Nonselective suppression of voltage-gated currents by odorants in the newt olfactory receptor cells

Abstract

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch-clamp technique. Under voltage clamp, membrane depolarization to voltages between -90 mV and +40 mV from a holding potential (Vh) of -100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na+ current: I(Na), T-type Ca2+ current: I(Ca), T, L-type Ca2+ current: I(Ca), L, delayed rectifier K+ current: I(KV) and Ca2(+)-activated K+ current: IK(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (INa), 0.15 mM (ICa,T), 0.14 mM (ICa,L), 1.7 mM (IKV), and 0.17 mM (IK(Ca)), and Hill coefficient was 1.4 (INa), 1.0 (ICa,T), 1.1 (ICa,L), 1.0 (IKV), and 1.1 (IK(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of INa was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were -53 mV at control, -66 mV at 0.01 mM, and -84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on INa in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.

Figure 1

Suppression by amyl acetate of membrane currents of an isolated newt olfactory receptor cell. (A) membrane currents were induced by depolarization from V_h of −100 mV. Command voltages were increased in 10 mV step from −90 mV to +40 mV. The cell was bathed in the normal Ringer solution and the recording pipette was filled with K⁺ solution. (B) membrane currents induced by the same depolarization as in A in the presence of 1 mM amyl acetate in the bath. (C) membrane currents measured after the wash out of amyl acetate. (D) membrane currents evoked by depolarization to 0 mV (V_h = −100 mV) in the normal Ringer solution. The pipette solution was exchanged from the standard (thin line) to the 1 mM amyl acetate-containing solution (thick line) by an intrapipette perfusion technique.

Figure 2

Effects of acetophenone and limonene on membrane currents of an isolated newt olfactory receptor cell. (A) membrane currents evoked by depolarization to 0 mV (V_h = −100 mV) in the normal Ringer solution (thin line) and in the Ringer solution containing 1 mM acetophenone (thick line). (B) membrane currents evoked by depolarization to 0 mV (V_h = −100 mV) in the normal Ringer solution (thin line) and in the Ringer solution containing 1 mM limonene (thick line). The effect of each odorant was examined in the same cell.

Figure 3

Effects of various concentrations of amyl acetate on a Na⁺ current (I_Na) of an isolated newt olfactory receptor cell. (A) I_Na evoked by depolarization to −40 mV (V_h = −100 mV) in a Ca²⁺-free solution containing 110 mM Na⁺ and amyl acetate of various concentration (0, 0.01, 0.03, 0.1, 0.3, 1 mM) recorded by using the pipette filled with Cs⁺ solution. (B) relation between the amount of suppression of I_Na and amyl acetate concentration (N = 11). Replotted from A. The continuous line is a mathematical fit of the data points to Eq. 1. Short vertical bars represent SEM (the standard error of the mean).

Figure 4

Effects of amyl acetate on a T-type Ca²⁺ current (I_Ca,T), an L-type Ca²⁺ current (I_Ca,L), a delayed rectifier K⁺ current (I_Kv) and a Ca²⁺-activated K⁺ current (I_K(Ca)) of an isolated newt olfactory receptor cell. (A) inhibition of I_Ca,T evoked by depolarization to −40 mV (V_h = −100 mV) in Na⁺-free solution containing 10 mM Ca²⁺ and 0.1 mM Cd²⁺ recorded by using pipettes filled with Cs⁺ solution (N = 6). Cd²⁺ was added to the solution to block I_Ca,L selectively. The continuous line shows a least squares fit of the data points to Eq. 1. Short vertical bars represent SEM. (B) inhibition of I_Ca,L evoked by depolarization to −10 mV (V_h = −100 mV) in Na⁺-free solution containing 10 mM Ba²⁺. To amplify the Ca²⁺ current, Ba²⁺ was substituted for Ca²⁺. 0.1 mM Ni²⁺ was added to the solution to block I_Ca,T selectively. Pipette was filled with Cs⁺ solution (N = 7). (C) inhibition of I_Kv evoked by depolarization to +50 mV (V_h = −100 mV) in Na⁺, Ca²⁺-free solution recorded by using pipettes filled with K⁺ solution (N = 5). (D) inhibition of I_K(Ca) evoked by depolarization to 0 mV (V_h = −100 mV). I_K(Ca) was obtained by subtracting the record in Na⁺, Ca²⁺-free solution containing 3 mM Co²⁺ from that in Na⁺-free solution containing 3 mM Ca²⁺ and 0.1 mM Ni²⁺. Recording pipette was filled with K⁺ solution (N = 6).

Figure 5

Time course of suppression of I_Na in an isolated newt olfactory receptor cell by amyl acetate. I_Na was evoked by repetitive depolarization to −40 mV (V_h = −100 mV) in Ca²⁺-free solution containing 110 mM Na⁺ and was recorded by using pipette filled with Cs⁺ solution. Voltage steps were applied to the cell for 20 ms every 200 ms. 1 mM amyl acetate was applied by pressure ejection for 600 ms between the onset of the fourth voltage pulse and onset of the seventh pulse.

Figure 6

Effects of amyl acetate on activation and inactivation curves of I_Na. I_Na was recorded in Ca²⁺-free solution containing 110 mM Na⁺. Recording pipette was filled with Cs⁺ solution. V_h was −100 mV in all recordings. (A) activation curves in the control solution (•) and in solutions containing amyl acetate (▪, 0.01 mM; ▴ 0.1 mM). Relative conductance at a specified membrane voltage was estimated as a ratio of the recorded current amplitude to that expected from the maximum conductance (the linear part of individual I-V curves near the reversal potential). Symbols represent mean of seven cells and vertical bars SEM. Lines represent a single Boltzmann function obtained by the least-squares nonlinear fit to the data. (B) inactivation curves in the control solution (•) and in the solution containing amyl acetate (▪, 0.01 mM; ▴ 0.1mM). Relative conductance was estimated as a ratio of the current amplitude induced by depolarization to −40 mV after a 1-s conditioning pulse of various voltages (−150 to + 50 mV) to that induced by the same depolarization without conditioning pulses. The relative conductance is plotted against the conditioning voltage. Each symbol represents mean of seven cells, and short vertical bars SEM. Lines represent a single Boltzmann function.

Figure 7

Effects of odorants on the action potentials of an isolated newt olfactory cell evoked by current injections of various intensities (2, 4, 6, 8, 10 pA). Recording were made under the current clamp condition by using pipette filled with K⁺ solution. (A) responses recorded in the normal Ringer solution. (B) responses in the Ringer solution containing 0.1 mM amyl acetate. (C) responses in the Ringer solution containing 1 mM amyl acetate.

Figure 7

Effects of odorants on the action potentials of an isolated newt olfactory cell evoked by current injections of various intensities (2, 4, 6, 8, 10 pA). Recording were made under the current clamp condition by using pipette filled with K⁺ solution. (A) responses recorded in the normal Ringer solution. (B) responses in the Ringer solution containing 0.1 mM amyl acetate. (C) responses in the Ringer solution containing 1 mM amyl acetate.

Figure 7

Effects of odorants on the action potentials of an isolated newt olfactory cell evoked by current injections of various intensities (2, 4, 6, 8, 10 pA). Recording were made under the current clamp condition by using pipette filled with K⁺ solution. (A) responses recorded in the normal Ringer solution. (B) responses in the Ringer solution containing 0.1 mM amyl acetate. (C) responses in the Ringer solution containing 1 mM amyl acetate.