CPT-cGMP (Fig. 2B). These results suggest that amyl acetate directly blocks the cone CNG channel as well. Dose-response relationships of amyl acetate against the photoreceptor CNG currents were examined by measuring the current-voltage (I–V) relationships. In the control solution without odorant, the rod CNG current induced by 0.1 mM 8-CPT-cGMP showed a linear relationship between 250 mV and 150 mV, which reversed at 14 mV (Fig. 3A). Similar reversal potentials were obtained from five cells (563 mV). Bath application of amyl acetate of several concentrations (0.1, 1 mM) made the slope of I–V curves less steep, and the I–V curves reversed at 14 mV, which was identical with the reversal potential in the
182
F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
Fig. 1. Amyl acetate suppresses the cyclic nucleotide-gated (CNG) current of an isolated newt rod photoreceptor cell. (A) The CNG inward current recorded at 2 and 5 min after rupture of the patch membrane at the holding potential (Vh ) of 250 mV. The recording pipette was filled with Cs 1 solution containing 1 mM cGMP. Puffer application of 1 mM amyl acetate for 10 ms (bottom bar) rapidly reduced the amplitude of the inward current. (B) Puffer application of 1 mM amyl acetate for 75 ms (bottom bar) also reduced the amplitude of the inward current induced by 0.1 mM 8-p-chlorophenylthio-cGMP (8-CPT-cGMP) from the recording pipette. (C) Summary of the effects of amyl acetate on the rod CNG current induced by various cGMP analogs in the recording pipette (1 mM [cGMP] p , 0.1 mM [8-CPT-cGMP] p or 0.2 mM [8-Br-cGMP] p ) and the bath solution (0.1 mM 8-CPT-cGMP). Each bin shows the mean ratio of the amplitude of the suppressed inward current to that of the sustained inward current before odor application, and error bar S.E.M. (n55). 1 mM amyl acetate was applied to a cell through the puffer pipette for 100 ms. Five min after rupture of the patch membrane, amplitude of the suppressed current was measured at 50 ms after the onset of puff application. (D) Summary of the effects of various odorants on the rod CNG current induced by 0.1 mM 8-CPT-cGMP in the recording pipette. Each bin shows the mean ratio of the suppression and error bar S.E.M. (n55). 1 mM amyl acetate, limonene or acetophenone was applied to a cell through the puffer pipette for 100 ms.
control condition (Fig. 3A). This observation suggests that amyl acetate decreases the conductance of the rod CNG channel.
Fig. 3B shows the dose-response curve of amyl acetate against the rod CNG current. The dose-response curve of 0.1 mM 8-CPT-cGMP induced current was fitted by the
F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
183
Fig. 2. Amyl acetate suppresses the CNG current of a cone photoreceptor. (A) The CNG inward current recorded at 2 and 5 min after rupture of the patch membrane at the Vh of 250 mV. The recording pipette was filled with Cs 1 solution containing 0.1 mM 8-CPT-GMP. Puffer application of 1 mM amyl acetate for 10 ms (bottom bar) rapidly reduced the amplitude of the inward current. (B) Summary of the effects of amyl acetate (1 mM) on the cone CNG currents induced by various cGMP analogs. Each bin was recorded similarly as Fig. 1C.
Hill equation with a half-blocking concentration (IC 50 ) of 72 mM and a Hill coefficient of 1.3 (Fig. 3B, n55 cells). Similar values were obtained from the cone CNG current. IC 50 of amyl acetate was 87 mM, and the Hill coefficient 1.2 (n54 cells, data not shown).
In the present study, we found that odorants suppress the photoreceptor CNG currents in a similar manner as in olfactory receptor cells [11]. The suppressive action of amyl acetate on the photoreceptor CNG currents was very rapid (|10 ms; Fig. 1, 2), which is consistent with the
Fig. 3. Concentration dependence of amyl acetate against the rod CNG current induced by 0.1 mM 8-CPT-cGMP in the recording pipette. (A) Effects of amyl acetate (concentration indicated to the right of each trace) on I–V curves. The cell was voltage clamped at 250 mV and a ramp depolarization (between –50 mV and 150 mV at a speed of 100 mV/ s) was applied in the presence and absence of amyl acetate (0.1, 1 mM). Note that the slope of I–V curves became less steep during bath application of amyl acetate, suggesting a conductance decrease. (B) Current amplitude at the command voltage of 250 mV in (A) was normalized and plotted as a function of amyl acetate concentration. Data points are mean ratios across cells (6S.E.M., n55). The continuous line shows a least squares fit of the data to the Hill equation.
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F. Kawai, E.-i. Miyachi / Brain Research 876 (2000) 180 – 184
observation of the olfactory CNG current [11]. In addition, the suppression was also observed even when hydrolysis of cGMP was inhibited. These results suggest that the second messenger system via PDE is not involved in the suppression. Chemical structures of the odorants used in the present experiment (Table 1) show the high lipid solubility and neutral charge. Therefore, it is likely that these molecules can readily cross the cell membranes and can bind to hydrophobic sites of CNG channels. Furthermore, it is known that odorants non-selectively suppress the voltage-gated currents in the olfactory cells [6–9] and the retinal horizontal cells [10]. Thus, we suggest that odorants directly block both olfactory and photoreceptor CNG channels as well as the voltage-gated channels.
Acknowledgements This work was supported by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, and Grant-in-Aid for Science Research from the Ministry of Education, Science and Culture (No. 12780620 to F.K.; No. 11680794 to E.M.).
References [1] H.A. Bakalyar, R.R. Reed, The second messenger cascade in olfactory receptor neurons, Curr. Biol. 1 (1991) 204–208.
[2] D.A. Baylor, T.D. Lamb, K.-W. Yau, The membrane current of single rod outer segments, J. Physiol. (Lond) 288 (1979) 589–611. [3] S. Firestein, F.S. Werblin, Gating currents in isolated olfactory receptor neurons of the larval tiger salamander, Proc. Natl. Acad. Sci. USA 88 (1987) 6292–6296. [4] G.H. Gold, T. Nakamura, Cyclic nucleotide-gated conductances: a new class of ionic channels mediates visual and olfactory transduction, Trends. Pharmacol. 8 (1987) 312–316. [5] O.P. Hamill, A. Marty, E. Neher, F.J. Sakmann, Sigworth, Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pflugers Arch. 391 (1981) 85–100. [6] F. Kawai, T. Kurahashi, A. Kaneko, Nonselective suppression of voltage-gated currents by odorants in the newt olfactory receptor cells, J. Gen. Physiol. 109 (1997) 265–272. [7] F. Kawai, T. Kurahashi, A. Kaneko, T-type Ca 21 channel lowers the threshold of spike generation in the newt olfactory receptor cell, J. Gen. Physiol. 108 (1996) 525–535. [8] F. Kawai, Odorant suppression of delayed rectifier potassium current in newt olfactory receptor cells, Neurosci. Lett. 269 (1999) 45–48. [9] F. Kawai, Odorants suppress T- and L-type Ca 21 currents in olfactory receptor cells by shifting their inactivation curves to a negative voltage, Neurosci. Res. 35 (1999) 253–263. [10] F. Kawai, E.-I. Miyachi, Odorants suppress voltage-gated currents in retinal horizontal cells, Neurosci. Lett. 281 (2000) 151–154. [11] T. Kurahashi, G. Lowe, G.H. Gold, Suppression of odorant responses by odorants in olfactory receptor cells, Science 265 (1994) 118–120. [12] T. Kurahashi, K.-W. Yau, Tale of an unusual chloride current, Curr. Biol. 4 (1994) 256–258. [13] K. Nakatani, Y. Koutalos, K.-W. Yau, Ca 21 modulation of the cGMP-gated channel of bullfrog retinal rod photoreceptors, J. Physiol. (Lond) 484 (1995) 69–76. [14] D. Restrepo, J.H. Teeter, D. Schild, Second messenger signaling in olfactory transduction, J. Neurobiol. 30 (1996) 37–48.