American Society of Anesthesiologists

Fig. 1.

$Activation of human (h) transient receptor potential vanilloid 1 (TRPV1) and human transient receptor potential cation channel, subfamily A, member 1 (TRPA1) results in a QX-314–independent reduction of sodium current peak amplitudes. (A) Typical traces of sodium currents of Nav1.7 expressed in human embryonic kidney 293t (HEK-293t) cells. Currents were activated at 0.1 Hz by 40 test pulses from the holding potentials of −120 (upper traces) or 70 mV (lower traces) to −10 mV. (B) The averaged normalized peak current amplitudes of current activated from traces 1 to 40. Note that holding at −120 mV generated currents with stable peak amplitudes throughout the protocol, whereas currents generated in cells held at −70 mV underwent a strong rundown. (C) Voltage dependency of fast inactivation of Nav1.7 expressed in HEK-293t cells. Inset: the recording protocol; cells were held at −120 mV and fast inactivation was induced by 100-ms-long depolarizing test pulses applied in steps on 10 mV between −150 and −10 mV followed by a test pulse to −10 mV. Current amplitudes were normalized to the value obtained at −150 mV and plotted against the membrane potential. The solid line represents the Boltzmann fit and the dotted lines are drawn to guide eye for estimation of the fraction of inactivated channels at −120 and −70 mV. (D) Scheme of the protocol applied to determine inhibition of sodium currents after activation of hTRPV1 or hTRPA1 ± QX-314. Sodium currents were activated by test pulses from −120 to −10 mV before and after a 60-s-long application of 1 μM capsaicin, 1 μM capsaicin + 5 mM QX-314, 1 μM capsaicin + 30 mM QX-314, 200 μM carvacrol, 200 μM carvacrol + 5 mM QX-314, 200 μM carvacrol + 30 mM QX-314, or 30 mM QX-314 alone. (E and F) Representative current traces obtained using the protocol described in D from cells expressing hTRPV1 (5 mM QX-314 together with 1 μM capsaicin, E) and hTRPA1 (5 mM QX-314 together with 200 μM carvacrol, F). (G) Bar diagrams displaying the inhibition of sodium currents following protocols described in D. The peak amplitude of sodium currents activated after application of agonist ± QX-314 were normalized to the peak amplitudes of sodium currents determined before the protocol. Note that 5 mM QX-314 did not induce a significant reduction of sodium currents compared with the application of the classical agonists alone. (H) Representative traces of lidocaine-induced tonic block of resting Nav1.7 channels. Cells were held at −120 mV and currents were activated by test pulses to −10 mV at 0.1 Hz, and increasing concentrations of lidocaine were applied to induce tonic block. (I) The dose–response curve of lidocaine-induced tonic block of Nav1.7-mediated sodium currents. Current amplitudes determined at each concentration were normalized to the current activated in control solution and then plotted against the lidocaine concentration. The solid line represents the Hill fit (see Materials and Methods). Data are mean ± SD. n.s. = not significant.$

Activation of human (h) transient receptor potential vanilloid 1 (TRPV1) and human transient receptor potential cation channel, subfamily A, member 1 (TRPA1) results in a QX-314–independent reduction of sodium current peak amplitudes. (A) Typical traces of sodium currents of Nav1.7 expressed in human embryonic kidney 293t (HEK-293t) cells. Currents were activated at 0.1 Hz by 40 test pulses from the holding potentials of −120 (upper traces) or 70 mV (lower traces) to −10 mV. (B) The averaged normalized peak current amplitudes of current activated from traces 1 to 40. Note that holding at −120 mV generated currents with stable peak amplitudes throughout the protocol, whereas currents generated in cells held at −70 mV underwent a strong rundown. (C) Voltage dependency of fast inactivation of Nav1.7 expressed in HEK-293t cells. Inset: the recording protocol; cells were held at −120 mV and fast inactivation was induced by 100-ms-long depolarizing test pulses applied in steps on 10 mV between −150 and −10 mV followed by a test pulse to −10 mV. Current amplitudes were normalized to the value obtained at −150 mV and plotted against the membrane potential. The solid line represents the Boltzmann fit and the dotted lines are drawn to guide eye for estimation of the fraction of inactivated channels at −120 and −70 mV. (D) Scheme of the protocol applied to determine inhibition of sodium currents after activation of hTRPV1 or hTRPA1 ± QX-314. Sodium currents were activated by test pulses from −120 to −10 mV before and after a 60-s-long application of 1 μM capsaicin, 1 μM capsaicin + 5 mM QX-314, 1 μM capsaicin + 30 mM QX-314, 200 μM carvacrol, 200 μM carvacrol + 5 mM QX-314, 200 μM carvacrol + 30 mM QX-314, or 30 mM QX-314 alone. (E and F) Representative current traces obtained using the protocol described in D from cells expressing hTRPV1 (5 mM QX-314 together with 1 μM capsaicin, E) and hTRPA1 (5 mM QX-314 together with 200 μM carvacrol, F). (G) Bar diagrams displaying the inhibition of sodium currents following protocols described in D. The peak amplitude of sodium currents activated after application of agonist ± QX-314 were normalized to the peak amplitudes of sodium currents determined before the protocol. Note that 5 mM QX-314 did not induce a significant reduction of sodium currents compared with the application of the classical agonists alone. (H) Representative traces of lidocaine-induced tonic block of resting Nav1.7 channels. Cells were held at −120 mV and currents were activated by test pulses to −10 mV at 0.1 Hz, and increasing concentrations of lidocaine were applied to induce tonic block. (I) The dose–response curve of lidocaine-induced tonic block of Nav1.7-mediated sodium currents. Current amplitudes determined at each concentration were normalized to the current activated in control solution and then plotted against the lidocaine concentration. The solid line represents the Hill fit (see Materials and Methods). Data are mean ± SD. n.s. = not significant.

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