Capsaicin as an amphipathic modulator of Na_V1.5 mechanosensitivity

Figures & data

Figure 1. Amphipathic compounds inhibit voltage-gated Na⁺ currents from Na_V1.5 channels expressed in HEK293 cells. (a), Molecular structures of the amphipaths (from left to right): amiodarone, capsaicin, propranolol, and Triton X-100. (b-c), Representative Na⁺ currents elicited by a step from −120 to the −35-mV test voltage (b), and peak Na⁺ current-voltage plots across all test voltages (c) with 10⁻⁹ to 10⁻[4] M (blue-red spectrum) of membrane-permeable amphipathic compounds in the extracellular solution. (d), Dose-response curves for maximum peak Na⁺ current of Na_V1.5 vs. amphipathic concentration; IC₅₀ values: amiodarone, 8.4 µM; capsaicin, 60.2 µM; propranolol, 7.6 µM; Triton X-100, 5.3 µM.

Figure 2. Capsaicin inhibits pressure- and shear-sensitivity of Na_V1.5. (a), Representative Na_V1.5 currents elicited by voltage ladders ranging −100 to 0 mV in a cell-attached patch (a) or −120 mV to −30 mV in a whole cell (b), recorded at rest (filled symbols) or with force (empty symbols), in the presence of 0 µM (black) or 20 µM capsaicin (red). Difference currents were constructed by subtracting the control Na⁺ currents from the pressure- (a) or shear-stimulated (b) currents. (c-d), Steady-state activation (c) and inactivation (d) curves of Na⁺ currents in cell-attached patches (left) or whole cells (right), recorded at rest (filled symbols) or with force (empty symbols), in the presence of 0 µM (black) or 20 µM capsaicin (red). (e-h), Maximum peak Na⁺ current (e), time constant of activation (f), and voltage dependence of activation (g, V_1/2A) or inactivation (h, V_1/2I), recorded with 0 or −30 mmHg pressure in the patch (left) and 0 or 10 mL/min flow rate in whole cells (right) in the presence of 0 µM (black) or 20 µM capsaicin (red). n = 12–24 cells, *P < 0.05 comparing 0 to −30 mmHg or 0 to 10 mL/min, †P < 0.05 comparing 0 to 20 µM capsaicin by a 2-way ANOVA with Tukey posttest.

Figure 3. Effects of capsaicin on mechanosensitivity of Na_V1.5 inactivation recovery time. (a-b), Representative Na_V1.5 currents at −20 mV in a cell-attached patch (a, ●) or −30 mV in a whole cell (b, ■), elicited after recovering from the control step for 3–300 ms at −120 mV (a) or 3–1000 ms at −130 mV (b). Na⁺ currents were recorded at rest (gray) or with force (black and red traces: a, −30 mmHg pressure; b, 10 mL/min shear stress) in the presence of 0 µM (top) or 20 µM capsaicin (bottom). (c-d), Normalized peak Na⁺ current versus recovery time in the presence of 0 µM (black) or 20 µM capsaicin (red), at 0 (●) or −30 mmHg pressure (○) in the patch (c) or at 0 (■) or 10 mL/min (□) shear stress in whole cells (d). (e-f), Inactivation recovery times (t_1/2) versus 0 or −30 mmHg pressure in the patch (e) and 0 or 10 mL/min shear stress in whole cells (f) with 0 µM (black) or 20 µM capsaicin (red). n = 8–11 cells, *P < 0.05 comparing 0 to −30 mmHg or 0 to 10 mL/min, †P < 0.05 comparing 0 to 20 µM capsaicin by a 2-way ANOVA with Tukey posttest.

Figure 4. Effects of capsaicin on mechanosensitivity of Na_V1.5 use-dependent inactivation. (a-b), Representative Na_V1.5 currents at the 20^th step to −20 mV in a cell-attached patch (A, ·) or to −40 mV in a whole cell (b, ■), elicited at intersweep frequencies 3–33 Hz (a) or 3–50 Hz (b). Na⁺ currents were recorded at rest (gray) or with force (black and red traces: a, −30 mmHg pressure; b, 10 mL/min shear stress) in the presence of 0 µM (top) or 20 µM capsaicin (bottom). (c-d), Use-dependent inhibition of peak Na⁺ current versus intersweep frequency in the presence of 0 µM (black) or 20 µM capsaicin (red), at 0 (●) or −30 mmHg pressure (○) in the patch (c) or at 0 (■) or 10 mL/min (□) shear stress in whole cells (d). (e-f), Maximum use-dependent inhibition (e) or frequency of use-dependent inhibition (f) versus pressure in the patch (left) and shear stress in whole cells (right) with 0 µM (black) or 20 µM capsaicin (red). n = 8–18 cells, *P < 0.05 comparing 0 to −30 mmHg or 0 to 10 mL/min, †P < 0.05 comparing 0 to 20 µM capsaicin by a 2-way ANOVA with Tukey posttest.

Table 1. Partition coefficients and IC₅₀ values for amphipathic agents. Partition coefficients denoted logP_OW for amiodarone, capsaicin, propranolol, and Triton-X100 were previously reported [34–37]. IC₅₀, concentration at which an amphipathic agent inhibited half of the maximum peak whole cell Na⁺ current from HEK293 cells transfected with Na_V1.5. Slope, the hill slope to mechanistically characterize drug behavior based on the slope of fit

	Partition coefficient (logPOW)	IC50 (µM)	Slope
Amiodarone	7.2	8.4	0.50
Capsaicin	3.04	60	0.64
Propranolol	3.48	7.6	1.01
Triton X-100	4.6	5.3	1.00

Table 2. Effect of capsaicin on pressure-induced Na_V1.5 mechanosensitivity in cell-attached patches. Effects of pressure (0 or −30 mmHg) on parameters of macroscopic Na⁺ currents without (0 µM) or with capsaicin (20 µM): maximum peak Na⁺ currents normalized to controls at 0 mmHg (I_MAX), voltage dependence of activation (V_1/2A) or inactivation (V_1/2I), time constant of activation (τ_A), time of inactivation recovery (t_1/2R), slope of inactivation recovery (slope), maximum use-dependent inhibition (block), frequency of use-dependent inhibition (ƒ_1/2). n = 8–24 cells, *P < 0.05, 0 to −30 mmHg or †P < 0.05, 0 to 20 µM capsaicin by a 2-way ANOVA with Tukey posttest

	0 mmHg	0 µM -30 mmHg	Change	n	0 mmHg	20 µM -30 mmHg	Change	n
IMAX (%)	100.0 ± 0.0	116.6 ± 2.4*	16.6 ± 2.4	24	100.0 ± 0.0	104.8 ± 3.0^†	4.8 ± 3.0	14
V1/2A (mV)	−41.9 ± 3.0	−46.4 ± 3.0*	−4.5 ± 0.6	24	−57.5 ± 2.8^†	−60.0 ± 3.1*^†	−2.4 ± 0.6	14
V1/2I (mV)	−54.1 ± 3.1	−60.1 ± 3.3*	−6.0 ± 0.9	24	−70.5 ± 3.3^†	−71.9 ± 3.5^†	−1.4 ± 1.2	14
τA (ms)	0.32 ± 0.04	0.26 ± 0.04*	−20.0 ± 5.3%	24	0.23 ± 0.04	0.21 ± 0.04	−11.0 ± 5.4%	14
t1/2R (ms)	13.2 ± 2.5	22.1 ± 4.9*	8.9 ± 3.9	11	29.5 ± 6.4^†	48.7 ± 9.4*^†	19.2 ± 8.6	11
Slope (ms^−[^1])	1.3 ± 0.1	1.2 ± 0.1	−0.10 ± 0.17	11	1.9 ± 0.3	2.3 ± 0.8	0.41 ± 0.87	11
Block (%)	80.9 ± 7.9	77.8 ± 8.6	3.2 ± 9.4	11	93.6 ± 4.5	85.5 ± 8.5	4.7 ± 3.8	8
ƒ1/2 (Hz)	22.3 ± 2.6	20.0 ± 2.1	−2.4 ± 3.3	11	19.1 ± 2.5	14.8 ± 1.4*	−4.2 ± 1.4	8

Table 3. Effect of capsaicin on shear-induced Na_V1.5 mechanosensitivity in whole cells. Effects of shear stress (0 or 10 mL/min) on parameters of whole cell Na⁺ currents without (0 µM) or with capsaicin (20 µM): maximum peak conductance (G_MAX), maximum peak current density (I_PEAK), voltage dependence of activation (V_1/2A) or inactivation (V_1/2I), time constants of activation (τ_A) and fast (τ_F) or slow inactivation (τ_S), time of inactivation recovery (t_1/2R), slope of inactivation recovery (slope), maximum use-dependent inhibition (block), frequency of use-dependent inhibition (ƒ_1/2). n = 8–18 cells, *P < 0.05, 0 to 10 mL/min or †P < 0.05, 0 to 20 µM capsaicin by a 2-way ANOVA with Tukey posttest

	0 mL/min	0 µM 10 mL/min	Change	n	0 mL/min	20 µM 10 mL/min	Change	n
GMAX (nS)	0.98 ± 0.13	1.24 ± 0.22*	0.26 ± 0.10	12	0.78 ± 0.12^†	0.85 ± 0.15^†	0.08 ± 0.05	12
IPEAK (pA/pF)	−66.8 ± 12.3	−86.6 ± 17.7*	16.0 ± 3.1%	12	−56.7 ± 58.5^†	−58.5 ± 11.6^†	3.1 ± 3.8%^†	12
V1/2A (mV)	−57.3 ± 1.7	−58.9 ± 1.4*	−1.5 ± 0.6	12	−58.9 ± 1.5^†	−59.2 ± 1.6	−0.3 ± 0.1	12
V1/2I (mV)	−90.3 ± 3.3	−92.7 ± 2.6*	−2.5 ± 0.9	12	−95.4 ± 2.6^†	−95.8 ± 2.7^†	−0.5 ± 0.6	12
τA (ms)	0.5 ± 0.05	0.39 ± 0.02*	−20.4 ± 3.3%	12	0.39 ± 0.04^†	0.38 ± 0.04	−1.3 ± 7.0%^†	12
τF (ms)	0.82 ± 0.08	0.57 ± 0.04*	−27.5 ± 4.0%	12	0.80 ± 0.08	0.56 ± 0.05*	−25.1 ± 5.9%	12
τS (ms)	4.7 ± 0.3	3.5 ± 0.2*	−24.2 ± 4.4%	12	3.8 ± 0.3^†	2.9 ± 0.2*^†	−20.5 ± 4.8%	12
t1/2R (ms)	18.8 ± 1.7	16.6 ± 1.8*	−2.2 ± 0.6	8	38.0 ± 4.5^†	28.4 ± 4.2*^†	−9.5 ± 0.9^†	8
slope (ms^−[^1])	−1.26 ± 0.04	−1.28 ± 0.05	−0.02 ± 0.02	8	−1.43 ± 0.02^†	−1.42 ± 0.06	0.01 ± 0.04	8
Block (%)	64.6 ± 2.4	64.7 ± 2.6	0.2 ± 1.7	18	89.0 ± 1.4^†	83.9 ± 2.0*^†	−5.1 ± 0.9^†	10
ƒ1/2 (Hz)	26.1 ± 2.0	27.3 ± 2.3	1.2 ± 0.9	18	18.9 ± 1.0^†	21.0 ± 1.7	2.1 ± 1.4	10

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