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Figure 1. Tetracaine (Ttc) inhibition of currents elicited by ACh (IAChs). (A) Molecular structure of Ttc, showing the amine group largely charged at the recording pH. (B) Superimposed IAChs elicited by 10 μM ACh either alone (Control) or co-applied with different Ttc concentrations, as stated on the right. Note that IAChdecay was accelerated at Ttc concentrations of 0.5 μM or higher. Unless otherwise stated, the holding potential was −60 mV, downward deflections represent inward currents and the bars above the recordings indicate the timing of drug application. (C) Ttc concentration-IACh inhibition relationship. Peak (Ip; black symbols) and steady state (Iss, measured 20 s after the peak; gray symbols) IACh amplitudes elicited in the presence of Ttc were normalized to the IACh evoked by ACh alone (Control) and represented against the logarithm of Ttc concentration. Solid and dashed lines are sigmoid curves fitted to Ip and Iss data, respectively. Note that both curves overlap up to 0.1 μM Ttc. Error bars indicate SEM. Each point is the average of 4–23 oocytes from 3 to 11 frogs.
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Figure 2. Voltage dependence of nicotinic acetylcholine receptor (nAChR) blockade by tetracaine (Ttc). (A)
IAChs (upper traces) elicited by 10 μM ACh either alone (A1,A2, black recordings), or in the presence of 0.1 μM (A1, orange), or 0.7 μM Ttc (A2, red) when the voltage protocol, indicated below the currents, was applied. (B) Plots of net i/v relationships for IAChs evoked, following the protocol shown in A. Control IAChs are represented by black symbols and lines (B1,B2), whereas those evoked in the presence of 0.1 μM (B1) and 0.7 μM Ttc (B2) are drawn in orange and red, respectively. Values were normalized as a percentage of current with reference to their control IACh at â60 mV. Each point is the average of 5 (N = 1) and 12 (N = 3) cells for 0.1 and 0.7 μM Ttc, respectively. (C) Kinetics of the voltage-dependent blockade of nAChRs at â60 mV. (C1)
IAChs were elicited by 10 μM ACh alone (control, black recordings), or together with either 0.1 μM (orange trace) or 0.7 μM Ttc (red recording) at â60 mV; during the IACh plateau, an 800 ms voltage jump to +40 mV was given (bottom trace shows the voltage protocol). Membrane leak currents (recorded in the absence of ACh) have been subtracted. (C2) Zoomed in view of the areas indicated by arrows in C1 (immediately after the voltage jump). Kinetics of the voltage-dependent blockade of nAChRs by 0.1 μM (orange trace) and 0.7 μM Ttc (red trace) were determined by fitting the net IACh decays to exponential functions (green curves over the recordings). The small, slow IACh changes evoked by the voltage pulse when the cell was bathed solely with ACh (black recordings in C1) have been subtracted. (C3) Time constant values of the voltage-dependent IACh blockade kinetics elicited by 0.1 and 0.7 μM Ttc. Asterisk indicates significant differences between both values (p < 0.05, t-test).
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Figure 3. Pharmacological profile of nicotinic acetylcholine receptor (nAChR) blockade by tetracaine (Ttc). (A)
IAChs evoked by different ACh concentrations (10, 100 μM, and 1 mM) either alone (A1,A2, black recordings), co-applied with 0.7 μM Ttc (A1, red recordings), or co-applied with 0.7 μM Ttc, after Ttc pre-application for 12 s at the same concentration (A2, red recordings). (B) Averaged ACh concentration-IAChamplitude relationship. IAChs were evoked by different ACh concentrations alone (filled black circles; n = 10â13, N = 3), or co-applied with 0.7 μM Ttc, either directly (open circles; n = 3â6, N = 2), or subsequent to its pre-application (open triangles; n = 4â7, N = 2). Data were normalized to the maximal IACh elicited by ACh alone and fitted to the Hill equation (solid and dashed lines). (C) Percentage of IACh inhibition when different ACh concentrations were directly co-applied with 0.7 μM Ttc (circles and solid line; n = 9â33, N = 4â13), or after pre-application of the same Ttc concentration for 12 s (triangles and dashed line; n = 11â21, N = 2â6). Asterisks indicate significant differences in the percentage of IACh inhibition between ACh-Ttc co-application alone, and pre- and co-application of Ttc at each ACh concentration (p < 0.05, t-test). ACh concentration effected no significant changes in the extent of inhibition by Ttc, either when Ttc and ACh were directly co-applied, or when this co-application was preceded by Ttc pre-application (p > 0.05, ANOVA; except at 3 μM ACh, indicated by the pound sign. However, IAChs at such low ACh concentration are too small for accurate determination of the percentage of inhibition).
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Figure 4. Effect of tetracaine (Ttc) application timing and holding potential on nicotinic acetylcholine receptor (nAChR) blockade. (A)
IAChs elicited at −60 mV (A1,A2,A3) and at +40 mV (A4,A5,A6) by co-application of 10 μM ACh and 0.7 μM Ttc (Co-app; A1,A4), sole Ttc pre-application before superfusion of the agonist (Pre-app; A2,A5), or Ttc pre-application followed by its co-application with ACh (Pre- and co-app; A3,A6). (B) Column graphs showing the percentages of IACh inhibition by Ttc at −60 mV (B1) and +40 mV (B2) at the Ip (red filled columns) and the Iss (red striped columns), when Ttc was applied as indicated in (A). Asterisks indicate significant differences between IACh inhibition by Ttc at Ip and Iss (p < 0.05, paired t-test). Pound signs indicate significant differences of Ip inhibition among Ttc application-timing protocols, as compared with the values for ACh and Ttc co-application (p < 0.05, ANOVA and Bonferroni t-test). Note that IACh decay was only accelerated (i.e., significant differences observed between Ip and Iss inhibition) when Ttc was either co-applied with ACh, or pre-applied and later co-applied with ACh, at −60 mV. Each column represents the average obtained from 12 to 20 oocytes (N = 4–9) for (B1), and from 6 to 11 cells (N = 2–3) for (B2).
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Figure 5. Effect of tetracaine (Ttc) application while nicotinic acetylcholine receptors (nAChRs) were activated by 10 μM acetylcholine (ACh). (A) Two superimposed IAChs elicited by 40 s pulses of ACh. The red recording shows the fast inhibitory effect of 0.7 μM Ttc, superfused when indicated by the red horizontal bar. The kinetics of IACh inhibition followed an exponential function (green trace) with a time constant similar to those found for membrane currents evoked by superfusion of a high-K+ (70 mM) solution (blue recording in B). Onset and decay of the K+ current were fitted to exponential functions (discontinuous green line). (C) Time constant values of the exponential functions fitted to the onset (On) and recovery (Off) of IACh blockade by Ttc and K+ currents. Note that the rate of IACh inhibition is conditioned by the solution exchange kinetics, but IACh recovery, after Ttc removal, exhibited slower kinetics. Asterisk indicates significant differences (n = 13, p < 0.05, ANOVA test).
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Figure 6. Acceleration of IACh decay by tetracaine (Ttc) is dependent on the concentration of acetylcholine (ACh). (A) Plot of averaged IACh decay (expressed in percentages) elicited by 10 μM ACh (A1; n = 12, N = 6) or 100 μM ACh (A2; n = 18, N = 6), either alone (black recordings) or co-applied with 0.7 μM Ttc (red traces). Green lines over the averaged recordings represent two-exponential functions fitted to the IACh decay. Insets are representative recordings of IAChs elicited by 10 μM ACh (A1) or 100 μM ACh (A2), either alone (black recordings), or co-applied with 0.7 μM Ttc (red traces). The Ip amplitudes in the presence of Ttc have been normalized to their control values for easier comparison of decay kinetics. (B) Averaged percentages of change in IACh decay elicited by 0.7 μM Ttc, computed as the difference between IAChs obtained in the absence and presence of Ttc, for currents evoked by 10 μM ACh (green line; n = 12; N = 6) or 100 μM ACh (blue line; n = 18, N = 6). Notice the earlier maximum decay acceleration (arrows) when IACh was evoked by 100 μM ACh. Zero time corresponds to the beginning of Ttc-ACh co-application and the downward deflections are due to the earlier Ip in the presence of Ttc (see inset of A1).
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Figure 7. Acceleration of the decay of currents elicited by ACh (IACh) is dependent on the concentration of tetracaine (Ttc). (A) Representative IAChs elicited by 10 μM ACh, either alone (black; A1-A6), or co-applied with 0.1 μM (orange; A1), 0.7 μM (red; A3), or 2 μM Ttc (purple; A5). The same IAChs are in the right-hand (A2,A4,A6), but their peak amplitudes were normalized to show more effectively the differences on IACh decay. (B) Normalized and averaged IACh decay elicited by 10 μM ACh, either alone (black trace; n = 43, N = 12), or plus 0.01 μM (brown trace; n = 12, N = 4); 0.1 μM (orange trace; n = 9, N = 3); 0.7 μM (red trace; n = 12, N = 6); or 2 μM Ttc (purple trace; n = 10, N = 4). Each averaged IACh decay was fitted by a two-exponential function (green lines overlapping each recording). Note that with Ttc concentrations of up to 0.1 μM, the IACh decay overlaps the control. (C) Percentages of IACh inhibition by 0.01, 0.1, 0.7, and 2 μM Ttc (same cells and color codes as in B) at different times after Ip. Low Ttc concentrations blocked nAChRs, but they did not modify IACh decay. In addition, note that the time-dependent increase in the percentage of IACh inhibition was already established 2 s after Ip. For each Ttc concentration, asterisks indicate significant differences among the percentages of IACh inhibition at different times, as compared with their respective Ip (p < 0.05, ANOVA, Bonferroni t-test).
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Figure 8. IACh desensitization increases with increasing tetracaine (Ttc) concentration. (A)
IAChs evoked by 10 μM ACh, either alone (black; A1,A2) or co-applied with 0.1 μM (orange recording; A1), or 0.7 μM Ttc (red trace; A2). Ip and Iss values are indicated by arrows in the IAChs elicited solely by ACh (Ip_Ctr and Iss_Ctr), or together with Ttc (Ip_Ttc and Iss_Ttc). Note that Ip_Ttc was reached earlier than Ip_Ctr. (B) Relationship between changes in IACh desensitization (see Equation 2) and extent of Iss inhibition evoked by different concentrations of Ttc (0.01â2 μM). The black discontinuous line is a reference indicating no change in desensitization and the blue line is the best linear fit to values falling below the reference line (0.1â2 μM Ttc). Each point represents the average obtained from 7 to 19 oocytes (N = 2â9), except for 0.5 μM Ttc, in which n = 3 and N = 1. Asterisks indicate significant differences from control desensitization (p < 0.05, one-sample t-test).
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Figure 9. Deactivation kinetics of currents elicited by ACh (IACh) are dependent on the concentration of tetracaine (Ttc). (A) Representative IAChs elicited by 100 μM ACh, either alone (black recording), or together with 0.1 μM (orange) or 0.7 μM (red) Ttc (A1). Superfusion of Ttc lasted 12 s after ACh washout. These recordings were normalized to the same Ip
(A2) to show changes in desensitization more effectively. (A3) Deactivation of IAChs shown in (A1). The black arrow indicates ACh washout and the red arrow indicates Ttc removal. (A4)
IACh deactivations shown in (A3) were scaled to the same amplitude to better compare their time course. (B) Relationship between desensitization changes (Equation 2) and the apparent deactivation time constant (Ïapparentâdeactivation) elicited by 0.1 μM (orange triangle) and 0.7 μM (red circle) Ttc. The black filled symbol corresponds to the Ïapparentâdeactivation of IAChs elicited by ACh alone, which is rate limited by the solution exchange kinetics. The green discontinuous line indicates the control desensitization ratio. Note the higher desensitization rate elicited by Ttc (values lower than 1 in the ordinate), and the slower deactivation rate (higher Ïapparentâdeactivation values in the abscissa) following a linear relationship (blue discontinuous line). Each point represents the average of 12â25 oocytes from eight donors. Asterisks indicate significant differences in desensitization and deactivation (p < 0.05, t-test), with respect to control values and the pound means differences in both parameters depending on the Ttc concentration used (p < 0.05, t-test).
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Figure 10. Idealization of two putative tetracaine (Ttc) binding sites within the channel pore. (A) Lateral (upper) and top (lower) views of the transmembrane domain (TMD), in the open nicotinic acetylcholine receptor (nAChR), showing Ttc (highlighted in cyan) bound at the higher affinity (at the middle of the pore; a1) and lower affinity (closer to the extracellular side; a2) sites. (B) Lateral view of the three nAChR domains (membrane bilayer in gray). The two Ttc binding loci within the channel pore are highlighted by a square. A zoomed in image of this frame, from the synaptic cleft, is shown on the right. Ttc molecules (in purple) were used to block the high-affinity site within the pore, to reveal the Ttc low-affinity binding site (Ttc interacting molecules shown in brown), which includes E262(α), N224(γ), K271(γ), and E274(γ) as key interacting residues.
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