Using lidocaine and benzocaine to link sodium channel molecular conformations to state-dependent antiarrhythmic drug affinity

Abstract

Rationale: Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in Na(V)1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block.

Objective: Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes.

Methods and results: The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed.

Conclusions: These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.

Figure 1

Gating charge-voltage (Q–V) relationships for WT (A) and F1759K (B) channels before and after exposure to 10 mM lidocaine. Insets show I_g for representative cells in step depolarizations to 0 mV in the absence and presence of 10 mM lidocaine. The Q_max from the Boltzmann fits to the individual cell's Q–V relationships in control solution (□) was used to normalize the Q_max in lidocaine (▲) and after wash (○). The protocols were done from a holding potential (V_hp) of −150mV. (A) For WT (n=2 cells) the Q_max after lidocaine was 0.67 ± 0.02 of control, V_½ shifted from −67 ± 4 mV to −80 ± 5 mV, and the slope factor flattened (−12 ± 3 mV to −20 ± 3 mV). All changes were significant at p<0.05. The inset shows that I_g was decreased in drug. (B) For F1759K channels (n = 4 cells) in lidocaine Q_max was 0.99 ± 0.01 of control, and the slope factor was unchanged after lidocaine (13 ± 1 mV in both groups). V _½ was significantly shifted from −72 ± 2 mV to −80 ± 1 mV. In the inset, both Ig traces appear superimposed.

Figure 2

Steady-state voltage-dependent Na channel availability protocol (A, bottom) showing data for a representative cell expressing F1759K channels in control (left) and after exposure to 1 mM lidocaine. Summary data for cells expressing WT (B) and F1759K (C) channels exposed to lidocaine. Means ± SEM in control solution are shown as open symbols, and in the presence of various concentrations of lidocaine as filled symbols. (B) Data for 0.5 mM (●, n=5) and 1 mM (♦, n=3) lidocaine are shown. V_hp was −150 mV. Mean ± SEM for parameters to fits with individual datasets: half-point −93 ± 1 mV (control), −122 ± 2.8 mV (0.5 mM), and −126 ± 4.3 mV (1 mM); slope factor: 5.3 ± 0.3 mV (control), 6.0 ±0.8 mV (0.5 mM), and 6.2 ± 1.2 mV (1 mM). Inset shows summary of shifts in V_½ of availability for cells exposed to four concentrations of lidocaine including cells exposed to 5 µM (n=7) and 50 µM (n=9), concentrations at which little reduction in I_max was evident (data not shown). (C) Data for F1759K exposed to 1 mM (♦, n=8), 3 mM (■ , n=4) and 10 mM (▲, n=3) lidocaine. Means ± SEM of parameters for fits to individual datasets were for half-point: −91 ± 1 mV (control), −104 ± 1 mV (1 mM), −103 ± 2 mV (3 mM), and −102 ± 1 mV (10 mM), and for slope factors: 6.2 ± 0.3 mV (control), 6.4 ± 0.6 mV (1 mM), 7.3 ± 0.8 mV (3 mM), and 9.3 ± 1.7 mV (10 mM). Inset shows concentration response relationship of tonic block, estimated from the fitted asymptotes of the availability relationships. ED₅₀'s were 0.78 mM ± 0.2 mM for WT, and 2.2 mM ± 0.2 mM for F7159K.

Figure 3

Q–V relationships for WT (A) and F1759K (B) channels exposed to 2 mM benzocaine. The Q_max from the Boltzmann fits to the individual cell's Q–V relationships in control solution (□) was used to normalize the Q_max in 2 mM benzocaine (◄) and after wash (○). V_hp was −150mV. (A) For WT (n=3) Q_max in benzocaine was 0.85 ± 0.03 of control, V_½ shifted from −62 ± 5 mV to −76 ± 4 mV, and s, the slope factor, flattened from −12 ± 2 mV to −17 ± 2 mV. All three changes were significant. Under similar experimental conditions used for these gating current recordings, peak I_Na was decreased by 58% ± 1% (n=3, data not shown). (B) For F1759K channels (n = 4 cells) Q_max in benzocaine was unchanged at 1.0 ± 0.01and the slope factor was unchanged (−10.9 ± 0.4 mV in control and −11.4 ± 0.2 mV in benzocaine). The shift in V _½ was significant from −69 ± 2 mV to −76 ± 2 mV.

Figure 4

Steady-state voltage-dependent Na channel availability of WT (A) and F1759K (B) channels exposed to benzocaine. Protocol as described in Methods. (A) WT in control (□), 0.3 mM (►, n=3), 0.5 mM (●, n=7), 1 mM (♦, n=6), and 2 mM (◄, n=4) benzocaine. V_hp was −135 or −150 mV. The dashed lines represent the solid lines fitted to mean data but scaled to a value of one, and adjusted for the background shift in kinetics based upon the difference in time between recordings. Means ± SEM of parameters to fits to individual datasets are for half-point: −88 ± 1 mV (control), −101 ± 1 mV (0.3 mM), - +106 ± 2 mV (0.5 mM), 117 ± 2 mV (1 mM), and −122 ± 4 mV (2 mM) and slope factor: 6.7 ± 0.2 mV (control), 6.6 ± 0.5 mV (0.3 mM), 9.0 ± 0.8 mV (0.5 mM), 8.8 ± 0.3 mV (1 mM), and 8.5 ± 0.5 mV (2 mM). Inset shows the concentration response for tonic block calculated from the means ± SEM of the I_max from the fits of the individual datasets by a Boltzmann (see Methods) normalized to the I_max in control. Single-site concentration-response fit (see Methods) estimated the ED₅₀ to be 0.32 ± 0.02 mM. (B) F1759K channels in control (□), 1 mM (♦, n=5) and 2 mM (◄, n=4) benzocaine. The dashed lines represent data scaled to a value of one and adjusted as described above. V_1/2 shifted −8.3 ± 0.7 mV (1 mM) and −9.7 ± 1.1 mV (2 mM). Means ± SEM of parameters from fits to individual datasets were for half-point: −91 ± 1 mV (control), −100 ± 2 mV (1 mM), and −99 ± 2 mV (2 mM), and for slope factor: 6.2 ± 0.2 mV (control), 5.4 ± 0.3 mV (1 mM), and 7.1 ± 0.3 mV (2 mM). Inset shows the concentration-response relationship calculated from the means ± SEM of the I_max from the fits of the individual datasets by a Boltzmann (see Methods) normalized to the I_max in control. Solid line shows the single-site concentration-response relationship; ED₅₀ was 0.66 ± 0.10 mM.

Figure 5

Proposed location for lidocaine (A,B) and benzocaine (C,D) in the closed (A,C) and open/inactivated Na channel (B,D). Lidocaine and benzocaine are shown as space-filled docking in the interface of IIIS6–IVS6 (green ribbons). Green color represents C atoms, blue represents N atoms, and red represents O atoms. Amino acid residues making contacts with drugs are shown by blue (IIIS6) and red (IVS6) colors. The optimized structures of the complexes with the inner pore were calculated with the Discover module of Insight II. It should be noted that because of the differences in sizes between residues in Na_V1.5 when compared to those in KcSA, construction of the homology model in Na channel with its bulky residues required additional optimization of the structure at the S6 crossing to avoid nonbonded repulsion between side chains. The tertiary amine of lidocaine and the primary amine of benzocaine are predicted to be oriented similarly in the open/inactivated channel where the drugs adopt a vertical orientation. The aromatic ring of benzocaine fits into the interface between DIIIS6 and DIVS6. (E, F) Lidocaine and benzocaine are shown aligned as they are oriented in the closed channel and in the open/inactivated channel. In the closed channel both are oriented more horizontally (E) with their aromatic rings aligned with Phe1759.