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Odorant suppression of delayed rectifier potassium current in newt olfactory receptor cells
Neuroscience Letters 269 (1999) 45±48
Odorant suppression of delayed recti®er potassium current in newt olfactory receptor cells Fusao Kawai a, b,* a
Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan b Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104±6058, USA Received 15 April 1999; received in revised form 11 May 1999; accepted 12 May 1999
Abstract Effects of odorants on a delayed recti®er potassium current (IK) in newt olfactory receptor cells (ORCs) were investigated using the whole-cell version of the patch-clamp technique. Under voltage clamp, odorants (amyl acetate, limonene and acetophenone) reversibly suppressed IK without shifting its I±V curve. An amyl acetate puff completely suppressed IK induced by the ®rst step pulse of repetitive depolarizations, suggesting that binding of an odorant molecule to the open channel is not required to block the channel. Although it is known that odorants suppress Na 1 and Ca 21 currents (INa, ICa) by shifting their inactivation curves to a negative voltage, odorants did not shift the inactivation curve of IK signi®cantly. This suggests that odorants suppress IK without changing its voltage dependence. Therefore, the blocking mechanisms by odorants of IK in ORCs are different from those of INa and ICa. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Olfactory receptor cell; Potassium channel; Suppression; Odorants; Patch clamp; Newt
Depolarizing receptor potentials of the olfactory receptor cell (ORC), which are induced by odorant biding to receptor proteins at the ciliary surface, are encoded into a train of action potentials (for review, see Refs. [1,4,11,16]). The action potentials of ORCs are triggered by the activation of both Na 1 and T-type Ca 21 currents [3,7,12±15,17,18] and repolarized by the activation of K 1 currents [3,13,14, 17]. Previously, our group reported that odorants such as amyl acetate, limonene and acetophenone (banana-, lemon-, and orange-blossom-like-odor) suppress spike generation induced by current injections in isolated newt ORCs [8]. This is caused by the nonselective suppression by odorants of voltage-gated currents on the somatic membrane of ORCs. The major currents measured in newt ORCs include a Na 1 current (INa), a T-type Ca 21 current (ICa,T), an L-type Ca 21 current (ICa,L), and a delayed recti®er K 1 current (IK) [7,8]. It is also known that odorants suppress the odorinduced transduction current in the ciliary membrane of ORCs [10]. In this last experiment, we showed that odorants * c/o Dr. Peter Sterling, 123 Anatomy/Chemistry Building, The Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104-6058, USA. Tel.: 11-215-898-9228; fax: 11610-259-1096. E-mail address: [email protected] (F. Kawai)
suppress INa, ICa,T and ICa,L by shifting their inactivation curves to a negative voltage [8]. However, it is unclear how odorants suppress IK, which contributes to the repolarization of the membrane potential in ORCs. In the present study, I investigated the mechanism underlying suppression by odorants of IK in newt ORCs. Receptor cells were dissociated enzymatically from the olfactory epithelium of the newt. Dissociation protocols were similar to those reported previously [9]. In short, the animal was anaesthetized by cooling on ice, decapitated and pithed. The mucosae excised from the olfactory cavity were incubated for 5 min at 308C in a Ringer solution containing 0.1% collagenase (Sigma, St Louis, MO) with no added Ca 21. The tissue was then rinsed twice and triturated with a normal Ringer solution (in mM): 110 Na 1, 3.7 K 1, 3 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. In the present experiment, I selected receptor cells which had lost their cilia to study IK of the somatic membrane. IK was recorded in the whole-cell-recording con®guration [5]. Superfusion saline contained 110 mM Choline chloride, 3.7 mM KCl, 1 mM CoCl2, 2 mM HEPES, 15 mM glucose, 10 ppm phenol red (pH adjusted to 7.4 with KOH). The
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 42 4- 3
46
F. Kawai / Neuroscience Letters 269 (1999) 45±48
Fig. 1. Amyl acetate completely and reversibly suppresses IK of isolated newt olfactory receptor cells (ORCs) without shifting its I±V curve. (A) IK evoked by depolarization to 120 mV (Vh 2100 mV) in the control solution (thin line), after addition of 1 mM (thick solid line) and 10 mM (thick dashed line) amyl acetate, and after washout (dotted line). (B) I±V relation of IK recorded from the cell shown in (A). Peak amplitude under the control (®lled square), 1 mM (®lled circle) and 10 mM (®lled triangle) amyl acetate conditions was measured and plotted against the voltage.
recording pipette was ®lled with pseudo-intracellular (K 1) solution (in mM): 119 KCl, 1 CaCl2, 5 EGTA, 10 HEPES (pH adjusted to 7.4 with KOH). The resistance of the pipette
was about 10 MV. For recording, the culture dish was mounted on the stage of an inverted microscope with phase contrast optics (Nikon, Diaphot TMD-2, Tokyo, Japan). A patch-clamp ampli®er (Axon, Axopatch-1D, Burlingame, CA), linked to an IBM-compatible PC, was used to measure membrane current. The voltage-clamp procedures were controlled by using the software pCLAMP (Axon, Burlingame, CA). Data were low-pass ®ltered (4pole Bessel type) with a cut-off frequency of 5 kHz and then digitized at 10 kHz by an analog-to-digital interface (Scienti®c Solutions, Lab Master DMA, Mentor, OH). All experiments were performed at room temperature (23±258C). 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 stream of superfusate was directed at the cell by placing the tip of the U-tube outlet approximately 1mm from the cell. The puffer pipettes (tip diameter about 5 mm) were placed approximately 30 mm from the cell. The time resolution (approximately 20 ms) of the onset and offset of the pressure ejection system was estimated by using puff application of 3 M KCl solution. In the experiment of Fig. 1A, a depolarizing step to 120
Fig. 2. Time course of suppression by amyl acetate of IK in isolated ORCs. (A) IK was evoked by repetitive depolarization to 120 mV (Vh 2100 mV). IK amplitude was normalized by its ®rst response amplitude. Voltage steps were applied to the cell for 50 ms every 1 s. Amyl acetate (10 mM) was applied by pressure ejection for 3 s shown in the top bar. (B) IK was evoked by depolarization from Vh of 2100 mV. Command voltages were increased in 10 mV steps from 220 to 120 mV. The cell was depolarized at 0.1 s, and 1 mM amyl acetate was applied by pressure ejection between 1.2 and 4 s (top bar). (C) I±V relation of IK recorded from the cell shown in (B) before and after the application of 1 mM amyl acetate. The IK amplitude at the peak (®lled square) and 3000 ms (®lled circle) in (B) was plotted against the command voltage.
F. Kawai / Neuroscience Letters 269 (1999) 45±48
47
Fig. 3. Hyperpolarization of the membrane do not relieve amyl acetate block of IK. (A) IK was induced by depolarization from Vh of 2100 mV in the control solution. Command voltages were increased in 10 mV steps from 230 to 120 mV. (B,C) IK was induced by depolarization from Vh of 2100 mV (B) and 2150 mV (C) under application of 10 mM amyl acetate. Each response was recorded from the same cell as (A). Command voltages were also the same as those in (A). (D) Inactivation curves of IK in the control solution (®lled square) and in the solution containing 1 mM amyl acetate (®lled circle). The relative conductance was estimated as a ratio of the current amplitude induced by depolarization to 140 mV after a 5-s conditioning pulse of various voltages (2150 mV to 140 mV) to that induced by the same depolarization from the conditioning pulse of 2150 mV. The relative conductance is plotted against the conditioning voltage. Each symbol represents the mean values from ®ve cells and vertical bars SEM.
mV (Vh 2100 mV) induced a slowly activating IK of about 1000 pA in the odorant-free solution. Bath application of amyl acetate suppressed IK with no apparent change in its kinetics (Fig. 1A). The current showed full recovery after washout of odorant. IK was suppressed over the entire voltage range without shifting its I±V relation (Fig. 1B, n 6), suggesting that amyl acetate did not change the voltage dependence of activation of IK. IK was also suppressed by bath application of 1 mM limonene and acetophenone (data not shown). The effects of amyl acetate on IK were dose-dependent; the dose-response relations of IK were ®tted by the Hill equation with a half-blocking concentration (IC50) of 1.7 mM and a Hill coef®cient of 1.0. Similar values were obtained for limonene and acetophenone; the IC50 was 1.6 mM (limonene) and 1.2 mM (acetophenone), and Hill coef®cients were 1.1 (limonene) and 1.0 (acetophenone). To elucidate the blocking mechanisms by odorants, the time course of the current suppression was measured for IK. Fig. 2A shows the effects of pressure ejection of 10 mM
amyl acetate on IK evoked by repetitive depolarizing pulses. Amyl acetate almost completely suppressed IK induced by the ®rst step depolarization during puff application. Similar results were obtained by 10 mM limonene and acetophenone. Thus, binding of an odorant molecule to the open channel is not required to block the channel. After cessation of the puff, the response amplitude recovered in 4 s (with time constant of approximately 700 ms), which was signi®cantly slower than the time resolution ((20 ms) for the present U-tube system. Suppression of IK was also observed, when amyl acetate was applied to a cell from a puffer pipette during command voltage steps (Fig. 2B). An amyl acetate puff (1 mM) deceased IK rapidly (time constant ,300 ms) without shifting its I±V curve (Fig. 2C). Hyperpolarization of the membrane relieves the odorant block of INa, ICa,T and ICa,L in ORCs [8]. To test the possibility that odorant suppression of IK is similar to the mechanism of INa and ICa suppression, the relation between the suppression of IK and the holding potential was examined. At Vh of 2100 mV, 10 mM amyl acetate almost completely
48
F. Kawai / Neuroscience Letters 269 (1999) 45±48
reduced the peak amplitude of IK in the all voltage range recorded (Fig. 3B). Shifting Vh to 2150 mV under the application of amyl acetate failed to recover IK amplitude (Fig. 3C, n 5). Similar results were obtained by 10 mM limonene (n 4) and acetophenone (n 4). These suggest that hyperpolarization of the membrane do not relieve the odorant block of IK signi®cantly. To analyze the mechanisms of suppression in more detail, inactivation curves of IK in the presence and absence of amyl acetate were measured (Fig. 3D). In the control solution the inactivation curve was steeply voltage dependent around the resting potential of the newt ORCs (270 mV; [7]), but for depolarized potentials the voltage dependence was weak and inactivation did not remove the last 50% of the current. Amyl acetate did not shift the inactivation curve signi®cantly. Similar results were obtained by 1 mM limonene and acetophenone. These suggest that odorants suppress IK without changing its voltage dependence. In the present study, effects of odorants on IK in the somatic membrane of newt ORCs were investigated using whole-cell recording. At the ®rst depolarization, the odorant puff had already suppressed IK, suggesting that binding of an odorant molecule to the open channel is not required to block the channel. Therefore, odorants such as amyl acetate, limonene and acetophenone are closed channel blockers of IK. This observation is similar to the action of odorants on INa, ICa,T and ICa,L in ORCs [8], and also similar to the action of the uncharged local anesthetics such as benzocaine on INa in various preparations [2,6]. Odorants suppress INa, ICa,T and ICa,L in ORCs by shifting their inactivation curves toward negative voltages [8]. In contrast, I showed that odorants suppressed IK without changing its voltage dependence of activation and inactivation. Although the detailed mechanism whereby odorants block IK is still unclear, the present data suggests that odorants suppress IK by reducing the total K 1 conductance. This could be the result of a decrease in single-channel conductance, a decreased probability of channel opening, or to a combination of these factors. Single-channel recording is needed to discriminate between these possibilities. I thank Drs. Akimichi Kaneko and Takashi Kurahashi for their advice and discussions; Dr. Robert Smith for critical reading of the manuscript. [1] Bakalyar, H.A. and Reed, R.R., The second messenger cascade in olfactory receptor neurons. Curr. Biol., 1 (1991) 204±208.
[2] Butterworth, J.F. and Strichartz, G.R., Molecular mechanisms of local anesthesia: a review. Anesthesiology, 72 (1990) 711±734. [3] Firestein, S. and Werblin, F.S., Gating currents in isolated olfactory receptor neurons of the larval tiger salamander. Proc. Natl. Acad. Sci. USA, 88 (1987) 6292±6296. [4] Gold, G.H. and Nakamura, T., Cyclic nucleotide-gated conductances: a new class of ionic channels mediates visual and olfactory transduction. Trends Pharmacol., 8 (1987) 312±316. [5] Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. P¯uÈgers Arch., 391 (1981) 85±100. [6] Hille, B., Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J. Gen. Physiol., 69 (1977) 497±515. [7] Kawai, F., Kurahashi, T. and Kaneko, A., 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] Kawai, F., Kurahashi, T. and Kaneko, A., Nonselective suppression of voltage-gated currents by odorants in the newt olfactory receptor cells. J. Gen. Physiol., 109 (1997) 265±272. [9] Kurahashi, T., Activation by odorants of cation-selective conductance in the olfactory receptor cells isolated from the newt. J. Physiol. (Lond.), 419 (1989) 177±192. [10] Kurahashi, T., Lowe, G. and Gold, G.H., Suppression of odorant responses by odorants in olfactory receptor cells. Science, 265 (1994) 118±120. [11] Kurahashi, T. and Yau, K.W., Tale of an unusual chloride current. Curr. Biol., 4 (1994) 256±258. [12] Maue, R.A. and Dionne, V.E., Patch-clamp studies of isolated mouse olfactory receptor neurons. J. Gen. Physiol., 90 (1987) 95±125. [13] Miyamoto, T., Restrepo, D. and Teeter, J.H., Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel cat®sh. J. Gen. Physiol., 99 (1992) 505±530. [14] Nevitt, G.A. and Moody, W.J., An electrophysiological characterization of ciliated olfactory receptor cells of the coho salmon Oncorhynchus kisutch. J. Exp. Biol., 166 (1992) 1± 17. [15] Rajendra, S., Lynch, J.W. and Barry, P.H., An analysis of Na 1 currents in rat olfactory receptor neurons. P¯uÈgers Arch., 420 (1992) 342±346. [16] Restrepo, D., Teeter, J.H. and Schild, D., Second messenger signaling in olfactory transduction. J. Neurobiol., 30 (1996) 37±48. [17] Schild, D., Whole-cell currents in olfactory receptor cells on Xenopus laevis. Brain Res., 78 (1989) 223±232. [18] Trombley, P.Q. and Westbrook, G.L., Voltage-gated currents in identi®ed rat olfactory receptor neurons. J. Neurosci., 11 (1991) 435±444.
Odorant suppression of delayed recti®er potassium current in newt olfactory receptor cells Fusao Kawai a, b,* a
Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan b Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104±6058, USA Received 15 April 1999; received in revised form 11 May 1999; accepted 12 May 1999
Abstract Effects of odorants on a delayed recti®er potassium current (IK) in newt olfactory receptor cells (ORCs) were investigated using the whole-cell version of the patch-clamp technique. Under voltage clamp, odorants (amyl acetate, limonene and acetophenone) reversibly suppressed IK without shifting its I±V curve. An amyl acetate puff completely suppressed IK induced by the ®rst step pulse of repetitive depolarizations, suggesting that binding of an odorant molecule to the open channel is not required to block the channel. Although it is known that odorants suppress Na 1 and Ca 21 currents (INa, ICa) by shifting their inactivation curves to a negative voltage, odorants did not shift the inactivation curve of IK signi®cantly. This suggests that odorants suppress IK without changing its voltage dependence. Therefore, the blocking mechanisms by odorants of IK in ORCs are different from those of INa and ICa. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Olfactory receptor cell; Potassium channel; Suppression; Odorants; Patch clamp; Newt
Depolarizing receptor potentials of the olfactory receptor cell (ORC), which are induced by odorant biding to receptor proteins at the ciliary surface, are encoded into a train of action potentials (for review, see Refs. [1,4,11,16]). The action potentials of ORCs are triggered by the activation of both Na 1 and T-type Ca 21 currents [3,7,12±15,17,18] and repolarized by the activation of K 1 currents [3,13,14, 17]. Previously, our group reported that odorants such as amyl acetate, limonene and acetophenone (banana-, lemon-, and orange-blossom-like-odor) suppress spike generation induced by current injections in isolated newt ORCs [8]. This is caused by the nonselective suppression by odorants of voltage-gated currents on the somatic membrane of ORCs. The major currents measured in newt ORCs include a Na 1 current (INa), a T-type Ca 21 current (ICa,T), an L-type Ca 21 current (ICa,L), and a delayed recti®er K 1 current (IK) [7,8]. It is also known that odorants suppress the odorinduced transduction current in the ciliary membrane of ORCs [10]. In this last experiment, we showed that odorants * c/o Dr. Peter Sterling, 123 Anatomy/Chemistry Building, The Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104-6058, USA. Tel.: 11-215-898-9228; fax: 11610-259-1096. E-mail address: [email protected] (F. Kawai)
suppress INa, ICa,T and ICa,L by shifting their inactivation curves to a negative voltage [8]. However, it is unclear how odorants suppress IK, which contributes to the repolarization of the membrane potential in ORCs. In the present study, I investigated the mechanism underlying suppression by odorants of IK in newt ORCs. Receptor cells were dissociated enzymatically from the olfactory epithelium of the newt. Dissociation protocols were similar to those reported previously [9]. In short, the animal was anaesthetized by cooling on ice, decapitated and pithed. The mucosae excised from the olfactory cavity were incubated for 5 min at 308C in a Ringer solution containing 0.1% collagenase (Sigma, St Louis, MO) with no added Ca 21. The tissue was then rinsed twice and triturated with a normal Ringer solution (in mM): 110 Na 1, 3.7 K 1, 3 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. In the present experiment, I selected receptor cells which had lost their cilia to study IK of the somatic membrane. IK was recorded in the whole-cell-recording con®guration [5]. Superfusion saline contained 110 mM Choline chloride, 3.7 mM KCl, 1 mM CoCl2, 2 mM HEPES, 15 mM glucose, 10 ppm phenol red (pH adjusted to 7.4 with KOH). The
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 42 4- 3
46
F. Kawai / Neuroscience Letters 269 (1999) 45±48
Fig. 1. Amyl acetate completely and reversibly suppresses IK of isolated newt olfactory receptor cells (ORCs) without shifting its I±V curve. (A) IK evoked by depolarization to 120 mV (Vh 2100 mV) in the control solution (thin line), after addition of 1 mM (thick solid line) and 10 mM (thick dashed line) amyl acetate, and after washout (dotted line). (B) I±V relation of IK recorded from the cell shown in (A). Peak amplitude under the control (®lled square), 1 mM (®lled circle) and 10 mM (®lled triangle) amyl acetate conditions was measured and plotted against the voltage.
recording pipette was ®lled with pseudo-intracellular (K 1) solution (in mM): 119 KCl, 1 CaCl2, 5 EGTA, 10 HEPES (pH adjusted to 7.4 with KOH). The resistance of the pipette
was about 10 MV. For recording, the culture dish was mounted on the stage of an inverted microscope with phase contrast optics (Nikon, Diaphot TMD-2, Tokyo, Japan). A patch-clamp ampli®er (Axon, Axopatch-1D, Burlingame, CA), linked to an IBM-compatible PC, was used to measure membrane current. The voltage-clamp procedures were controlled by using the software pCLAMP (Axon, Burlingame, CA). Data were low-pass ®ltered (4pole Bessel type) with a cut-off frequency of 5 kHz and then digitized at 10 kHz by an analog-to-digital interface (Scienti®c Solutions, Lab Master DMA, Mentor, OH). All experiments were performed at room temperature (23±258C). 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 stream of superfusate was directed at the cell by placing the tip of the U-tube outlet approximately 1mm from the cell. The puffer pipettes (tip diameter about 5 mm) were placed approximately 30 mm from the cell. The time resolution (approximately 20 ms) of the onset and offset of the pressure ejection system was estimated by using puff application of 3 M KCl solution. In the experiment of Fig. 1A, a depolarizing step to 120
Fig. 2. Time course of suppression by amyl acetate of IK in isolated ORCs. (A) IK was evoked by repetitive depolarization to 120 mV (Vh 2100 mV). IK amplitude was normalized by its ®rst response amplitude. Voltage steps were applied to the cell for 50 ms every 1 s. Amyl acetate (10 mM) was applied by pressure ejection for 3 s shown in the top bar. (B) IK was evoked by depolarization from Vh of 2100 mV. Command voltages were increased in 10 mV steps from 220 to 120 mV. The cell was depolarized at 0.1 s, and 1 mM amyl acetate was applied by pressure ejection between 1.2 and 4 s (top bar). (C) I±V relation of IK recorded from the cell shown in (B) before and after the application of 1 mM amyl acetate. The IK amplitude at the peak (®lled square) and 3000 ms (®lled circle) in (B) was plotted against the command voltage.
F. Kawai / Neuroscience Letters 269 (1999) 45±48
47
Fig. 3. Hyperpolarization of the membrane do not relieve amyl acetate block of IK. (A) IK was induced by depolarization from Vh of 2100 mV in the control solution. Command voltages were increased in 10 mV steps from 230 to 120 mV. (B,C) IK was induced by depolarization from Vh of 2100 mV (B) and 2150 mV (C) under application of 10 mM amyl acetate. Each response was recorded from the same cell as (A). Command voltages were also the same as those in (A). (D) Inactivation curves of IK in the control solution (®lled square) and in the solution containing 1 mM amyl acetate (®lled circle). The relative conductance was estimated as a ratio of the current amplitude induced by depolarization to 140 mV after a 5-s conditioning pulse of various voltages (2150 mV to 140 mV) to that induced by the same depolarization from the conditioning pulse of 2150 mV. The relative conductance is plotted against the conditioning voltage. Each symbol represents the mean values from ®ve cells and vertical bars SEM.
mV (Vh 2100 mV) induced a slowly activating IK of about 1000 pA in the odorant-free solution. Bath application of amyl acetate suppressed IK with no apparent change in its kinetics (Fig. 1A). The current showed full recovery after washout of odorant. IK was suppressed over the entire voltage range without shifting its I±V relation (Fig. 1B, n 6), suggesting that amyl acetate did not change the voltage dependence of activation of IK. IK was also suppressed by bath application of 1 mM limonene and acetophenone (data not shown). The effects of amyl acetate on IK were dose-dependent; the dose-response relations of IK were ®tted by the Hill equation with a half-blocking concentration (IC50) of 1.7 mM and a Hill coef®cient of 1.0. Similar values were obtained for limonene and acetophenone; the IC50 was 1.6 mM (limonene) and 1.2 mM (acetophenone), and Hill coef®cients were 1.1 (limonene) and 1.0 (acetophenone). To elucidate the blocking mechanisms by odorants, the time course of the current suppression was measured for IK. Fig. 2A shows the effects of pressure ejection of 10 mM
amyl acetate on IK evoked by repetitive depolarizing pulses. Amyl acetate almost completely suppressed IK induced by the ®rst step depolarization during puff application. Similar results were obtained by 10 mM limonene and acetophenone. Thus, binding of an odorant molecule to the open channel is not required to block the channel. After cessation of the puff, the response amplitude recovered in 4 s (with time constant of approximately 700 ms), which was signi®cantly slower than the time resolution ((20 ms) for the present U-tube system. Suppression of IK was also observed, when amyl acetate was applied to a cell from a puffer pipette during command voltage steps (Fig. 2B). An amyl acetate puff (1 mM) deceased IK rapidly (time constant ,300 ms) without shifting its I±V curve (Fig. 2C). Hyperpolarization of the membrane relieves the odorant block of INa, ICa,T and ICa,L in ORCs [8]. To test the possibility that odorant suppression of IK is similar to the mechanism of INa and ICa suppression, the relation between the suppression of IK and the holding potential was examined. At Vh of 2100 mV, 10 mM amyl acetate almost completely
48
F. Kawai / Neuroscience Letters 269 (1999) 45±48
reduced the peak amplitude of IK in the all voltage range recorded (Fig. 3B). Shifting Vh to 2150 mV under the application of amyl acetate failed to recover IK amplitude (Fig. 3C, n 5). Similar results were obtained by 10 mM limonene (n 4) and acetophenone (n 4). These suggest that hyperpolarization of the membrane do not relieve the odorant block of IK signi®cantly. To analyze the mechanisms of suppression in more detail, inactivation curves of IK in the presence and absence of amyl acetate were measured (Fig. 3D). In the control solution the inactivation curve was steeply voltage dependent around the resting potential of the newt ORCs (270 mV; [7]), but for depolarized potentials the voltage dependence was weak and inactivation did not remove the last 50% of the current. Amyl acetate did not shift the inactivation curve signi®cantly. Similar results were obtained by 1 mM limonene and acetophenone. These suggest that odorants suppress IK without changing its voltage dependence. In the present study, effects of odorants on IK in the somatic membrane of newt ORCs were investigated using whole-cell recording. At the ®rst depolarization, the odorant puff had already suppressed IK, suggesting that binding of an odorant molecule to the open channel is not required to block the channel. Therefore, odorants such as amyl acetate, limonene and acetophenone are closed channel blockers of IK. This observation is similar to the action of odorants on INa, ICa,T and ICa,L in ORCs [8], and also similar to the action of the uncharged local anesthetics such as benzocaine on INa in various preparations [2,6]. Odorants suppress INa, ICa,T and ICa,L in ORCs by shifting their inactivation curves toward negative voltages [8]. In contrast, I showed that odorants suppressed IK without changing its voltage dependence of activation and inactivation. Although the detailed mechanism whereby odorants block IK is still unclear, the present data suggests that odorants suppress IK by reducing the total K 1 conductance. This could be the result of a decrease in single-channel conductance, a decreased probability of channel opening, or to a combination of these factors. Single-channel recording is needed to discriminate between these possibilities. I thank Drs. Akimichi Kaneko and Takashi Kurahashi for their advice and discussions; Dr. Robert Smith for critical reading of the manuscript. [1] Bakalyar, H.A. and Reed, R.R., The second messenger cascade in olfactory receptor neurons. Curr. Biol., 1 (1991) 204±208.
[2] Butterworth, J.F. and Strichartz, G.R., Molecular mechanisms of local anesthesia: a review. Anesthesiology, 72 (1990) 711±734. [3] Firestein, S. and Werblin, F.S., Gating currents in isolated olfactory receptor neurons of the larval tiger salamander. Proc. Natl. Acad. Sci. USA, 88 (1987) 6292±6296. [4] Gold, G.H. and Nakamura, T., Cyclic nucleotide-gated conductances: a new class of ionic channels mediates visual and olfactory transduction. Trends Pharmacol., 8 (1987) 312±316. [5] Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. P¯uÈgers Arch., 391 (1981) 85±100. [6] Hille, B., Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J. Gen. Physiol., 69 (1977) 497±515. [7] Kawai, F., Kurahashi, T. and Kaneko, A., 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] Kawai, F., Kurahashi, T. and Kaneko, A., Nonselective suppression of voltage-gated currents by odorants in the newt olfactory receptor cells. J. Gen. Physiol., 109 (1997) 265±272. [9] Kurahashi, T., Activation by odorants of cation-selective conductance in the olfactory receptor cells isolated from the newt. J. Physiol. (Lond.), 419 (1989) 177±192. [10] Kurahashi, T., Lowe, G. and Gold, G.H., Suppression of odorant responses by odorants in olfactory receptor cells. Science, 265 (1994) 118±120. [11] Kurahashi, T. and Yau, K.W., Tale of an unusual chloride current. Curr. Biol., 4 (1994) 256±258. [12] Maue, R.A. and Dionne, V.E., Patch-clamp studies of isolated mouse olfactory receptor neurons. J. Gen. Physiol., 90 (1987) 95±125. [13] Miyamoto, T., Restrepo, D. and Teeter, J.H., Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel cat®sh. J. Gen. Physiol., 99 (1992) 505±530. [14] Nevitt, G.A. and Moody, W.J., An electrophysiological characterization of ciliated olfactory receptor cells of the coho salmon Oncorhynchus kisutch. J. Exp. Biol., 166 (1992) 1± 17. [15] Rajendra, S., Lynch, J.W. and Barry, P.H., An analysis of Na 1 currents in rat olfactory receptor neurons. P¯uÈgers Arch., 420 (1992) 342±346. [16] Restrepo, D., Teeter, J.H. and Schild, D., Second messenger signaling in olfactory transduction. J. Neurobiol., 30 (1996) 37±48. [17] Schild, D., Whole-cell currents in olfactory receptor cells on Xenopus laevis. Brain Res., 78 (1989) 223±232. [18] Trombley, P.Q. and Westbrook, G.L., Voltage-gated currents in identi®ed rat olfactory receptor neurons. J. Neurosci., 11 (1991) 435±444.